AP Bio Unit 6 Gene expression and regulation ultimate notes

DNA and RNA structure 

discovery of DNA structure

• in the 1950’s Rosalind Franklin performed X-ray Crystallography of DNA

  • her work revleaed a pattern that was regular and repetitive

• during the same time, Edwin Chargaff analyzed DNA samples from different species

  • he found the following rule held true for all species

    • the amount of adenin equals the amount of theyme (2 H bonds)

    • the amount of cytosine equals the amounts of guanine (3 H bonds)

Nucleotide structure

• purines

  • double ring structure (A,G)

• Pyrimidines

  • single ring structure (C,U,T)

nucleotide pairing

• the base pairs are held together by hydrogen bonds

• adenine and thymine have two hydrogen bonds

• cytosine and guanine have three hyrdrogen bonds

discovery of DNA structure

• watson and crick combined the findings of franklin (helix shape) and chargaff (base pairing) to create the first 3D, double helix model of DNA

  

key features of DNA structure

1. DNA is a double stranded helix

   a. backbone:

• sugar-phosphate

b. center:

• nucleotides pairing

2. DNA strands are antiparallel

a. one strand runs 5’ to 3’, other strand runs in opposite, upside-down direction 3’ ti 5’

b. 5’ end: free phosphate group

c. 3’ end: free hydroxyl group

key function of DNA

• DNA is the primary source of heritable information

  • genetic information is stored in and passed from one generation to the next through DNA

    • exception: RNA is the primary source of heritable information in some viruses

prokaryotice vs eukaryotic DNA

plasmids

• plasmids replicate independently from the chromosomal DNA

  • primarily found in prokaryotes

    • contain genes that may be useful to the prokaryote when it is in a particular environment, but may not be required for survivial

• plasmids can be manipulated in laboratories

  • plasmids can be removed from bacteria, then a gene of interest can be inserted into the plasmid to form recombinant plasmid DNA

  • when the recombinant plasmid is inserted back into the bacteria the gene will be expressed

• bacteria can exchange genes found on plasmids with neighboring bacteria

  • once DNA is echanged the bacteria can express the genes acquired

  • helps with survival of prokaryotes

DNA Replication

models of DNA Replication

• there were 3 alternative models for DNA replication

• conservative model

  • the parental strands direct synthesis of an entirely new double stranded molecule

    • the parental strands are fully “conserved”

• semi conservative model

  • the two parental strands each make a copy of itself

    • after one round of replication the two daughter molecules each have one parental and one new strand

• dispersive model

  • the material in the two parental sterands is dispersed randomly between the two duaghter molecules

    • after one round of replication the daughter molecules contain a random mix of parental and new DNA

which model is correct?

• in 1954 meselson and stahl performed an expirment using bacteria

  process:

  1. bacteria was cultured with a heavy isotope, 15^N

  2. bacteria was transferred to a medium with 14^N, a light isoptope

  3. DNA was centrifuged and analyzed after each replication

semi conservative model

• by analyzing samples of DNA after each generation, it was found that the parental strands were following the semi-conservative model

steps in DNA Replication

1. DNA replication begins at sites called origins of replication

   • various proteins attach to the origin of replication and open the DNA to form a replication fork

2. helicase will unwind the DNA strands at each replication fork

   • to keep the DNA from re-bonding with itself, proteins called single strand binding proteins (SSBPs) bind to the DNA to keep it open

   • topoisomerase will help prevent strain ahead of the replication fork by relaxing supercoiling

3. the enzume primase initiates replication by adding short segments of RNA, called primers, to the parental DNA strand.

   • the enzyme that synthesize DNA can only attach new DNA nucleotides to an existing strand of nucleotides

   • primers serve as the foundation for DNA synthesis

4. antiparallel elongation

   • DNA polymerase III (DNAP III) attaches to each primer on the parental strand and moves in the 3’ to 5’ direction

     • as it moves, it adds nucleotides to the new stran in the 5’ to 3’ direction

     • the DNAP III that follows helicase is known as the leading strand and it only requires one primer

     • the DNAP III on the other parental strand that moves away from helicase is known as the lagging strand and requires many primers

5. the leading stranf is synthesized in one continous segment, but since the llagging strand moves away from the replication fork it is synthesized in chunks

   • okazaki fragments: segments of the lagging strand

6. after DNAP III forms an okazaki fragment, DNAP I replaces RNA nucleotides with DNA nucleotides

   • DNA ligase: joins the okazaki fragments forming a continous DNA strand

putting it all together video:

problems at the 5’ end

• since DNAP III can only add nucleotides to a 3’ end, there is no way to finish replication on the 5’ end of a lagging strand

  • over many replication, this would mean that the DNA would become shorter and shorter

  • how arer the genes on DNA protected from this?

    • telomeres: repeating units of short nucleotide sequences that do not code for genes

      • form a cap at the end of DNA to help postpone erosion

      • the enzyme telomerase adds telomeres to DNA

proofreading and repair

• as DNA polymerase adds nucleotides to the new DNA strand, it proofreads the bases add

  • if errors still occur, mismatch repair will take place

    • enzymes remove and replace the incorrectly paired nucleotide

    • if segments of DNA are damaged, nuclease can renove segments of nucleotides and DNA polymerase and ligase can replace the segments

Transcription and RNA Processing

proteins

• proteins are polypeptides made up of amino acids

  • amino acids are linked by peptide bonds

• Gene expression: the process by which DNA directs the synthesis of proteins

  • includes two steag: transciption and translation

• DNA → RNA → protein

  (transciption) (translation)

Transciption and Translation

• transciption: the synthesis of RNA using information from DNA

  • allows for the “message” of the DNA to be transcribed

  • occurs in the nucleus

• translation: the synthesis of a polypeptide using information from RNA

  • occurs at the ribosome

  • a nucleotide sequence becomes an amino acid sequence

types of RNA

1. Messenger RNA (mRNA)

2. Ribosomal RNA (rRNA)

3. Transfer RNA (tRNA)

Messenger RNA (mRNA)

• Messenger RNA is synthesized during transcription using a DNA template

  • mRNA carries information from the DNA (at the nucleus) to the ribosomes in the cytoplasm

Transfer RNA (tRNA)

• transfer RNA molecules are important in the process of transltion

  • each tRNA can carry a specific amino acid

  • can attach to mRNA via their anticodon

    • a complementary codon to mRNA

  • allow information to be translated into a peptide sequence

Ribosomal RNA (rRNA)

• rRNA helps form ribosomes

• helps link amino acids together

The Genetic Code

• DNA contains the sequence of nucleotides that codes for proteins

  • the sequince is read in groups of three called the triplet code

• during transcription, only one DNA strand is being transcribed

  • known as the template strand (also known as the noncoding strand, minus strand, or antisense strand)

• mRNA molecules formed antiparallel and complementary to the DNA nucleotides

  • Base pairing: A = U and C = G

  • the mRNA nucleotide triplets are called codons

    • codons code for amino acids

• 64 different codon combinations

  • 61 code for amino acids

  • 3 are stop codons

  • universal to all life

• redundancy: more than one codon code for each amino acid

reading frame: the codons on the mRNA must be read in the correct groupings during translation to synthesize the correct proteins

example: the fat cat ate the rat

• if the reading fram shifts even one letter, it will produce a completely different outcome

  • Hef atc ata tet her at

Steps of Transcription

there are three steps

1. initation

2. elongation

3. termination

Step 1: Initiation

• transcription begins when RNA polymerase molecules attach to a promoter region of DNA

  • do not need a primer to attach

  • promoter regions are upstream of the desired gene to transcribe

• Eukaryotes:

  • promoter region is called TATA box

  • transcription factors help RNA polymerase bind

• Prokaryotes:

  • RNA polymerase can bind directly to promoter

Step 2: Elongation

• RNA polymerase opens the DNA and reads the triplet code of the template strand

  • moves in the 3’ to 5’ direction

    • the mRNA transcript elongates 5’ to 3’

• RNA polymerase moves downstream

  • only opens small sections of DNA at a time

    • pairs complementary RNa nucleotides

    • the growing mRNA strand peels away from the DNA template strand

      • DNA double helix then reforms

• a single gene can be transcribed simultaneously by several RNA polymerase molecules

  • helps increase the amount of mRNA synthesized

    • increases protein production

Step 3: Termination

Prokaryotes:

• transcription proceeds through a termination sequence

  • causes a termination signal

    • RNA polymerase detaches

    • mRNA transciption is released and proceeds to translation

      • mRNA does NOT need modifications

Eukaryotes:

• RNA polymerase transcribes a sequence of DNA called the polyadenylation signal sequence

  • codes for a polyadenylation signal (AAUAAA)

    • releases the pre-mRNA from the DNA

      • must undergo modification before translation

Pre-mRNA modifications

there arre three modification that must occur to eurkaryotic pre-mRNA before it is ready for translation

1. 5’ cap

2. Poly-A tail

3. RNA splicing

1. 5’ cap (GTP): the 5’ end of the pre-mRNA recieves a midified guanone nucleotide “cap”

2. Poly-A tail: the 3’ end of the pre-mRNA revieces 50-250 adenin nucleotides

• both the 5’ cap and the poly-A tail function to:

  • help the mature mRNA leave the nucleus

  • help protect the mRNA from degradation

  • help ribosomes attach to the 5’ end of the mRNA when it reaches the cytoplasm

3. RNA Splicing: sections of the pre-mRNA, called introns, are removed and then expns are joined together

   • a. introns: intervening sequence, do not code for amino acids

   • b. exons: expressed sections, code for amino acids

why does splicing occur?

• a single gene can code for more than one kind of polypeptide

  • known as alternative splicing

• snRNPs: small nuclear RNA

• Spliceosome: several snRNPs that recognize splice site sequence and cut the gene

once all modifications have occured, the pre-mRNA is now considered mature mRNA and can leave the nucleus and procees to the cytoplasm for translation at the ribosomes

Translation

• Translation: the synthesis of a polypeptide using information from the mRNA

  • occurs at the ribosome

  • a nucleotide sequence becomes an amino acid sequence

• tRNA is a key player in translating mRNA to an amino acid sequence

Transfer RNA

• tRNA has an anticodon region which is complementary and antiparallel to mRNA

• tRNA carries the amino acid that the mRNA codon codes for

  • ACU codes for Thr

• the enzyme aminoacyl-tRNA synthetase is responsible for attaching amino acids to tRNA

• when tRNA carries an amino acid it is “charged”

Ribosomes

• translation occurs at the ribosome

  • ribosomes have two subunits: small and large

  • prokaryotic and eukaryotic ribosomal subunits differ in size

    • prokaryotes: small subunits (30s) large subunit (40s)

    • eukaryotes: small subunit (40s) large subunits (60s)

• the large subunit has three sites: A, P, and E

  • A site: amino acid site

    • holds the next tRNA carrying an amino acid

  • P site: polypeptide site

    • holds the tRNA carrying the growing polypeptide cahin

  • E site: exit site

Translation

translation occurs in three stages:

1. initiaition

2. elongation

3. termination

Step 1: Initation

• translation begins when the small ribosomal subunit binds to the mRNA and a charged tRNA binds tot he start codon, AUG, on the mRNA

  • the tRNA carries methionine

  • next, the large subunit binds

  • note: the first tRNA carrying Met will go to the P site, every other tRNA will go to the A site first

Step 2: Elongation

• elongation starts when the next tRNA comes into the A site

• mRNA is moved through the ribosome and its codons are read

• each mRNA codon codes for a specific amino acid

  • codon charts are used to determine the amino acid

• since all organisms use the same genetic code, it supportd the idea of common ancestry

Elongation occurs in steps:

1. codon recognition: the appropriate anticodon of the next tRNA goes to the A-site

2. peptide bond formation: peptide bonds are formed that transfer the ploypeptide to the A site tRNA

3. translocation: the tRNA in the A site moves to the P site, the tRNA in the P site goes to the E site. The A site is open for the tRNA

Step 3: Termination

termination occurs when a stop coodn in the mRNa reaches the A site of the ribosome

• stop codons do not code for amino acids

• the stop codon signals for a release factor

  • hydrolyzes the bond that holds the polypeptide to the P site

  • polypeptide releases

  • all translational units disassemble

Protein Structures

primary: chain of amino acids

secondary: coils and folds due to hydrogen bonds forming

tertiary: side chain interaction

quaternary: 2+ polypeptide chains interacting

Protein folding

• as translation takes place, the growing polypeptide chain begins to coil and fold

  • genes determine the primary structure

    • primary structure determines the final shape

  • some polypeptide require chaperone proteins to fold correctly and some require modification before it can be functional in the cell

Virus Vocabulary

• parasites: need host “machinery”

• capsids: crystal-like protein shell

• assimilation: virus takes over host and reprograms host cells to produce the viral proteins

• bacteriophages: viruses that infect bacteria

• lytic: actively reproduce virus in bacteria and release virus by rupturing host

• lysogenic: integrate viral DNA into bacterial DNA

• prions: misfolded proteins transmitted

  • central dogma violation

Lytic cycle

Lysogenic cycle

Retroviruses

• retroviruses, like HIV, are an exception to the standard glow of genetic information

• information flows from RNA to DNA

  • uses an enzyme known as reverse transcriptase

    • couples viral RNA to DNA

    • DNA then becomes part of the RNA

Prions

• infectious proteins that caus other proteins to misfold, leading to severe neurodegenerative diseases. Unlinke bacteria, viruses, or fungi, prions lack DNA or RNA and are purely protein based.

Regulation of Gene expression & Cell Specialization

Gene Expression

• prokaryotes and eukaryotes must be able to regulate which genes are expressed at any given time

  • genes can be turned “on” or “off” based on environmental and internal cues

    • on/off refers to whether or not transcriptioin will take place

  • allows for cell specialization

Bacterial Gene Expression

• Operons: a group of genes that can be turned on or off

  • operons have 3 parts:

    • promoter: where RNA polymerase can attach

    • operator: the on/off switch

    • genes: code for related enzymes in pathway

• operons can be repressible or inducible

  • repressible (on to off): transcription is usually on, but can be repressed (stopped)

  • inducible (off to on): transcription is usually off, but can be induced (started)

• Regulatory gene: produces a repressor protein that binds to the operator to block RNA polymerase from transcribing the gene

  • always expressed, but at low levels

  • binding of a repressor to an operator is reversible

Allosteric Regulation

• allosteric activator: substrate binds to allosteric site and stabilizes the shape of the enzyme so that the active sites remain open

• allosteric inhibitor: substrate binds to allosteric site and stabilizes the enzyme shape so that the active sites are closed (inactive form)

repressible operons

• example: the Trp operon

  • the trp operon in bacteria controls the synthesis of tryptophan

  • since it is repressible, transcription is active

    • it can be switched off by a trp repressor

      • allosteric enzyme that is only active when tryptophan binds to it

• when too much tryptophan builds up in bacteria, tryptophan is more likely to bind to the repressor turning it active, which will then temporarily shut off transcription for tryptophan.

  

Inducible operon

• example: the lac operon

  • the lac operon controls synthesis of lactase, an enzyme that digests lactose (milk sugar)

  • since it is inducible, transcription is off

    • a lac repressor is bound to the operatore (allosterically active)

  • the inducer for the lac repressor is allolactose

    • when present it will bind to the lac repressor and turn the lac repressor off (allosterically inactive)

      • the genes can now be transcribed

Eukaryotic Gene Expression

• the phenotype of a cell or organism is determined by a combination of genes that are expressed and the levels that they are expressed

  • differences between cell types is known as differential gene expression

• eukaryotic gene expression is regulated at different stages

• chromatin structure:

• if DNA is tightly wound it is less accessible for transciption

• how can it be modified?

  • histone acetylation adds acetyl groups to histones, which loosens the DNA

  • DNA methylation adds methyl groups to DNA, which causes the chromatin to condense

• epigenetic inheritance:

• chromatin modifications do not alter the nucleotide sequence of the DNA, but they can be heritable to future generations

  • modifications can be reversed, unlinke mutations

  • explains why one identical twins may inherit a disease while the other does not

• transcription initiation:

• once chromatin modifications allow the DNA to be more accessible, specific transcription factors bind to control elements

  • sections of non coding DNA that serve as binding sites

  • gene expression can be increased or decreased by binding of activators or repressors to control elements

• RNA processing:

• alternative splicing of pre-mRNA

  translation initiation

• translation can be activated or repressed by initiation factors

• MicroRNA’s and small interfering RNA’s (siRNA) can bind to mRNA and degrade it or block translation

Eukaryotic Development

• during embryonic development, cell division and cell differentiation occurs

  • cells become specialized in their structure and function

  • morphogenesis: the physical process that gives an organism its shape

• how do cells differentiate during early development?

  • cytoplasmic determinants: substances in the maternal egg that influence cells

  • inductioni: cell to cell signals that can cause a change in gene expression

• both cytoplasmic determinants and induction influence pattern formation

  • a “body plan” for the organism

    • homeotic genes map out the body structures

• as cells differentiate, apoptosis plays a critical role

  • apoptosis: programmed cell death

    • allows structures to take their form

      • example: if apoptosis did not occur during the development of human hands and feet we would be born with webbed fingers and toes

      • ubiquitin: “death tag”

        • marks unwanted proteins with a lab which are broken does in proteasomes (waste disposers)

Mutations and Biotechnology

Mutations

• Mutations: changes in the genetic material of a cell, which can alter phenotypes

  • primary source of genetic variation

  • normal function and production of cellular products is essentialn

    • any disruption can cause new phenotypes

  • changes can be large scale or small scale

    • large scale: chromosomal changes

    • small scale: nucleotide substitutions, insertions, or deletions

Small Scale Mutations

• point mutations: change a single nucleotide pair of a gene

  • substitution: the replacement of one nucleotide and its partner with another pair of nucleotides

    • silent: change still codes for the same amino acid (remember: redundancy in the genetic code)

    • missense: change results in a different amino acid

    • nonsense: change results in a stop codon

Small Scale Mutations

• frameshift mutation: when the reading frame of the genetic information is altered

  • disastrous effects to resulting proteins

    • insertion: a nucleotide is inserted

    • deletion: a nucleotide is removed

Large Scale Mutations

mutations that affect chromosomes

• nondisjunction: when chromosomes do not deperate properly in meiosis

  • results in the incorrect number of chromosomes

    • example: down syndrome- trisomy 21

• translocation: a segment of one chromosome moves to another

• inversions: a segment is reversed

• duplications: a segment is repeated

• deletions: a segment is lost

Natural selection

• any time mutations occur, they are subject to natrual selection

  • genetic changes can sometimes enhance the survival and reproduction of an organism

Increasing Genetic Variation

• prokaryotes can exchange genetic material through horizontal gene transfer

• if there is a mutation that is beneficial to the survival and reproduction of that prokaryote then it can also be transferred

  • transformation: uptaking of DNA from a nearby cell

  • transduction: viral transmission of genetic material

  • conjugation: cell to cell transfer of DNA

  • transposition: movement of DNA segments within and between DNA molecules

Transformations

• will DNA or proteins transform bacteria?

  • DNA

• first evidence that DNA is the genetic material

Hershey & Chase

• grew two viruses with radioactively labeled macromolecules in either proteins or DNA

Biotechnology

• both DNA and RNA can be manipulated through genetic engineering

• Gel Electrophoresis: a technique used to separate DNA fragments by size

• DNA is loaded into wells on one end of a gel and an electric current is applied

  • DNA fragments are negatively charged so they move towards the positive electrode

• PCR (polymerase chain reaction): a method used in molecular biology to make several copies of a specific DNA segment

• segments of DNA are amplified

• results can be analyzed using gel electrophoresis

• DNA Sequencing: the process of determining the order of nucleotides in DNA

• Sanger Sequencing: determines DNA sequences by selectifvely incorperating chain-terminating dideoxynucleotides (ddNTPs), generating NDA fragments of varying lengths that are analyzed using capillary electrophoresis.

• Modern Sequencing: uses massively parallel sequencing to generate millions of short reads simultaneously, making it faster, cheaper, and more scalable for whole-genome and high-throughput sequencing applications

Biotechnology

Biotechnology Today

• genetic engineering

  • maniupulation of DNA

  • if you are going to engineer DNA & genes & organisms, then you need a set of tools to work with\

Bacteria

• Bacteria review

  • one-celled prokaryotes

  • reproduce by mitosis

    • binary fission

  • rapid growth

    • generation every 20 minutes

    • 10^8 (100 million) colony overnight

  • dominant form of life on earth

  • incredibly diverse

• Bacterial genome:

  • single circular chromosme

    • haploid

    • naked DNA

      • no histone proteins

      • 4 million base pairs

      • 4300 genes

      • 1/1000 DNA in eukaryote

• transformation:

  • bacteria are opportunists

    • pick up naked foreign DNA wherever it may be hanging out

    • have surface transport proteins that are specialized for the uptake of naked DNA

  • imports bits of chromosomes from other bacteria

  • incorperate the DNA bits into their own chromosome

    • express genes

    • transformation

    • form of recombination

• Plasmids

  • small supplemental circles of DNA

    • 5000-20000 base pairs

    • self-replicating

  • carry extra genes

    • 2-30 genes

    • genes for antibiotic resistance

  • can be exchanged between bacteria

    • bacterial sex

    • rapid evolution

  • can be imported from environment

  • how can plasimids help us?

    • a way to get genes into bacteria easily

      • insert new gene into plasmid

      • insert plasmid into bacteria = vector

      • bacteria now expresses new gene

        • bacteria makes new proteins

  • biotechnology

    • plasmids used to insert new genes into bacteria

How do we cut DNA?

• restriction enzymes

  • resitriction endonucleases

  • discovered in 1960’s

  • evolved in bacteria to cup up foreign DNA

    • “restrict” the action of the attacking organism

    • protection against viruses & other bacteri a

      • bacteria protect their own DNA by methylation & by not using the base sequences recognized by the enzymes in their own DNA

Restriction enzymes

• action of enzyme

  • cut DNA at specific sequences

    • restriction site

  • symmetrical “palindrome”

  • produces protrudinf ends

    • sticky ends

    • will bind to any complementary DNA

  • many different enzymes

    • named after organism they are found in

      • ex: EcoRI, HindIII, BamHI, SmaI

• discovery of restriction enzymes

  • restriction enzymes are named for the organism they come from: EcoRI = 1st restriction enzyme found in E.Coli

• restriction enzymes:

  • cut DNA at specific sites

    • leave “sticky ends”

• Sticky ends

  • cut other DNA with same enzymes

    • leave “sticky ends” on both

    • can glue DNA (genes) together at “sticky ends”

    • why mix genes together? gene produces protein in different organism or different individual

    • the code is universal

      • since all living organisms use the same DNA, use the same code book, and read their genes the same way

  Copy (& Read) DNA

  • tranformation

    • insert recombinant plasmid into bacteria

    • grow recombinant bacteria in agar cultures

      • bactiera make lots of copies of plasmid

      • “cloning” the plasmid

    • production of many copies of inserted gene

    • production of “new” protein

      • transformed phenotype

Uses of genetic engineering

• genetically modified organisms (GMO)

  • enabling plants to produce new proteins

    • protect crop from insects: BT corn

      • corn produces a bacterial toxin that kills corn borer (caterpillar pest of corn)

    • extend growing season: fishberries

      • strawberries with an anti-freezing gene from flounder

    • improve quality of food: golden rice

      • ricde producing vitamin A improves nutritional value

Cut, Paste, Copy, Find……

• word processing metaphor

  • cut

    • restriction enzymes

  • paste

    • logase

  • copy

    • plasmids

      • bacterial transformation