bimm final

Topic 1: Introduction to BIMM 100

Molecular Biology: study of macromolecules essential for life (nucleic acids and proteins)

Central Dogma: DNA makes RNA makes Proteins

• DNA makes more DNA via replication

• DNA makes RNA via transcription

• RNA makes DNA via reverse transcription

• RNA makes proteins via translation

Topic 2: DNA Structure & Heredity

Learning Objectives:

1. Describe and diagram the basic structure of DNA & RNA, including similarities and differences.

2. Describe the fundamental discoveries that led to the field of molecular biology.

3. Explain the three experiments that discovered and verified that DNA is the molecule of heredity

4. Explain the double helix structure of DNA and how it was determined

5. Describe the directionality of nucleic acids, in terms of synthesis and overall structure.

Heredity: the passing on of physical or mental characteristics genetically from one generation to another

• Concept of heredity begins with Aristotle in 380 BCE as heredity in relation to the motions in the blood

  • Specifically, the man’s body provides instructions for development, while a woman’s body provides materials

• John Hershel utilized thoughts of cause and effect in the universe and in physics, allowing Charles Darwin to apply this thought process to living organisms in the 1830s

  • Observed diversity in populations and generated his theory of evolution by natural selection:

    • Each species shows variation

    • There’s competition for resources

    • The better adapted individual survives

    • Survivor passes on traits to offspring

  • The concept of species changing over time was accepted, but not natural selection

  • Individuals in populations exhibit variations, some of which are heritable

  • Individual organisms do not exhibit genetic change during their lifespans

  • Natural selection is partially based on the fact that not all offspring will survive due to limit resources

  • Natural selection can lead to the appearance of new species

  • Factors in the environment mean some organisms will have better reproductive success than others

  • Concept: there’s some “unit” of heredity that’s carrying traits from parent to offspring

• Gregor Mendel did experiments with pea plants in the 1850s

  • Crossed two true-breeding plants, and observed that over multiple generations traits from both parent generations appeared

  • Now we know that Mendel’s experiments were so successful because this was a very simple case of heredity, and most things don’t work out like this

  • Concept: there’s some “unit” of heredity that’s carrying these traits but we don’t know what it is

• Darwin and Mendel both provide evidence for a physical “unit” that can carry traits, but their work supports contradictory units

  • Darwin: needs to be mutable so that it can change according to best fit

  • Mendel: needs to be constant and stable so that it can pass down parental traits

Frederick Griffith - 1928 - Bacterial Transformation

• Goal: develop a vaccine for the spanish flu

• R strain bacteria: R bacteria forms colonies with a rough appearance and are nonvirulent, so they do not cause death

• S strain bacteria: S bacteria forms colonies with a smooth appearance and were virulent, killing mice

• Experiment: Griffith used four trials

1. Living S strain: mouse died

2. Living R strain: mouse lives

3. Dead S strain: mouse lives

4. Living R strain with dead S strain: mouse dies

• Conclusion: in order for the bacterial strand to be lethal, the strain must be alive, there is some transforming principle from the dead S bacteria allowing the R strain to transform into S strain

• Question: what happened in the fourth trial?

  • Something from the dead S strain is converting R strain bacteria to S strain bacteria

  • Modern solution: Bacterial plasmid, released when the cell dies and taken up by living cells

Friedrich Miescher - 1860s - Discovery of Nucleic Acids

• Goal: What’s inside the nucleus?

• Experiment: studying the cell nucleus by extracting neutrophils (white blood cells) off of bandages

• Results: discovers that it contains phosphorus and nitrogen, but no sulfur

  • Nucleotides are made of pentose sugars, triphosphate, and a variable nitrogenous base

• Modern Conclusion: nucleotides build nucleic acids through covalent bonds between the sugar of one nucleotide and the phosphate of another nucleotide

  • 3’ OH of one nucleotide attached to the 5’ phosphate of the next nucleotide

    • We consider the 5’ end the top/front and the 3’ OH the bottom/end, nucleic acids are synthesized in the 5 → 3’ direction, with new nucleotides being added onto the 3’ end

DNA vs RNA Structural Differences

• RNA has a ribose sugar: additional OH group

  • Makes RNA much more reactive

• DNA has a deoxyribose sugar: missing an oxygen

• RNA and DNA both have adenine, guanine, and cytosine

  • Purine: 2 ring structure (Adenine/Guanine)

  • Pyrimidine: 1 ring structure (uracil, thymine, cytosine)

• RNA has uracil

• DNA has thymine

• Cytosine & Guanine: 3 hydrogen bonds

• Adenine & Thymine: 2 hydrogen bonds

Nucleotide Nomenclature (NTP)

• Nucleotide = Nucleoside Tri-Phosphate = NTP

• DNA has dNTPs

  • dATP

  • dGTP

  • dCTP

  • dTTP

• RNA has NTPs

  • ATP

  • GTP

  • CTP

  • UTP

Heredity: DNA vs Proteins

• DNA has only 4 nucleotides, compared to 20 variations of amino acids in proteins, so it was commonly thought that proteins carried hereditary information

Avery, McCarty, MacLeod - 1944 - Identifying the transforming principle

• Goal: Identify Griffith’s transforming principle

• Experiment: heat-kill a bunch of S strain bacteria and centrifuge it so that the cells settle at the bottom of the tube, but we can extract filtrate with the stuff the cells released post-death

  • Mix this filtrate with the R strain and see if they become S cells

• Conclusion: firstly, there is something in the S cells that turns R cells into S cells

• Experiment 2: 3+ trials with different enzymes to kill specific cellular components

1. Protease (kills proteins): yes transform

2. RNase (kills RNA): yes transform

3. DNase (kills DNA): no transform

• Conclusion 2: DNA must be what allows these cells to transform

• Question: What is DNA and how does it encode the information of heredity?

  • → Hershey-Chase

Hershey-Chase Experiment - 1952 - Confirming DNA as the transforming principle

• Goal: Confirm DNA as the transforming principle

• Bacteriophage: viruses that attack bacteria, here specifically, simple phages composed of only an outer protein shell and inner DNA

• Prior Knowledge: these phages attached to the surface of host bacterial cells and injected a substance (either protein or DNA) into the host cell, which caused the host bacteria to make more phages

• Experiment: two different phage batches infect two identical bacteria cultures and then centrifuged

  • Centrifugation caused bacteria to form a pellet at the bottom of the tube, while lighter material including phage parts remained in the filtrate and could be removed

1. Sample 1 was produced in the presence of radioactive sulfur as sulfur is found in proteins but not in DNA (only proteins are labeled)

   1. Radioactive sulfur was found in the supernatant

2. Sample 2 was produced in the presence of radioactive phosphorus, which is found in DNA and not in proteins (only DNA is labeled)

   1. Radioactive phosphorus was found in the pellet

• Conclusion: DNA was injected into host cells and made up the genetic material of the phage

Erwin Chargaff - 1947

• Chargaff’s Rule: # purines = # pyrimidines

  • A = T

  • C = G

Rosalind Franklin - 1952 - X-Ray Diffraction

• Use an extremely pure sample in a specific salt concentration and let the liquid evaporate to get a crystal of your molecule

• She determines that DNA’s structure has 2 sugar phosphate backbones connected by nitrogenous bases with a helical twist: double helix

• “Antiparallel” strands of DNA thus means that the 5’ → 3’ of one strand runs counter to the        5’ → 3’ direction of the other strand

Topic 3: DNA Replication

Learning Objectives:

1. Describe the experiment that determined semi-conservative replication of DNA

2. Describe the process of DNA replication, including the function of each enzyme/component

3. Contrast leading and lagging strand synthesis and explain why lagging strand synthesis is a more involved process

4. Explain the role of telomeres and how they are maintained

Q. What about the structure of DNA suggests a possible copying mechanism for the genetic material?

1. Complementary base pairing, specifically advocates for semiconservative replication as there seemed to be a template strand

Potential Modes for DNA

• Semi-Conservative: one brand new strand, one original molecule strand

• Conservative: one molecule is entirely the original, one new molecule that’s entirely new

• Dispersive: patchwork

Meselson-Stahl Experiment - Semi-Conservative Mode of Replication

• Experiment: Grow E. coli in a heavy ¹⁵N broth and grow many generations to the point where we have a bacterial generation with entirely heavy ¹⁵N bacterial DNA, then switch the nitrogen isotope to the normal ¹⁴N kind

  • Centrifuge bacterial cells, the heavier something is the further down in the tube it will settle

• Results: each new generation of bacteria, with new replicated DNA, became less and less dense each time, implying a mix of old and new DNA

  • If semi-conservative: multiple bands with an increase in the % of lighter band to darker band

  • If conservative: two bands, % of lighter increases with each generation

  • If patchwork: many bands, % stays the same throughout generations

Arthur Kornberg - 1956 - DNA Polymerase synthesizes new DNA

• Experiment: combined different cellular components with a template DNA and observed which enzymes were needed to produce replicated DNA

Gyurasits & Wake - 1973 - Bidirectional Replication

• Treated bacteria with radioactive thymine

• If replication were unidirectional, we’d expect the coloring to be light in the middle and dark at the end (radioactive at one end)

• If replication were bidirectional, we’d expect there to be a dark → light → dark coloring

Eukaryotic cells have multiple origins of replication

• Bacterial cells have one origin, oriC

• DNA gets replicated in S phase, which is the longest section of interphase because chromosomes are all of different lengths/sizes

  • Having multiple origins of replication allows replication of all chromosomes to finish at the same amount of time, so more efficient, but the coordination of replication is the more complicated issue

DNA Replication

• Topoisomerase: Travels in front of helicase and smooths coiling and strains

• Helicase: travels down DNA and separates the strands

• Single-Stranded Binding Proteins: prevent DNA from closing

• Primase: places RNA primer for DNA polymerase to bind to

• DNA polymerase III: synthesizes the bulk of DNA by latching onto RNA Primer and proofreads wrong base pairing during replication

• DNA polymerase II: DNA damage repair outside of replication

• DNA polymerase I: removes and replaces RNA primers with DNA

• Ligase: glues together nicks and fragments on new DNA strands

Steps:

1. Replication Fork Formation: helicase disrupts base pair hydrogen-bonding to separate DNA strands into the replication fork, topoisomerase prevents supercoiling ahead of helicase and single-stranded binding proteins prevent the strands from closing ahead of polymerase

2. Primer Binding: RNA Primase binds to 3’ end of DNA strand and places primers

3. Elongation: DNA polymerase III binds to the primer and begins adding new base pairs complementary to the template strand

   1. Lagging Strand Synthesis: Multiple primers are placed along the lagging strand, with DNA polymerase adding new pieces of DNA between the primers to create Okazaki fragments

4. Termination: DNA polymerase I replaces the primer segments with DNA, and ligase joins together Okazaki fragments, telomerase adds telomeres

   1. The RNA Primer Issue: the last RNA primer has no 3’ end, so DNA polymerase I is unable to replace it with DNA, thus, with every round of cell division and replication, the chromosome would get shorter and shorter

   2. Solution: telomerase places telomeres, series of repeats on chromosomes that replicate often (stem/germ cells)

      1. Telomerase is a reverse transcriptase and uses a small RNA template to build out and lengthen DNA

Topic 4: DNA Repair

Learning Objectives: 

1. Distinguish between exo- and endo-nucleases

2. Describe mechanisms for fixing errors during the replication process

3. Describe mechanisms for repairing various types of DNA damage, including when each type would be used

Nucleases: cleave phosphodiester backbone in nucleotide chains

• Exonucleases: cleave on nucleotide at a time starting from the end of a chain

• Endonucleases: cleave in the middle of a polynucleotide chain

Replication Errors

• DNA polymerase has proofreading abilities as a result of a 3’ → 5’ exonuclease complex attached to polymerase

• Mismatch Repair (MMR): incorrect base pairings during DNA replication that polymerase III did not catch

1. Damage Detection proteins (MSH2, MSH6) float around in the cell during DNA replication and detect a mismatch

2. Endonuclease (MLH1) makes an internal cut in the phosphodiester backbone of the strand

3. Helicase pulls out the incorrect portion of DNA out, an endonuclease will come and break the phosphodiester bind and remove the incorrect nucleotide

4. DNA polymerase II will bind to the 3’ end and place the correct base

5. Ligase glues the pieces together

• Replication Fork Collapse: break in the phosphodiester backbone of one strand

1. DNA polymerase III detects the break, the replication fork collapses

2. 5’ exonuclease will cut off some of the broken 5’ end, leaving a 3’ overhang on the other strand

3. Lagging strand is ligated to the original strand that was broken

4. The chewed back 5’ end uses the original lagging strand’s template as its own template

5. The holliday structure junction is cut in half by an endonuclease, and ligase seals strands together

• Base Excision Repair (BER): incorrect base pairings where one base pair has turned into another, only the base itself is removed

1. Proteins within the nucleus detect a mismatch

2. DNA glycosylase removes the nitrogenous base, but leaves the phosphate backbone in tact

3. Endonuclease (AP Lyase picks it up) leaves a cut where the nitrogenous base was removed and flips out the base-less portion

4. DNA polymerase II inserts the proper base

5. Ligase seals the junction

• Nucleotide Excision Repair (NER): fixing bulky adducts like thymine-thymine dimers, the whole nucleotide is taken out

1. XP-C proteins recognize the distorted DNA helix

2. TFIIH, RPA, and XP-G open the DNA double helix and hold the strands apart

3. Two endonucleases remove either side of the thymine-thymine dimer and remove 20-30 base pairs more than the actual issue

4. DNA polymerase II fills in the gaps

5. Ligase closes the gap

• Nonhomologous End Joining: fast method of preparing double stranded breaks

1. DNA-PK complexes recognize the damage because of lack of telomeres

2. Fill in the gap on either side of the break or chew back the broken ends to make them blunt

3. Ligase ligates the sides together

• Homologous Recombination: accurate yet inefficient method of preparing double stranded breaks

1. 5’ exonuclease chews back 5’ end, 3’ strand forms an overhang

2. 3’ strand end is extended by DNA polymerase II

3. The 3’ overhang end invades sister chromatid, and new DNA is synthesized across the damaged region

4. Endonuclease cleaves phosphodiester bonds

5. Ligase ligates the gaps

• HDR can be done if a sister chromatid is present, NHEJ is done when it’s just that one chromosome present

Topic 5: Molecular Cloning & Sequencing

Learning Objectives: 

1. Describe the process of PCR including necessary components and why it is a useful tool

2. Describe the function, origin, and uses of restriction enzymes in nature and research

3. Explain the concept behind gel electrophoresis and what information it can provide

4. Describe the overall experimental approach of molecular cloning, including the function of each component and step

Gene: a DNA sequence expressed to form a functional RNA or polypeptide product

Paul Berg - Viruses DNA insertion into humans

• There must be some mechanism of cutting open a DNA strand, adding DNA, and closing it up to form a functional strand

Boyer and Mertz: plasmid and restriction enzyme discoveries

• Plasmids: small, circular pieces of DNA that replicate independently from host chromosomal DNA

• Restriction Enzymes: types of endonucleases that recognize specific initiation sequences and cut on either end of the sequence

  • Cutting on either end leaves “sticky ends”, which will hydrogen bond with each other when close together

  • Primary purpose in prokaryotes is to prevent against viruses

  • Methylation of synthesized strands prevents restriction enzymes from cutting the prokaryote’s own DNA

Gel Electrophoresis: DNA Size

• An electrical current runs through a pad of gel agarose, DNA is loaded into the gel on the negative end and is slowly drawn to the positive end

  • Smaller pieces of DNA travel further and faster than larger pieces

• Useful for making sure a restriction enzyme worked

• Ladders are segments of DNA where we know their size, and can use them for relative sizing

Berg & Cohen - Viral genes into bacterial plasmids

• First intentional process of transformation by using restriction enzymes to cut up DNA and transfer it to another cell

• Selectable Markers allow for distinctions of bacteria with the new plasmid

  • Use a drug resistance gene on the plasmid, and grow the cells on the drug so that the cells that do not have the drug die immediately

• Inefficient process

Polymerase Chain Reaction (PCR): make copies of DNA efficiently and quickly with a specific genome target

• Requires: dNTPs, DNA primers (not RNA), DNA polymerase, template DNA, buffer

1. Denaturing (separates the strands)

2. Annealing (so that the primers will land on template)

3. Extension (new synthesis)

Sanger Sequencing: Reading a Nucleotide Sequence

• ddNTPs (dideoxynucleotides) are missing an oxygen on the 3’ OH (OH → H), stopping further replication as polymerase cannot build upon the 3’ end

  • Nucleotides are connected 3’ OH → 5’ phosphate

• Sanger Sequencing gels load a lot of ddNTPs for every single nucleotide, so at every base there’s a fragment cut somewhere

• Running a gel sorts the fragments by size, and using computers and a chromatogram, you can see which base pair was cut when, and can sequence DNA like so

Topic 6: Transcription

Learning Objectives: 

1. Define transposons and the two general types

2. Describe the function of promoters and identify common promoter elements in prokaryotes and eukaryotes

3. Explain the process of transcription, including all necessary components

4. Contrast the different RNA polymerases in terms of goals, function, and associated factors

5. Describe techniques used to study protein binding to DNA and analyze the results

Transcription Overview: making an RNA copy from a DNA template

1. Initiation

2. Elongation

3. Termination

• RNA Polymerase: transcription’s main enzyme

  • Only works from 3’ → 5’, but there is no preset “template” and “coding” strand

    • The direction the polymerase sits is what determines which strand is which

  • Build off 3’OH, this bonds to first phosphate group of nucleotide and cleaves off the other two phosphate groups (ATP), which gives it the energy to keep going

• RNA polymerase works by opening the DNA strands in a small bubble at a time

  • This means that the RNA molecule does not stay attached to the DNA, and instead hangs off the polymerase like a tail

• RNA polymerase does not proofread: this is primarily because it is unnecessary, an incorrect protein likely won’t fold properly and will be disposed of later as the mRNA degrades

• RNA polymerase does not need a primer and can act as its own helicase

Mobile Elements

• The majority of our genomes are repeat regions of which we do not know what they all do

• Transposons are kinds of mobile elements that are repeat regions

Barbara McClinock - genes have physical positions

• Experiment: Corn kernels in a specific species could be white, purple, or white with purple specks

• Result: identifies specific positioning of genes on chromosomes

  • C locus gene: codes for an enzyme that produces purple pigment

    • A broken C locus gene produces white pigment (absence of enzyme)

  • Ds: dissociation element, exists on the chromosome and can move

    • Ds separate from C locus produces a fully functional C locus gene

    • Ds close to C locus can move (mobile element) and disrupt the C locus gene, creating the white kernel

      • When they move they leave a footprint and we can tell where they are

  • Ac: accelerator locus

    • If Ac is near the gene being disrupted, the mobile element moves more often

      • This causes the speckling because in certain cells the purple is expressed, and in some it is colorless

• Idea: the location of a gene is relevant

Transposons/Transposable Elements: cut-and-paste mobile elements

• Transposons are segments of DNA that can move around to different genome positions within a singular cell

  • Two kinds: DNA based Transposons and Retrotransposons

• DNA Based Transposons

  • cut itself out and reinsert somewhere else

  • Transposase: exonuclease (cut) and ligase (glue) functions

    • Blunt cuts are made on either side of the transposon

    • At the sequence specific target location for the transposon, a staggered cut is made that leaves two 9-base pair overhang on the 5’ end

    • Transposase will ligate the 5’ ends to the transposon

    • Normal repair mechanisms detect the gaps left over and ligate them shut

  • DNA Transposons CAN Duplicate

    • If, during replication, the transposon region has already been copied, the transposon can cut itself out and move ahead of the replication fork before reinserting

    • Now, in the new copy there are two transposons

    • This is infrequent but does happen

• Retrotransposons utilize reverse transcriptase so they have both an RNA and DNA intermediate

  • The retrotransposon will be expressed and transcribed (RNA), this piece turns into DNA, and reinserts into the genome

  • Sometimes you make the DNA and then put it in, in others you make RNA and turn it to DNA

  • These replicate, you’re making an additional copy and reinserting, not cutting it out and pasting

    • This is why they make up such a large part of our genome

  • LINE: Long Interspersed Element, a 2-protein-coding-region (the second region is what codes for reverse transcription, the first is largely not necessary and some retrotransposons do not have them)

    • ORF1 and ORF2 are examples of the regions (ORF = open reading frame, so a protein coding sequence)

    • ORF2 protein holds onto an RNA template that is the sequence of the full retrotransposon (this is the LINE)

    • ORF2 is a reverse transcriptase, it’s a general name for the proteins that do this and sometimes can ligate sometimes cannot

      • Identifies its target sequence and creates two nicks, one on each strand on the 3’ end

      • Flip out the end like a flap, and this starts to base pair to the RNA template

      • We grow our DNA strand so that it matches the RNA template until we get to the end of the LINE RNA

      • At the end, we reinsert the flap and copy from the original template strand until that end is closed

      • Remember: there’s still an overhang on the other strand because the LINE RNA started base pairing, so cellular repair mechanisms will fix

    • Sequences tend to look like —AATACT–[LINE DNA] –AATACT—

    • 
      

Prokaryotes have a Sigma Factor Protein

• The Sigma Factor helps position and orient RNA polymerase in the correct spot and direction

  • The Sigma Factor has a U-shaped interaction with the DNA, interacting at the -35 and -10 box on the promoter

  • There are specific sequences in these spots the sigma factor targets and sticks to, and because it’s already bound to RNA polymerase, now polymerase is in the correct spot and ready for transcription

• Consensus sequence: the most frequent letter for each position, this can tell us how conserved/important it is

  • In terms of sigma factor: the consensus sequence will allow for the strongest reaction between the sigma factor and promoter, and this will allow the most binding and thus transcription

  • Having a purposefully weaker/more different consensus sequence might be so that we only transcribe a little

  • Ex: T₆₉A₇₉ means that thymine makes up 69% of all nucleotides in this position, and adenine makes up 79% of all nucleotides in that position

Prokaryote Transcription

• Once the sigma factor binds to the promoter, RNA polymerase will begin transcription and the sigma factor will leave and rebind so the cycle can restart

• Almost every gene use the -10 and -35 boxes with the same consensus sequence and same sigma factor

• There are a couple more specific sigma factors for specific/emergency situations, but they are used more sparingly

Eukaryotic Promoter Elements: 

• Transcription elements are highly variable, nearly everything is different for every gene

• TATA box is very highly conserved, but is not in that many genes, only the very important ones

  • Being highly conserved means that the sequence doesn’t vary a lot, so any mutations in the sequence is generally selected against

Eukaryotic Transcription Initiation

• Lots of transcription factors involved, some bound to polymerase, some bound to promoter

  • Same general function: direct and orient RNA polymerase and make sure it sits long enough so that it can do its job

Eukaryotic RNA Polymerases

• RNA Polymerase I

  • pre-rRNA (ribosomal RNA is generally transcribed as one big chain and is later chopped up to form the ribosome)

• RNA Polymerase II

  • mRNA (messenger RNA for protein coding)

  • snRNA (splicing RNA)

  • siRNA, miRNA (regulation RNAs)

• RNA Polymerase III

  • tRNA (amino acid transport)

  • 55 rRNA (ribosomal RNA)

  • Other small stable RNAS (splicing, signal recognition, other various unknown functions)

• RNA Polymerase II attaches at the most different promoters/has the greatest variety of promoters

  • This is because mRNAs are so highly variable

• RNA Polymerase I is the most active at transcription

  • This is because about 80% of the RNA produced in the cell is rRNA, ribosomes are constantly needed, so polymerase I must be the most active

• RNA Polymerase Subunits

  • RNA Polymerase II has a CTD (C-Terminal Domain): repeat string of amino acids that hangs off the polymerase as a tail

RNA Polymerase II’s General Transcription Factors

• TFIIA: associated with building connections between TBP/TFIID and stabilization

• TFIIB: building a bridge for TFIID and Polymerase II connection

• TFIID: large complex made up of TATA-box binding protein (TBP) and TAFs (TBP Associated Factors)

  • Identifies promoter and recruits additional transcription factors

• TFIIE: involved in DNA melting by separating DNA

• TFIIF: binds to Polymerase II for stabilization

• TFIIH: complex with a DNA helicase and polymerase II CTD kinase function

  • Helicase cuts and is involved in excision repair, separating DNA so polymerase can work

  • Kinase activity means phosphorylation, in this situation adding phosphate to polymerase II’s CTD tail

Eukaryotic Transcription Process

1. TFIID (made up of TATA box binding protein (TBP) and its associated actors (TAFs)) bind to the TATA box

   1. Nothing can bind to the promoter region until TFIID has recognized and bonded to the promoter

2. TFIIB is recruited to additionally bind to the promoter region

3. RNA Polymerase II and TFIIF will bind to the promoter region

4. Assembly of closed preinitiation complex (PIC) prior to TFIIH binding

5. TFIIH’s helicase function opens a transcription bubble, and opens the PIC

6. Polymerase II begins transcription in a very slow manner at first, making a short 20-30 string of nucleotides 

7. At this point, TFIIH phosphorylates the CTD tail and triggers polymerase to start transcribing quickly until its termination sequence

8. RNA polymerase transcribes the DNA template strand moving in a 3’ → 5’ direction

Electrophoretic Mobility Shift Assay: EMSA

• Goal: detect protein-DNA interactions, usually transcription factors in this situation

• Run on an agarose gel with electric charge

  • Same general principle as gel electrophoresis: smaller DNA moves through the gel faster, when DNA binds to proteins it moves through the gel slower because it becomes large and bulky

• Probe: piece of DNA that’s being investigated

  • Instead of using a dye like in electrophoresis, EMSA utilizes radioactivity because they are easily detected and can allow for more precise DNA nucleotide identification

• Cold Probe: the probe piece of DNA but not radioactively labeled

  • Not used traditionally

  • Used to prove specificity of binding because it competes with the normal probe

• Lane 1: the control lane, usually holds the probe on its own

• Lane 2-4: trial lanes, protein + probe combinations

  • DNA probes that bind to proteins do not move as far as they do in the control lane

  • This is known as a “shift” if successful binding happens

• Lane 5: final lane, antibody + protein + probe combination

  • A complementary antibody to the protein causes the antibody to bind to the protein already bound to DNA

  • This is known as a “supershift” if successful because the new complex is even bulkier than the other trials, and moves even slower

DNA Footprinting

• Goal: analyze the exact sequence a protein binds to

• DNAse: enzymes that cut DNA into pieces, have some sequence specificity, but can also be random

• A protein bound to a piece of DNA does not show up on the run

  • This specific protein is called a “protection asset”

• DNA is labeled on one side with a radioactive label so we can see from which end DNAse cut in from

• When reading the footprinting: take the labeled side of the DNA with nucleotide positions and place the label at the bottom of the footprint graph, estimate the base pairs

RNA Polymerase I: pre-rRNA for ribosomes

• Ribosomes are made up of two subunits, and three rRNA subunits

• Two rRNA subunits join together with proteins to form the large ribosome subunit

• One rRNA subunit joins together with proteins to form the small ribosome subunit

• There is only one rRNA gene, and this gets transcribed into one long pre-rRNA strand which is chopped up later to form subunits

  • We have very many copies of this one gene in our genome, and they are generally sequenced one after another, so RNA Polymerase I can just keep transcribing

RNA Polymerase III: tRNA for translation

• Relies on TFIIIA, TFIIIB, TFIIIC, does not require TATA Box Binding Protein (TBP)

• tRNAs are single stranded RNAs that can fold in on themselves with modified nucleotides for other purposes

Key: Proper ribosome function relies on all three RNA Polymerases

Topic 7: mRNA Processing & Splicing

Learning Objectives:

1. Explain the process of capping, polyadenylation, splicing, and nuclear export, including necessary components

2. Describe why each of the processing steps are necessary for mRNA function, but not for all RNA

3. Explain the experiment that demonstrated the two step process of splicing

4. Describe alternative splicing as a concept and how it increases protein diversity

General mRNA Processing Steps

1. Transcription + 5’ capping

2. Cleavage at poly(A) site via endonuclease

3. Polyadenylation/formation of polyA tail via PAP (PolyA polymerase) and ATP

4. RNA splicing of introns to produce mature mRNA

mRNA Capping Process with 7-methylguanosine

• A 7-methylguanosine is added to the 5’ end of an mRNA by linking the triphosphate group of the mRNA to the phosphates of the 7-methylguanosine

3’ [7-methylguanosine]-ppp  ppp-[mRNA] 3’ ↴

3’ [7-methylguanosine]-ppp  pp-[mRNA] 3’ ↴

3’ [7-methylguanosine]-ppp-[mRNA] 3’ 

p = phosphate

• This produces a triphosphate bridge

• Normal nucleases are unable to remove this nucleotide because it is not a normal phosphodiester bond

• 5’ cap purpose: prevent mRNA degradation prior to reaching the ribosome, ribosome identification of mRNA

  • 7-methylguanosone is added specifically to mRNAs, ribosomes will only translate RNAs with the cap

• Capping enzymes attach to phosphorylated CTD tails

  • In binding to the tail they become activated and can begin the capping process

  • Because only RNA Polymerase II has a CTD tail, only mRNAs get capped, and because phosphorylation happens at the beginning of the transcription process, the cap can get added to the beginning

Polyadenylation (can happen either before or after RNA splicing)

• Poly = many; Adenylate = adding adenine

• 3’ polyA tail purpose: prevent mRNA degradation prior to reaching the ribosome, ribosome identification of mRNA

  • Exonuclease will need to cut through a lot of adenine before reaching actual codon information

• Polymerase transcribes past the point where the tail will be added and reaches a termination sequence

• At two specific sequence where the tail will be added, nucleases will come and cut the additional nucleotides

  • On one side of the cut, there is an AAUAAA sequence, and a G/U rich region on the other side of the cut

  • Proteins recognize these components and cut in between them before discarding the G/U rich region

  • This AAUAAA sequence needs to be relatively conserved, all in the upper 90% for each nucleotide

• CPSF and CStF recognize the signal locations

• CFII and CFI complex cleaves in between the sequence

• PAP can start a slow adenylation of adenines onto the now free 3’ OH group

  • PAP begins slowly but gets faster as it continues adding

• CTD phosphorylation is required for both cleavage and polyadenylation

  • Once the cap is added, enzymes that recognize the polyA sequence attach to the CTD tail after capping enzymes leave and wait for polymerase to reach the end

mRNA Splicing

• Small transcripts with few exons will wait for capping/polyadenylation for splicing

• Large transcripts with lots of exons will cap and polyadenylate prior to splicing

• Introns: nucleotide sequences not necessary for protein synthesis instructions

  • Ribosomes cannot differentiate between introns and exons, so introns must be spliced out prior 

• At the end of each intron is a conserved adenine that attacks the phosphodiester bond between the last nucleotide of the exon and the first nucleotide of the intron (where the cut will be)

  • The 2’OH of the adenine is what does the “attack”

  • It breaks the bond between those nucleotides and creates a "lariat" or “loop” attached to both the conserved adenine and the first nucleotide of the intron

5’ —[intron]-[A] —3’   ↴

5’ —      +    ር^[A]_—3’        ↴

5’ —।—3’    +    ር^[A]     

• This occurs via transesterification, where the spliceosome mediates and holds onto both the lariat and the exons at the same time

  • It also attaches the two exons together at the end

Studying mRNA Splicing via Radioactive Isotopes

• Every nucleotide in mRNA is made radioactive so they appear on a gel

• Mix a pre-mRNA with nuclear components (stuff in the cell to make the pre-mRNA a mature mRNA)

• Let this incubate, taking a sample at different points in time to see how the mixture develops over time

• A: intermediate product

  • Becomes strong but disappears at the end

  • Likely lariat + exon because it is at the top of the gel, and lariats are big and bulky

• B: final product

  • The only band that got more visible over time and is present at 60 minutes

  • Likely just the lariat

• C: pre-mRNA

  • Gets gradually smaller/fainter over time, but as we see the control at 0 minutes it is the only band present, and must be the pre-mRNA

• D: final product

  • Increases over time, but still faint, meaning not much product has been produced and it must be the final step

• E: intermediate product

  • Slightly fainter at 60 minutes than at 15 minutes, so it must be a final step but not a final product

  • Likely just an exon by itself because it has moved further down in the gel and is the smallest

• Splicing sequences are not very conserved, there are some very common ones, and a general trend

  • Start with a G/U end, end with an A/G pyrimidine-rich region and adenine branch point 

snRNAs: mediating splicing

• Coded by polymerase II and III

• Spliceosomes are formed of a giant complex labeled U# (U1, U2, ect.)

• U1 snRNA: complementary to the front end of the intron

• U2 snRNA: identifies the branch point and is near complementary to this sequence

  • It is complementary to surrounding nucleotides, just not the specific 2’OH attacking A

  • This is so that it distorts the mRNA structure by pushing the A out, letting it attack the intron easier

• The rest of the spliceosome will assemble with the first and second transesterification before splicing

• Compensatory Mutation: the exact sequence of the intron/exon area doesn’t necessarily impact if splicing will happen, it’s the complementary aspect of the snRNA to the mRNA that dictates whether the cut region will be identified, and thus spliced

Alternative Splicing

• There are alternate ways mRNAs can be spliced, it is the norm for there to be multiple splicing pathways

  • EX: 5 exons, exons 1, 2, and 5 are always spliced out, exons 3 and 4 are not always spliced

  • Splicing or not splicing certain things allow for specific binding patterns of the resulting protein that can result in different additional functions (like nonpolar/polar)

• Boxes: Exons

• Lines: Introns

• Up/Down Lines: Splicing Options, how things could be connected

• Splicing Silencers: proteins that can be present, recognize splicing binding sites and bind to them

  • By binding to them, the spliceosome cannot recognize the sequence

Dscam in Drosophila

• Exon 4, 6, 9, and 17 have a lot of alternative splicing patterns

• This leads to 95 exons that can be spliced to create 38,016 slightly different proteins

• Thus, on each neuron we have slightly different neurons, whose purpose is to branch and make connections with other branches

  • This allows it to identify what cell it is, and make connections only with cells that are different and new

Nuclear Export

• RNAs have many reasons for leaving the nucleus

  • In mRNA case: ribosomes are in cytoplasm

  • Some RNAs never need to leave the nucleus depending on their function

• Nuclear Pore Complexes require recognition of a lot signifying factors before something can exit or enter the nucleus

  • They’re very complex and very highly controlled

• After splicing is over, REF (RNA Export Factor) is at exon junction where splicing occurred, and can interact with and recruit other proteins for export

  • RNAs are encoded with proteins everywhere, some will leave before export, others show that the mRNA is done

  • CBC: bound at cap, shows capping occurred

  • PABP: shows polyA tail is attached

  • NXF, Nxt: shows splicing occurred

• NPL3 is a species specific version of REF class of proteins

  • They bind as a part of the spliceosome and are left behind as a tag that indicates splicing has happened

  • They are phosphorylated

  • An enzyme will come at the end of polyadenylation (dephosphorylated), which indicates that capping and polyadenylation has occurred

  • Export factors can only bind to REFs after the REF has been dephosphorylated, so it cannot leave

  • In the cytoplasm an additional protein will rephosphorylate the REF, causing the REF to detach and go back to the nucleus for re-use, and the mRNA will be translated

Topic 8: Translation

Learning Objectives:

1. Determine the reading frame of an mRNA

2. Translate DNA and RNA sequences into amino acid sequences

3. Describe how tRNA and amino acids are correctly attached

4. Explain the process of translation including all three stages and important components

5. Identify and classify small-scale mutations in protein coding regions

Holley, Khorana, Nirenberg - 1968 - Determining the Genetic Code

• Synthesized mRNAs with specific nucleotide sequences and radioactive amino acids to see what amino acids get sequenced with what sequence

  • “What happens with a string of As, Gs, AGA, ect.”

• In the universal codon table, read left-top-right for codon 1-codon 2-codon 3

Understanding the Reading Frame

• The ribosome will always begin with the AUG on the mRNA

• Methionine is thus the first amino acid incorporated

• A stop codon will always end termination

• AUG and the stop codon are not at the direct beginning or end of the mRNA, there are UTR regions at the beginning and end that are untranslated

Translation with tRNAs

• Ribosome builds peptide bonds between amino acids, whose sequence is determined by the mRNA

• tRNAs determine which amino acid is used, they are responsible for specificity

  • tRNAs have an anticodon with complementary amino acid

  • The 3’ end has an acceptor stem where the amino acid becomes present

  • tRNAs can have different nucleotides that we don’t usually side, like inosine

    • Inosine has a broader base pairing ability that adenine does not

    • Inosine is also at the anticodon for tRNA because the genetic code is redundant and has multiple possibilities

• Aminoacyl-tRNA Synthetase: enzyme specific for each amino acid

  • Two binding sites, one to bind to the amino acid, one for the tRNA

  • Responsible for matching the two and creating a high-energy ester bond

• Inosine being able to bind to multiple things has to do with the wobble theory, where the third nucleotide does not matter

Ribosome Structure

• Ribosome structure: 

  • E: exit for tRNA

  • P: polypeptide site

  • A: aminoacylated site

Translation Steps

1. Initiation: (assembling ribosome at start codon)

   1. Polya tail and 5’ cap both need to be present and checked by the ribosome

   2. Preinitiation complex: small ribosomal subunit attached to some initiation factor

   3. tRNAs always have a protein attached

   4. eIF2: eukaryotic initiation factor, specific protein that binds to methionine tRNA

      1. Methionine has 2 tRNAs, normal ones for normal elongation, and tRNA_i^MET which is the start codon tRNA (special)

      2. eIF2 is bound to this special initiation tRNA

   5. This creates the preinitiation complex: small ribosomal subunit with eIF2-GTP attached to special tRNA_i^MET

   6.  Initiation-assembly on mRNA

      1. Small subunit only attaches if the EIF4F (cap-binding complex) and PABP (polyA binding protein) bind to both and hold them in a loop?

      2. Now the small subunit will slide down mRNA until it gets to AUG, and the large ribosomal subunit will slide on top

      3. Reorients so that the MET is at the P site

2. Elongation: (Cycle for every amino acids that needs to be added)

   1. tRNA lands at the A site

      1. Random tRNAs go in, it’s likely reaching blindly for the correct one, if you do get the correct one, it’ll bind, and that energy from the GTP creates the amino acids to tilt, which causes the ribosome to break the connection between the amino acid and its tRNA and puts them between the old amino acid and the new amino acid in the A site

         1. Now both are in the P site as the ribosome moves over one codon

         2. tRNA in the P site will leave, tRNA in A site moves to P site, repeat

3. Termination: (reach a stop codon, how do we break everything apart)

   1. There are three stop codons, there is no tRNA that binds to these codons

   2. We have ERF1 and ERF3 that work together as eukaryotic release factors

      1. Triggers cleavage of polypeptide from tRNA and releases the tRNA

      2. Triggers the large and small ribosomal subunits to release from mRNA, so everything can be reused

   3. A particular triplet of bases in the coding sequence of DNA is AAA. anticodon on tRNA that binds to the mRNA codon is:

      1. Coding seq: AAA

      2. Template strand: TTT

      3. mRNA: AAA

      4. tRNA: UUU

• mRNA loop helps with translation efficiency, we want lots of copies of the protein

  • This makes sense because we can reattach the components really easy cause we’re right back to the start

• Coupled transcription/translation

• Can only be used to describe mutations in the reading frame:

  • Substitution mutations:

    • Silent mutation: doesn’t change anything

    • Missense mutation: changes the amino acid

      • We don’t know if this alters the protein bad or good or no effect, we figure this out through experimentation

    • Nonsense mutation: mutation changes codon to a stop codon

      • Basically always destroys the function

  • Frameshift mutation

    • insertion/deletion in non-multiples of three, usually causing extensive missense mutations in a destructive manner

Topic 9: Regulation of Expression

Learning Objectives:

1. Explain the function and regulation of the Trp and Lac operon

2. Explain the function of enhancers in eukaryotic gene regulation

3. Describe the role of mRNA degradation in regulation of gene expression

4. Explain how ncRNAs can regulate gene expression

Cell Diversity

• Regulation of gene expression is fundamental to making the diverse range of cells we have work

  • Ex: neuron cells are long, cardiovascular cells are big and bulky

• But all of our cells have the same DNA, so how do we get these different shapes and functions?

  • Regulation of transcription of certain genes/traits

• Regulation of gene expression usually comes in levels of transcription rather than a binary off/on switch

• If every gene was always active, we wouldn’t be able to control resources

Metabolic Regulation

• Inhibitors can turn enzymes on and off in order to prevent/use substrate resources

• You can also control whether or not you make the enzyme itself

Prokaryotic Transcription

• Sigma factor recognizes two sites on the promoter to bring in RNA polymerase and begin transcription

• In prokaryotes the same sigma factor regulates the expression of most genes

  • Some specific genes/environments initiate the presence of specific sigma factors

    • Ex: low iron, flagella assembly

• Because prokaryotes don’t have lots of transcription factors, they use a different kind of gene regulation from eukaryotes (our vast variety of transcription factors allows for more control of gene expression in this way)

  • Prokaryotes have operons (eukaryotes do too but it’s not common)

Operons

• Operons are stretches of DNA that include the operator, promoter, and genes that they control

• For every operator is a repressor protein that can bind to the operator and turn the operon off, preventing transcription of the gene

• Operons are inducible (usually off, but able to be repressed) or repressible (usually on, but able to be repressed)

• Structurally: promoter + operator + the gene being transcribed

  • We make one big mRNA that contains the coding sequences for all three proteins in one

    • This is why this happens in prokaryotes and not really in eukaryotes (we don’t have these series of start and stop codons on a single mRNA)

    • This is called polycistronic: multiple genes being transcribed on one mRNA

• What’s the advantage of having genes grouped into operons?

  • Efficiency with turning on all of the genes at once (usually the genes will have a related function, so we likely need all when we need one)

• What’s the usual relationship between the genes in the operon and the molecule that regulates it?

  • Generally, the genes on the operon are either making the substrate that’s going to repress the operon, or when the substrate is present the operon can use it to turn on the gene

  • So the substrate being produced by the genes in the operon can act as an inducer and repressor

Lac Operon - making the enzymes that use lactose (inducible)

• Codes for three genes: LacZ (breaks down lactose to release them for glycolysis), LacY (encodes for the transmembrane protein that lets lactose into the cell), LacA 

  • Codes for enzymes that use lactose in metabolism

• The lac repressor is always present and active on its own

  • It’s turned off by the presence of lactose, which allows for the genes to be transcribed

• As soon as the repressor is produced it will bind go the operator region and cause a physical blockade that prevents the gene from being transcribed

• When lactose becomes present, lactose will bind the repressor protein and cause a conformational change that inactivates the regulatory protein and allow polymerase to successfully transcribe the gene

• Glucose represses transcription of lac operon

  • In the presence of glucose: adenylate cyclase is inhibited by glucose and prevents cAMP from being made

  • No cAMP means CAP activator protein cannot bind to CAP site near lac promoter

• Structure: CAP site (increase transcription) + promoter + operator + lac genes

  • CAP site is bound by CAP proteins, but these can’t bind on their own and require a transcriptional activator (cAMP)

• Low glucose: CAP binds upstream and activates transcription

• High glucose: CAP dissociates from lac operon and inhibits transcription and metabolism of lactose

Trp Operon - making the enzymes that make tryptophan (repressible)

• It’s repressible, usually on, but we can turn it off

  • This makes sense because tryptophan is an essential amino acid

• Tryptophan is only synthesized when there are none available outside the cell

  • Usually genes for enzymes that make things their default states are on

• Tryptophan helps the repressor protein to bind the operator and switch the operon off

• Operon structure: promoter + operator + genes of interest

  • Operator only works if it’s in between the promoter and genes of interest

  • Before the operon there’s a regulatory gene that’s always being transcribed at a low level

  • This regulatory gene codes for a repressor

• In the absence of tryptophan:

  • Polymerase translates the entire operon with no issue

• In the presence of tryptophan:

  • Tryptophan is a corepressor, when it binds to the repressor protein the repressor undergoes a conformational change into an active shape, which is now able to bind to the operator sequence

  • This stops polymerase from being able to transcribe because the repressor protein is large, and physically blocks the polymerase from continuing transcription past the operator

  • Once we have enough tryptophan, we don’t want to make anymore, so it becomes a corepressor

• What’s the result of a mutation that inactivates the regulatory gene of a repressible operon in a cell?

  • Deactivating the regulatory gene of a repressible operon implies that the operon is able to be repressed, and is usually on. Thus, disabling the regulatory gene would prevent it from stopping being transcribed, and there would be continuous transcription of the structural gene controlled by that regulator

Eukaryotic Gene Regulation

• Can regulate lots of things:

  • how tightly chromatin is wrapped around histones

  • mRNA exportation

  • mRNA degradation

• There are many kinds of transcription factors

  • General transcription factors like TFIIH or TFIID are necessary for transcription of ALL genes, so if they aren’t present there will be low rates of transcription for all genes

  • Specific transcription factors are transcription factors required for the transcription of just a few genes

    • They can activate/inhibit transcription

    • They bind specific DNA sequences/other proteins

• General gene structure: upstream enhancer + proximal control + promoter + exons/introns + poly-A sequence + stop codon

  • Enhancers can be really far away from things/the promoter

    • The reason why this still works is because the DNA is just a string that can be moved around

  • Proximal control units are enhancers that are next to the promoter

• CPG Island is the most common eukaryotic promoter (not TATA box!)

  • But the TATA box is super highly conserved, why is that?

    • TATA box is very inflexible (positioning + sequence is very precise)

      • The binding with its proteins is pretty strong, so when it’s present things will probably be transcribed

    • CPG islands are more flexible, so we can make some that are strong or weak, ect.

      • We have more versatility in expression

Gene & Transcript Structure with Modularity

• Most proteins binding to DNA have 2 domains: DNA binding domain + activation domain (interacts with transcription factors and polymerase to activate/enhance expression)

  • These proteins usually have to dimerize, two must come together in order to work

    • One on its own is usually very weak

    • You can use different combinations of just a handful of these proteins and already have so many ways to bind to DNA, allows for more specific regulation

• Visually, because DNA can bend the enhancer elements don’t need to be close to the genes they’re regulating because DNA can bend, so they can still mediate the reactions

• Enhancers can also help mediate alternative splicing

  • They can bind to different locations depending on where in the body the cell is

    • This is because different cells have different proteins being made

• What is the functional difference between enhancers and promoter proximal elements?

  • Enhancers can be located far from the promoter they regulate and are orientation distance independent

  • Proximal elements are located closer to the promoter and are often position and orientation dependent

• How could you experimentally determine whether a specific sequence is a promoter or an enhancer?

  • Take the sequence of DNA (promoter/enhancer), move it far away and see if transcription occurs and what was transcribed

mRNA Degradation Pathways

• Degrading mRNA early would reduce how much of a protein gets made

• Deadenylation-dependent mRNA decay

  • Removing the polyA tail first

  • Deadenylase (enzyme complex) chews through mRNA tail

    • Better at removing then a standard exonuclease

  • Two pathways:

    • Decappnig enzyme removes the cap, exonuclease chews away 5’ end

    • Large nuclease complex (exosome) works through the side where the polyA tail has been removed

    • We don’t know which happens when

• Deadenylation-independent mRNA decay

  • Proteins trigger initiation of decapping enzyme

  • Exonuclease comes and degrades

• Endonuclease mediated mRNA decay

  • mRNA is cleaved via endonuclease into two fragments

  • 5’ cap fragment is degraded from 3’ end by exosome

  • polyA tail fragment is degraded from 5’ end by exonuclease

Linker Scanning Experiment: allows us to identify what regions within a DNA sequence are important for transcriptional activity

• Two different cell types compared to wild-type levels of transcription

  • We check modifications at roughly each section of DNA nucleotides, and see if the levels of transcription drop from the wild-type once we get rid of that region

RNAi (RNA interference)

• In the cytoplasm we have some structure of dsRNA or shRNA (double stranded RNA or short-haired RNA which is dsRNA but with a hairpin on one end)

• Dicer will recognize it, bind it, and cut it into 20-30 base pair fragments

• RISC complex will recognize the strands and split the double strands into single strands

  • One strand gets thrown away

  • The other gets used as a template/”wanted poster” for other dsRNA/shRNA in the cytoplasm

• Once RISC finds a match for the RNA within the cytosol, certain things happen:

  • Perfect match: RNA gets snapped in half and is no longer functional

  • Pretty good match: inhibition of translation, RISC holds onto the RNA so ribosome cannot bind

• RNA based viruses will have an RNA genome, so at some stage during transcription/translation/replication there will exist at some point dsRNA

  • This evolved as a viral defense mechanism, to recognize dsRNA from being transcribed and infecting cells

miRNAs (rather than relying on viral DNA to be double stranded, we make our own dsRNA)

• Micro mRNAs are processed from longer precursor RNA molecules that form hairpin structures

  • They can be encoded from introns of other genes

• In our genome we have sections of DNA that when transcribed, are complementary and fold into dsRNA for dicer to chop up

  • This is used for nonviral RNA that we still want to degrade/stall the translation process

• Three ways these can occur:

  • mRNA as its own gene, it has a promoter and gets transcribed normally

  • Introns from another gene form a microRNA

    • This allows for an immediate degradation of an mRNA, so very low translation levels

RNAi in Lab

• We can design on a plasmid a piece of DNA that’s internally complementary, so it’ll fold and destroy expression of that gene

• Helpful because we can see what stopping a gene’s translation would do to a cell without the cell just dying

Topic 10: Measurement of Gene Expression

Learning Objectives:

1. Describe the use of reporter genes, including GFP, luciferase, and LacZ

2. Describe blotting techniques, especially northern blots and FISH

3. Explain PCR based methods to detect gene expression, including roles of RT-PCR, qPCR, and RNA-Seq

4. Identify the use cases, advantages, and disadvantages of each of these techniques

5. Interpret data and results from each of these techniques

Fluorescent Proteins: GFP (green fluorescent protein)

• GFP can be mutated to make a bunch of different colors (RFP, BFP, ect.)

• Requires some sort of laser to view the actual color, they don’t glow on their own

• Basic Promoter Studying

  • If we want to know when and where a gene is being transcribed we can use PCR to copy the promoter of a gene, but GFP coding sequence after the gene

  • Now anytime the gene gets transcribed naturally, GFP will also be transcribed naturally

  • Ex: during a heat stroke, we see glowing green on this part of the body, which means they’re related

  • Helps us see at what conditions the gene is expressed

• GFP can tag the protein itself rather than activating it alongside a protein

  • You do this by taking the coding sequence of GFP next to the coding sequence of the protein

  • So when the protein is transcribed it carries around a little GFP tag, and as it moves around the cell you can see it

  • Your sample can be alive, and watch the protein move through the cell in real life

• GFP Fusions

  • Create a fusion where you fuse a protein that you know where it locates + tag it with one color

  • Tag your protein of interest with a different color

  • Take a photo and merge the photos to see where the colors overlap

• GFP is costly bc lasers cost a lot of money, but it can get very precise (subcellular)

Luciferase

• Enzyme that produces glowing when substrate happens

• Cheap + quick way to see something on a a tissue (can’t get to cellular/subcellular level)

XGal/GUS Staining

• Relies on LacZ gene

• If you give this enzyme an XGal substrate instead of lactose, it will cleave it in half and release a dark blue molecule

• Cheapest and the fastest

• You can’t get subcellular, but you can get cellular specificity

• Sample needs to be dead + you need to do clarifying to make the sample clear

Blotting Techniques

• Isolate desired molecule from samples → run gel to separate out molecules by size → transfer molecules from gel to membrane → use antibodies or radiography to visualize molecules of interest 

• Northern Blot: used to detect and analyze specific RNA molecules within a sample

  • RNA molecules from the sample are separated based on size, smaller molecules migrate faster through the gel

  • RNA molecules are transferred from the gel onto a solid membrane, which preserves the relative positions of the RNA molecules from the gel onto the membrane

  • Membrane is exposed to a single-stranded DNA/RNA probe complementary to the RNA sequence of interest, and the probe will bind to its complementary RNA sequence

  • The membrane is examined to see where the probe has bound (usually has a tag of some sort) that allows visualization of the presence and amount of target RNA

  • Intense signal = abundant target RNA in original sample

  • Would it be possible to look for alternative splicing mRNAs using a northern blot?

    • We need to already know which exon is being spliced differently + its sequence

    • Otherwise, yes alternative splicing can result in mRNAs of different lengths, which will appear as distinctive bands

FISH: fluorescence in situ hybridization - used to identify and visualize specific genetic material

• Fixation: exposing cells to chemicals to cause cross linking between the chemicals and cellular components so nothing moves

  • Freeze everybody in place

• Permeabilization: make holes in the cell membrane to get probes inside

• Soak with a probe solution, they’ll bind to their target, wash away excess probes  

RT-PCR: Reverse Transcriptase PCR

• Take isolated RNA, take primer (usually a poly-T primer) that will bind, reverse transcriptase will make a DNA copy of the RNA

  • Called a cDNA because it’s complementary to the DNA

• Degrade all the RNA, use normal PCR to make the DNA a double strand 

• Random hexamers are mixtures of all possible combinations of six nucleotide sequences, which can then bind randomly across the RNA template

  • This allows for cDNA synthesis from the entire RNA population

  • This is often done because some mRNAs are without poly-A tails, so if you’re focused on these kinds mRNAs

QPCR: Quantitative PCR

• Get data at the end of each cycle of PCR, compare across

  • We do this via fluorescence that only glows when bound to double stranded DNA, so the more DNA we have, the brighter it’ll go

• Amplification plot: 

  • Y-axis is brightness

  • X-axis is cycle

  • The more DNA started = the sooner we can detect the brightness, so samples that begin with more DNA will appear at an earlier cycle 

    • 2^{cycles difference between samples} = relative gene expression

      • There’s __ as much in this as compared to this sample

cDNA Library

• When making cDNA library, standard cDNA is made with polyA tail to copy things to DNA, and then we design primers for a specific sequence

• But to build a cDNA library, we need a primer than can bind to everything

  • Before we begin this process, we ligate a known sequence of RNA to the beginning, and create a primer that will target that sequence

  • Same steps continue, now we can sequence our cDNA

RNA-Seq: used to study RNA gene expression 

• Isolate total RNA from sample

• Fragment into uniform pieces for efficient sequencing

• Adapter ligation: attach short DNA sequences (adaptors) to both ends of cDNA fragments

  • This is because we don’t know what exactly we’re targeting 

    • Previously, primers were designed for the specific sequence we’re targeting

• Convert to cDNA via random hexamers + normal PCR amplification and sequencing

• How many reads did you find for each gene in each sample?

  • Much more absolute for how often something was detected

• For single-cell RNA-seq to track mRNA across different kinds of cells we add specific adapter tags to each kind of cell

  • One way to do this: microfluidics - using very small small setups in order to separate out cells

  • You can do this with high-resolution mapping, where the different cell types are clustered together

Topic 11: Chromatin & ChIP

Learning Objectives:

1. Describe the structure of DNA packaging into chromatin

2. Describe the process of chromatin remodeling

3. Explain the effect of chromatin on gene expression

4. Explain the process and purpose of chromatin immunoprecipitation

DNA Packaging

• While DNA is more stable than RNA, we wrap it around proteins for additional protecting and also to control how much space it takes up + where it’s located

• DNA wrapped around nucleosomes twice (protein complexes)

  • “Beads on a string”

  • There are gaps in between each nucleosome

  • looser

• We can pack the DNA further to pack them into a really tightly coiled fiber

  • This is the tighter packaging

• Tighter packed DNA is transcribed/used less, looser packed DNA is transcribed more/used more

Nucleosomes

• Each nucleosome is made of 8 histones, DNA wraps around each nucleosome twice

  • DNA in between each histone is called “linker DNA”

• Each histone has a tail hanging off the end of it 

• DNA really tightly packaged is associated with no transcription or gene expression

• DNA really loosely packaged with exposed linker stretches is associated with active transcription and gene expression

Globin Gene

• Beta globin gene is responsible for forming hemoglobin (carries oxygen into red blood cells)

  • This gene is only needed for red blood cell types, so chromatin is decondensed

  • In non-red blood cells the gene is not really needed at all, so chromatin is highly condensed

• We can test the decondensed/condensed idea through DNase footprinting

• Globin gene is cut out via restriction enzyme + site that gets us a workable starting section of our gene

  • DNase enzyme can access the gene and chop it up if it’s decondensed

  • If the DNA is condensed, DNase can’t cut it as well, so the fragments will be large or not cut up at all

  • MSB is non-red blood cell, so we use it as a control

  • The thick band at 4.6 kb indicates that on the 4.6 kb gene (globin gene), there was no cutting, so DNase couldn’t get to the gene well and it was tightly coiled

    • Larger in size = higher up on the gel assay

  • Columns starting from 0 to 1.5 are red blood cells with different concentrations of DNase

  • Columns 0 and 0.01 had very small concentrations of DNase, so they look similar to MSB data

  • As we increase DNase concentration, our bands around 4.6 disappear and migrate throughout the gel column, this is because DNase enzyme was able to cut at it

Histones Tails & Marks

• Every histone has a tail, so each nucleosome has 8 amino acid sequence tails

• Each tail can be modified at different positions for whatever specific purpose

  • We don’t quite know how this works

  • This is epigenetics, by changing how DNA is packaged can change DNA expression

• Generally, trimethylation is associated with heterochromatin (tightly packed chromatin), acetylation is associated with euchromatin (loosely packed chromatin)

  • Trimethylation has effects but can generally go either way, acetylation is always loose packaging

• Lysine acetylation specifically is associated with decondensed chromatin

  • Lysine is positively charged normally, so it sticks strongly to negatively charged DNA

  • When you acetylated lysine, it becomes neutral, so it binds less strongly to negatively charged DNA

  • This is why acetylation → looser binding

Telomeres are Exceptions

• Telomeres just need to exist, they don’t need to be accessible, so they’re always very tightly packaged

  • Once produced, proteins come and pack it really tightly into a heterochromatin structure

• Ends of chromosomes are super densely packed to prevent fraying and degeneration throughout life cycle

Heterochromatin can spread

• Heterochromatin associated proteins can recruit enzymes that deacetylate enzymes near them + also recruit more of themselves

  • Removing these acetyl groups → heterochromatin

• Regions near heterochromatin will become heterochromatin and continue to recruit

• HP1 (Histone 1) has an additional job, it’s only present in heterochromatin

  • DNA wrapped around nucleosome has two flexible ends to link to the next chromatin clump

  • This is problematic for condensed chromatin because it allows them to move around and be loosely packed, potentially unwinding

  • HP1 grabs onto the two end strands and pull them tightly together, in this way they are no longer flexible

  • HP1 can also recruit more of itself, so it furthers the spread of heterochromatin to hold everything into place

Boundary Elements Stop Heterochromatin Spread

• Boundary elements are specific DNA sequences that can bind structural proteins to physically block

Going from heterochromatin → euchromatin → heterochromatin

• Transcription factors can help alter chromatin packing

  • They help recruit Histone Acetylase (HAT), to acetylase nucleosome lysines and decondense chromatin

    • Activation domain of the transcription factor is recruiting HAT until other TFs can get to the promoter for transcription

  • When going back to heterochromatin, instead of an activation domain the transcription factor will have a repression domain to recruit HDACs (histone deacetylase complex)

    • This will package chromatin tightly

ChIP: Chromatin Immunoprecipitation 

• Immunoprecipitation = use antibodies to tag specific modifications on proteins to study a specific target

• At what position on the chromosome contain what marker

• Steps:

  • Cross link DNA and associated proteins (nucleosomes) so that when we remove other cellular components, they don’t get unlinked

  • Sonication: stick a metal wand into sample, which blows up the cell and snaps DNA in the linker stretches

  • Design an antibody that will bind to one specific marking on one specific tail

    • They’re extremely specific, you do one antibody at a time

    • Mix into sample, anything with this marker will be tagged

    • Wash away everything without the marker

  • Undo crosslinking, remove nucleosomes, sequence DNA fragments

• Analyzing results: high points mean we had lots of sequences, low points mean we had no sequences

  • The x-axis depicts what was present where

Dsup (damage suppressor protein) & DNA Damage

• Protects DNA from damage that comes particularly from radiation via binding to DNA and acting as a shield

• Experimentally: 

  • Two cultures, one with no Dsup cells one with Dsup, after radiation there was mass viability in cells with Dsup

• This doesn’t quite work in humans, just in really basic cell samples

Topic 12: Genomes

Learning Objectives:

1. Explain how Next-Gen sequencing modifies Sanger sequencing for higher efficiency

2. Understand how sequencing provides advantages in studying genes and genomes

Biotechnology to Sequencing: Berg + Gilbert + Sanger + Mullis → the fundamental basics of sequencing

• The Human Genome Project was not the first genome to be sequenced, but this was significantly larger and more complex

  • Early on the development, it was thought to approach the sequencing task via a very controlled method where you sequence each region in order

  • John Craig Venter wanted a different approach, left the project, and founded the Solara Project

    • He finished sequencing before the original project via a faster and cheaper process (called shotgun sequencing, where a computer will puzzle everything together after sequencing everything in tons of tiny little pieces)

• Before Sequencing: if we wanted to identify a region of a gene that controls some trait, we use restriction enzymes to chop up DNA into a bunch of different little pieces and put them into different bacteria colonies

  • So each bacteria has different piece of the genome

  • Now we screen all of them and see what the end product is

  • End result is that you still don’t necessarily know the sequence, you just know where it is

• Sanger sequencing can get roughly 2000 base pairs in a row at a time if everything works perfectly (usually 1500-1800)

  • This is called primer walking, where you insert primers and just let everything keep going

  • You can't do multiple at a time, you have to let the prior one go before you can keep going

• Shotgun Sequencing: Venter’s Idea

  • Take genome and break it into a bunch of random small pieces

  • Sequence each of the little pieces

  • Have a computer puzzle piece them back together

    • This was the primary challenge, getting good technology to do so

    • You use a few copies of the same genome, and because the pieces are chopped up somewhat randomly, the computer will compare each sequence to every other sequence and see if there’s any identical component

  • We usually do short read technology for this (75-200 base pairs long)

    • This works fine everywhere except repeat regions

Next-Gen Sequencing

• Library prep → bridge pcr → modified sanger

• Library prep:

  • Break DNA into lots of small fragments roughly 75 base pairs long

  • Ligate a consistent sequence adaptor onto either end so that we can attach a primer to bind to 

• Bridge PCR

  • DNA fragments into sequencing machine’s flow cell (piece of glass with sequences complementary to the adaptors)

  • You wash your single stranded DNA on the flow cell, and they’ll stick to the sequences on the flow cell

  • Modified PCR will occur to amplify the amount of DNA and make them complementary to the original DNA strand

  • Wash the flow cell again to denature the now double stranded DNA and get rid of what was the original strand because it’s not locked in place

  • The adaptor at the other end will bend over (“bridge”) to bind to another complementary adaptor sequence

  • We want each sequence to have the same orientation, so we remove the ones that are like 5’ → 3’ instead of 3’ → 5’ or vice versa

    • So now we have an amplified sequence

• Modified Sanger Sequencing

  • We use differently colored fluorescent tags

    • Via a modified nucleotide that is tagged, but prevents future nucleotides from binding to it

    • This is a modified 3’ OH where the oxygen is missing

  • Wash it off, shine laser, take picture after each nucleotide incorporation, repeat

  • You remove this modified nucleotide after each image and then wash a new one on, take another photo, remove, repeat

• Long read sequencing: HiFi (High-Fidelity) and ONT (Oxford Nanopore)

  • HiFi (High-Fidelity): put DNA into a circular form and loop it around for reprocessing

    • High accuracy and ability to produce long reads

  • ONT (Oxford Nanopore): passing single-stranded DNA into a nanopore and measuring changes in electrical current as each nucleotide passes through

    • Allows for real-time sequencing

    • Can produce long reads

    • Prone to errors, but is better now

• Minimal Genome Project: aims to identify essential genes required for a living organism (specifically in Mycoplasma)

  • Systemically deleted genes one by one and observed the effects on cell viability

Topic 13: CRISPR

Learning Objectives: 

1. Describe the natural origins of the CRISPR system

2. Explain how the CRISPR system can be used for gene editing, and the role of each component required

3. Describe the concerns around CRISPR use in humans and how it has been applied

CRISPR: a tool for targeted genome editing

• Clustered regularly interspaced short palindromic Repeats + Cas9 is the Crispr associated protein 9

• Short palindromic repeats (spelled the same forwards and backwards), so once produced, it can base pair with itself

  • In between the repeats were “spacers” which had nothing in common except that they were in between palindromic repeats

  • The spacers are variable

CRISPR Timeline

• We’re doing genome editing by inducing double stranded homologous breaks, and using enzyme repair mechanisms to manipulate how it gets fixed

  • CRISPR helped revolutionize the damage

• Zinc Finger Nuclease recognize specific DNA sequences

  • Attach one to a nuclease, and that nuclease will go and cut that sequence the zinc finger recognizes

  • We can’t invent new ones, we can only do what the zinc finger has

  • Additionally, these proteins aren’t meant to be stuck together, so they don’t work very well when squished together

• TALENs is next step

  • RNA based approach for creating targeted damage where nucleases are being targeted by RNA molecules

  • Not easy to make, takes a long time + money to target just one specific time in the genome

• CRISPR/Cas9 is used in eukaryotes in roughly 2013

  • CRISPR specifically refers to a specific sequence in the bacterial genome (short palindromic repeat) that isn’t studied very well but first found in the 80s

    • Starts to be studied more heavily in the early 2000s

  • We discover that the spacer regions match little pieces of viral genomes (an extremely simplistic form of memory in an immune system)

  • We discover that Cas9 is very crucial for viral defense

  • So we think about inducing breaks in the eukaryotic genome at a specific position and from there do genome editing

CRISPR as virus defense in prokaryotes

•  In the bacterial chromosome we have the Cas locus (all the different genes necessary for the enzymes/proteins needed for the process) + crispr array (short palindromic repeats + spacers)

  • This whole thing gets expressed as one big RNA, but because the repeats are palindromic, they can fold in and form a small double stranded section (hairpin)

• The bulk of the Cas proteins come to the RNA and cut each hairpin out

  • The double stranded part is the palindromic repeat that folds in on itself, and then there’s a little “tail” or section that sticks out that’s the spacer

  • This structure is a guide RNA

  • This hairpin structure is highly conserved, and is what Cas9 will bind to and recognize it as a guide RNA

• Will drag the guide RNA and scan along different pieces of DNA and see if they match

  • If they match, it gets chopped in half

• This is how CRISPR acts in nature

CRISPR in Lab

• We only need two genes: Cas9 protein + something to be transcribed for the guide RNA

  • They’ll go into my genome and induce the double stranded break

  • Then we can mutate whatever we want

• Very easy to retarget something

Limitations for Where we can send guide RNA

• The target 20 base pairs need to be next to a PAM site

  • PAM sites tend to be “NGG” where N is any nucleotide

    • This roughly every 42 base pairs, so you can basically do this anywhere you want

HDR with Template

• You have a template DNA with two homologous sequences on either end of a desired insertion or modification

  • The homologous regions are complementary to sequences around the target site in the genome

• Using CRISPR-Cas9, you create a double-stranded break at the desired location, and the cell will initiate HDR and use homologous sequences in template DNA to repair the break, which incorporates the desired modification or insertion

  • To knock-out a gene: you could induce a stop codon or frameshift

  • To knock-in a gene: you substitute the nucleotide with what it should be

    • You can insert a ton of base pairs and the cell will just fix it

Wide Application of CRISPR-Cas9 System

• If I put this into the cell, I don’t want to remove the Cas9 and CRISPR genes in there

• Why is this? How do we achieve this?

  • CRISPR could just keep cutting, if your mutation isn’t that different from what it used to be, CRISPR could undo what you do

    • Also, if you do this in an organism, as the organism reproduces, CRISPR can be wrong sometimes, which multiples with how often the mistakes happen

  • Could check with fluorescent proteins + blotting + ect.

  • We’re usually doing this in eukaryotes, and CRISPR-Cas9 is a plasmid, so eventually most cells will stop replicating

Cas9 Tagging

• You add an extra marker on your plasmid, which usually is some fluorescence

  • If you want to know if Cas9 is present, then you shine a laser and see which has which

Two gRNAs (guide RNAs)

• When trying to knock something out sometimes it can mean breaking a bunch of genes

• Instead of just mutating each one, you could you two guide RNAs (one for the beginning of the region we want to get rid of, one for the end of the region we want to get rid of)

  • Then the middle gets taken out, we can do HDR or NHEJ and repair the break

CRISPR

• We can approach getting our designed system into the plasmid in three ways: DNA, RNA, Protein

  • DNA: put the actual DNA that codes for these genes into the cells, the cell’s natural machinery will take care of it

  • RNA: outside of target cell we just inject into the cells the mRNA for the Cas9 and the guide RNA, so we cut out the DNA step

  • Protein: we directly put in the Cas9 protein + the guideRNA

• RNA and Protein systems are nicer because they aren’t as stable but we have lots of control

  • Good for a clinical approach because we don’t need to worry about removing at the end

• Efficiency: DNA is least efficient, Protein is most efficient

• Cost: DNA costs the less, Protein costs the most

• Specificity: RNA and Protein have better specificity

CRISPR Variance

• We’ve been mutating Cas9 to do things other than cutting

• Normal CRISPR: Cas9 uses guide RNA to break it, we rely on NHEJ or HDR to fix with our desired sequence

• What if we don’t want to break a gene, just temporarily disable expression?

  • Mutate Cas9 protein → dCas (dead Cas)

    • Cas9 has two nuclease domains to cut each strand, mutating one disables Cas9 from cutting DNA, but it can still bind DNA

    • So it’ll find its target spot and not cut it but just bind and block polymerase from transcribing

  • CRISPRi (CRISPR interference) uses dCas to interfere with gene expression

  • CRISPRa: crispr activator, to spike the expression of a gene 

• CRISPR’s broad variance and effectiveness means that it has great potential for treating a variety of genetic disease because of how good it is at targeting

CRISPR in Humans

• Cancer:

  • Issue with cancer is that lots of cancers will hide from the immune system so that T cells can’t find them and degrade them

  • The idea with CRISPR is that you take out the T cells and use CRISPR to remodify the cells to target an individual’s specific tumor

• Human Embryos:

  • Fix a human’s entire genome while the human is a zygote

  • Specifically target a dominant mutation that would cause heart disease

  • Issue with this is that in the human, you can’t use federal funding for human embryo research

    • So you’d need private funding, but this is hard to get

  • Injected eggs and sperm as they were fusing with a Cas9 and guide RNA that were covalently linked

    • They had a huge success

• Sickle Cell: one nucleotide mutation

  • Craft correct bone marrow to implement into human

• The reason we started with sickle cell and cancer was because we aren’t injecting CRISPR into the human actually, we take care of it all outside of the body

  • We’re also fixing somatic cells, not germline cells, so you can’t pass on the edit