BIOL 106 Final Exam

Frederick Griffith- 1928

Frederick Griffith- 1928

• Studied Streptococcus pneumoniae, a pathogenic bacterium causing pneumonia

• 2 strains of Streptococcus

  • S strain is virulent (causes pneumonia)

  • R strain is nonvirulent

• Griffith infected mice with these strains hoping to understand the difference between the strains

• Transformation

  • Information specifying virulence passed from the dead S strain cells into the live R strain cells

• Out modern interpretation is that genetic material was actually transferred between the cells

Avery, MacLeod, & McCarty-1944

• Repeated Griffith’s experiment using purified cell extracts

• Removal of all protein from the transforming material did not destroy its ability to transform R strain cells

• DNA-digesting enzymes destroyed all transforming ability

• Supported DNA as the genetic material

Hershey & Chase-1952

• Investigated bacteriophages

  • Viruses that infect bacteria

• Bacteriophage was composed of only DNA and protein

• Wanted to determine which of these molecules is the genetic material that is injected into the bacteria

• Bacteriophage DNA was labeled with radioactive phosphorus (32P)

  • DNA contains phosphorus, proteins do not

• Bacteriophage protein was labeled with radioactive sulfur (35S)

  • Some amino acids contain sulfur, DNA does not

• Radioactive molecules were tracked

• Only the bacteriophage DNA (as indicated by the 32P) entered the bacteria as was used to produce more bacteriophage

• Conclusion: DNA is the genetic material

DNA structure

• DNA is a nucleic acid

• Polymer of nucleotides

  • 5-carbon sugar called deoxyribose

  • Phosphate group (PO4)

    • Attached to 5’ carbon of sugar

  • Nitrogenous base

    • Adenine, thymine, cytosine, guanine

  • Free hydroxyl group (-OH)

    • Attached at the 3’ carbon of sugar

• Phosphodiester bond

  • Bond between adjacent nucleotides

  • Formed between the phosphate group of one nucleotide and the 3’-OH of the nucleotide

• The chain of nucleotides has a 5’-to –3' orientation

Chargaff’s Rules (1950)

• Erwin Chargaff determined that

  • Amounts of A, G, T, C varied between different species, but not within tissues of same individual (or members of the same species)

  • Amount of adenine =amount of thymine

  • Amount of cytosine =amount of guanine

  • Always an equal proportion of purines (A and G) and pyrimidines (C and T)

Rosalind Franklin

• Performed X-ray diffraction studies to identify the 3-D structure

  • Discovered that DNA is helical (“a spiral structure in a macromolecule that contains a repeating pattern”)

  • Using Maurice Wilkins’ DNA fibers, discovered that the molecule has a diameter of 2 nm and makes a complete turn of the helix every 3.4 nm

James Watson and Francis Crick-1953

• Deduced the structure of DNA using evidence from Chargaff, Franklin, and others

• Did not perform a single experiment themselves related to DNA

• Proposed a double helix structure

Double helix

• 2 strands are polymers of nucleotides

• Phosphodiester backbone-repeating sugar and phosphate units until joined by phosphodiester bonds

• Wrap around 1 axis

• Antiparallel

• Complementarity of bases

• A forms 2 hydrogen bonds with T

• G forms 3 hydrogen bonds with C

• Gives consistent diameter (which was noted w/X-Ray diffraction)

DNA Replication

• 3 possible models

  • Conservative model

    • Produces one entirely new molecule, while conserving the old

  • Semiconservative model

    • Produces two hybrid molecules of old and new strands

  • Dispersive model

    • Produces two hybrid molecules with each strand a mixture of old and new

Meselson and Stahl- 1958

• Bacterial cells were grown in a heavy isotope of nitrogen, 15N

• All the DNA incorporated 15N

• Cells were switched to media containing lighter 14N

• DNA was extracted from the cells at various time intervals- mixed with CsCI & spun down in a centrifuge (resulting in heaviest DNA at the bottom)

Conservation model=rejected

2 densities were not observed after round 1

Semiconservative model=supported

• Consistent with all observations

• 1 band after round 1

• 2 bands after round 2

Dispersive model=rejected

• 1st round results consistent

• 2nd round- did not observe 1 band

DNA replication require 3 things

• Something to copy

  • Parental DNA molecule

• Something to do the copying

  • Enzymes

• Building blocks to make copy

  • Nucleotide triphosphates

• DNA replication include

  • Initiation: replication begins

  • Elongation: new strands of DNA are synthesized by DNA polymerase

  • Termination: replication is terminated

DNA polymerase

• Matches existing DNA bases with complementary nucleotides and links them

• All have several common features

  • Add new bases to 3’ end of existing strands

  • Synthesize in 5’ to 3’ direction

  • Require a primer of RNA

Prokaryotic Replication

• E. coli model

• Single circular molecule of DNA

• Replication begins at one origin of replication (oriC)

• Proceeds in both directions around the chromosome

• Replicon- DNA controlled by an origin

• E. coli has at least 3 DNA polymerases

  • DNA polymerase 1 (pol 1)

    • Acts on lagging strand to remove primers and replace them with DNA

  • DNA polymerase 2 (pol 2)

    • Involved in DNA repair processes

  • DNA polymerase 3 (pol 3)

    • Main replication enzyme

  • All 3 have 3’ to 5” exonuclease activity- proofreading

  • DNA pol 1 has 5’ to 3’ exonuclease activity

• Unwinding DNA causes torsional strain

  • Helicases: use energy from ATP to unwind DNA

  • Single-strand-binding proteins (SSBs) coat strands to keep them apart

  • Topoisomerases prevent supercoiling

    • DNA gyrase is used in replication

Endonucleases

Nucleases which cuts DNA internally

Exonucleases

Nucleases which chew away at an end of DNA

Semidiscontinous

• DNA polymerase can synthesize only in 1 direction

• Leading strand synthesized continuously from an initial primer

• Lagging strand synthesized discontinuously with multiple priming events

  • Okazaki fragments

• DNA is antiparallel (one strand is 5’ to 3’ & other is 3’ to 5’)

• DNA polymerase adds nucleotides only at the 3’ end

• Partial opening of helix forms replication fork

• DNA primase: RNA polymerase that makes RNA primer

  • RNA will be removed and replaced with DNA

Leading-strand synthesis

• Single priming event

• Strand extended by DNA pol 3

  • Processivity- β subunit forms “sliding clamp” to keep it attached

Lagging-strand synthesis

• Discontinuous synthesis

  • DNA pol 3

• RNA primer made by primase for each Okazaki fragment

• All RNA primers removed and replaced by DNA

  • DNA pol 1

• Backbone sealed

  • DNA ligase

• Termination occurs at specific site

  • DNA gyrase unlinks 2 copies

Replisome

• Enzymes involved in DNA replication form a macromolecular assembly

• 2 main components

  • Primosome

    • Primase, helicase, accessory proteins

  • Complex of 2 DNA pol 3

    • One for each strand

Replication fork

1. A DNA polymerase 3 enzyme is active on each strand. Primase synthesizes new primers for lagging strand

2. The “loop” in the lagging-strand template allows replication to occur 5’ to 3’ on both strands, with the complex moving to the left

3. When the polymerase 3 on the lagging strand hits the previously synthesized fragment, it releases the β clamp and the template strand. DNA polymerase 1 attaches to remove the primer

4. The clamp loader attaches the β clamp and transfers this to polymerase 3, creating a new loop in the lagging-strand template. DNA ligase joins the fragments after DNA polymerase 1 removes the primers

5. After the β clamp is loaded, the DNA polymerase 3 on the lagging strand adds basses to the next Okazaki fragment

Eukaryotic replication

• Complicated by

  • Larger amount of DNA in multiple chromosomes

  • Linear structure

• Basic enzymology is similar

  • Requires new enzymatic activity for dealing with ends only

• Multiple replicons: multiple origins of replications for each chromosome

  • Not sequence specific; can be adjusted

• Initiation phase of replication requires more factors to assemble both helicase and primase complexes onto the template, then load the polymerase with its sliding clamp unit

  • Primase includes both DNA and RNA polymerase

  • Main replication polymerase is a complex of DNA polymerase epsilon (pol ε) and DNA polymerase delta (pol δ)

Telomeres

• Specializaed structures found on the ends of eukaryotic chromosomes

• Protect ends of chromosomes from nucleases and maintain the integrity of linear chromosomes

• Gradual shortening of chromosomes with each round of cell division

  • Unable to replicate last section of lagging strand

• Replication potentially shortens the ends of eukaryotic chromosomes

• The ends of linear chromosomes are maintained by the action of the telomerase enzyme

• Telomeres composed of short repeated sequences of DNA

• Telomerase: enzyme makes telomere section of lagging strand using an internal RNA template (not the DNA itself)

  • Leading strand can be replicated to the end

• Telomerase developmentally regulated

  • Relationship between senescence and telomere length

• Cancer cells generally show activation of telomerase

DNA repair

• Mistakes during replication are detected and repaired by DNA polymerase proofreading activity

• Proofreading by DNA polymerase corrects errors during replication

• Mutagens: any agent that increases the number of mutations above background level

  • Radiation and chemicals

• Importance of DNA repair indicated by the multiplicity of repair systems that have been discovered

• Falls into 2 general categories

  • Specific repair

    • Targets a single kind of lesion in DNA and repairs only that damage

  • Nonspecific

    • Use a single mechanism to repair multiple kinds of lesions in DNA

Photo repair

• Specific repair mechanism

• For one particular form of damage caused by UV light

• Thymine dimers

  • Covalent link of adjacent thymine bases in DNA

• Photolyase

  • Absorbs light in visible range

  • Uses this energy to cleave thymine dimer

Excision repair

• Nonspecific repair

• Damaged region is removed and replaced by DNA synthesis

• 3 steps

  • Recognition of damage

  • Removal of the damaged region

  • Resynthesis using the information on the undamaged strand as a template

Uncorrected replication errors result in mutations

• Replication is not a perfect process. Errors occur and if uncorrected produce mutations

• Mutations are changes in the nucleotide sequence of the DNA strands

• Mutations can lead to changes in the protein sequence encoded by the DNA

• There are several types of mutations

• Point mutations

  • Silent

  • Missense

  • Nonsense

• Frameshift mutations

  • Insertions

  • Deletions

• Chromosome mutations

  • Insertions, deletions, translocations, inversions, fusions, duplications

The nature of genes

• Early ideas to explain how genes work came from studying human disease

• Archibald Garrod- 1902

  • Recognized that alkaptonuria is inherited via a recessive allele

  • Proposed that patients with the disease lacked a particular enzyme

• These ideas connected genes to enzymes

Beadle and Tatum- 1941

• Deliberately set out to create mutations in chromosomes and verify that they behaved in a Mendelian fashion in crosses

• Studied Neurospora crassa

  • Used X-rays to damage DNA

  • Looked for nutritional mutations

    • Had to have minimal media supplemented to grow

• Beadle and Tatum looked for fungal cells lacking specific enzymes

  • The enzymes were required for the biochemical pathway producing the amino acid arginine

  • They identified mutants deficient in each enzyme of the pathway

• One-gene/one-enzyme hypothesis has been modified to one-gene/one-polypeptide hypothesis (even this is overly simple)

Central dogma

• Information typically flows from DNA->RNA->protein

• Transcription= DNA-> RNA

• Translation= RNA-> protein

• Retroviruses & some mobile elements violate this order using reverse transcriptase to convert their RNA genome into DNA

Transcription

• DNA-directed synthesis of RNA

• Only template strand of DNA used (the other is called the “coding strand” or “sense strand”)

• U (uracil) in DNA replaced by T (thymine) in RNA

• MRNA used to direct synthesis of polypeptides

Translation

• Synthesis of polypeptides

• Takes place at ribosome

• Requires several kinds of RNA

Prokaryotic transcription

• Single RNA polymerase

• Initiation of mRNA synthesis does not require a primer (short stretch of nucleic acids)

• Requires: (transcription unit)

  • Promoter

    • Forms a recognition and binding site for the RNA polymerase

    • Found upstream of the start site

    • Not transcribed

    • Asymmetrical-indicate site of initiation and direction of transcription

  • Start site

  • Termination site

RNA

• All synthesized from DNA template by transcription

• Messenger RNA (mRNA)

• Ribosomal RNA (rRNA)

• Transfer RNA (tRNA)

• Small nuclear RNA (snRNA)

• Signal recognition particle RNA (SRP RNA)

• Micro-RNA (miRNA)

Genetic Code

• Francis Crick and Sydney Brenner determined how the order of nucleotide in DNA encoded amino acid order

• Codon: block of 3 DNA nucleotides corresponding to an amino acid

• Introduced single nucleotide insertions or deletions and looked for mutations

  • Frameshift mutations

  • Indicates importance of reading frame

• Marshall Nirenberg identified the codons that specify each amino acid (1961-66)

• Stop codons

  • 3 codons (UUA, UGA, UAG) used to terminate translation

• Start codon

  • Codon (AUG) used to signify the start of translation

• Code is degenerate, meaning that some amino acids are specified by more than one codon

Spaced codons

Codon sequence in a gene punctuated

Unspaced

Codons adjacent to each other

Code practically universal

• Strongest evidence that all living things share common ancestry

• Mitochondria and chloroplasts have some differences in “stop” signals

Elongation

• Grows in the 5’ to 3” direction as ribonucleotides are added

• Transcription bubble: contains RNA polymerase, DNA template, and growing RNA transcript

• After the transcription bubble passes, the now-transcribed DNA is rewound as it leaves the bubble

Termination

• Marked by sequence that signals “stop” to polymerase

  • Causes the formation of phosphodiester bonds to cease

  • RNA-DNA hybrid within the transcription bubble dissociates

  • RNA polymerase releases the DNA

  • DNA rewinds

• Hairpin

Prokaryotic transcription is coupled with translation

• MRNA begins to be translated before transcription is finished

• Operon

  • Grouping of functionally related genes

  • Multiple enzymes for a pathway

  • Can be regulated together

Eukaryotic Transcription

• 3 different RNA polymerases

  • RNA polymerase 1 transcribes rRNA

  • RNA polymerase 2 transcribes mRNA and some snRNA (most genes are transcribed mRNA)

  • RNA polymerase 3 transcribes tRNA and some other small RNAs

• Each RNA polymerase recognizes its own promoter (e.g., RNA pol 2 recognizes TATA box at –10nt)

• Initiation of transcription

  • Requires a series of transcription factors

    • Necessary to get the RNA polymerase 2 enzyme to a promoter and to initiate gene expression

    • Interact with RNA polymerase to form initiation complex at promoter

• Termination

  • Termination sits not as well defined

MRNA modifications

• In eukaryotes, the primary transcript must be modified to become mature mRNA

  • Addition of a 5’ cap

    • Protects from degradation; involved in translation initiation

  • Addition of a 3’ poly-A tail

    • Created by poly-A polymerase; protection from degradation

  • Removal of non-coding sequences (introns)

    • Pre-mRNA splicing done by spliceosome

Eukaryotic pre-mRNA splicing

• Introns: non-coding sequences

• Exons: sequences that will be translated

• Small ribonucleoprotein particles (snRNPs) recognize the intron-exon boundaries

• SnRNPs cluster with other proteins to form spliceosome

  • Responsible for removing introns

Alternative Splicing

• Single primary transcript can be spliced into different mRNAs by the inclusion of different sets of exons

• ~ 25% of known human genetic disorders are due to altered splicing

• Up to 95% of human multi-exon genes undergo alternative splicing to encode proteins with different functions

• Explains how 25,000 genes of the human genome can encode the more than 80,000 different mRNAs

TRNA and Ribosomes

• TRNA molecules carry amino acids to the ribosome for incorporation into a polypeptide

  • Aminoacyl-tRNA synthetases add amino acids to the acceptor stem of tRNA

  • Anticodon loop contains 3 nucleotides complementary to mRNA codons

TRNA charging Reaction

• Each aminoacyl-tRNA synthetase recognizes only 1 amino acid but several tRNAs

• This reaction is called the tRNA charging reaction

• Charged tRNA-has an amino acid added using the energy from ATP

• Ribosomes do not verify amino acid attached to tRNA-so correct attachment of amino acid to tRNA is important

• The ribosome has multiple tRNA binding sites

  • P (peptidyl) site: binds the tRNA attached to the growing peptide chain

  • A (aminoacyl) site: binds the tRNA carrying the next amino acid

  • E (exit) site: binds the tRNA that carried the last amino acid

• The ribosome has two primary functions

  • Decode the mRNA

  • Form peptide bonds

• Peptidyl transferase

  • Enzymatic component of the ribosome (RNA-based)

  • Forms peptide bonds between amino acids

Translation: Overview

• MRNA is threaded through the ribosome

• TRNAs carrying amino acids bind to the ribosome

• TRNAs interact with mRNA by base-paring with the mRNA’s codons

• The ribosome and tRNAs position the amino acids such that peptide bonds can be formed between each new amino acid and the growing polypeptide

Translation

• In prokaryotes, initiation complex includes

  • Initiator tRNA charged with N-formylmethionine

  • Small ribosomal subunit

  • MRNA strand

• Ribosome binding sequence (RBS) of mRNA positions small subunit correctly

• Large subunit now added

• Initiator tRNA bound to P site with A site empty

• Initiations in eukaryotes similar except

  • Initiating amino acid is methionine

  • More complicated initiation complex

  • Lack of an RBS- small submit binds to 5’ cap of mRNA

• Elongation adds amino acids

  • 2nd charged tRNA can bind to empty A site

  • Requires elongation factor called EF-Tu to bind to charged tRNA and GTP

  • Peptide bond can then form

  • Addition of successive amino acids occurs as a cycle

• There are fewer tRNAs than codons

• Wobble pairing allows less stringent pairing between the 3’ base of the codon and the 5’ base of the anticodon

• This allows fewer tRNAs to accommodate all codons

• Termination

  • Elongation continues until the ribosome encounters a stop codon

  • Stop codons are recognized by release factors which release the polypeptide from the ribosome

Protein targeting

• In eukaryotes, translation may occur in the cytoplasm or the rough endoplasmic reticulum (RER)

• Signal sequences at the beginning of the polypeptide sequence bind to the signal recognition particle (SRP)

• The signal sequence and SRP are recognized by RER receptor proteins

• Docking holds ribosome to RER

• Beginning of the protein- trafficking pathway

Wobble pairing

• There are fewer tRNAs than codons

• Wobble pairing allows less stringent pairing between the 3’ base of the codon and the 5’ base of the anticodon

• This allows fewer tRNAs to accommodate all codons

DNA and RNA Extraction

• Lysis buffer (mostly detergent) breaks down lipids in the cell and its membranes

• After the cells are broken down, they are treated with enzyme proteases and/or ribonucleases to further break down material

  • The processes and enzymes depend on the desired outcome

• Remaining material is centrifuged to separate it. The supernatant (liquid) containing the DNA/RNA is extracted

• Liquid is precipitated with ethanol to create strands

DNA Manipulation

• Restriction endonucleases revolutionized molecular biology

• Enzymes that cleave DNA at specific sites

  • Used by bacteria against viruses

• Restriction enzymes significant

  • Allow the creation of recombinant DNA molecules (from two different sources)

3 types of restriction enzymes

• Type 1 and 3 cleave with less precision and are not used in manipulating DNA

• Type 2

  • Recognize specific 4-12 bp DNA sequences

  • Cleave at specific site within sequence

  • Can lead to “sticky ends” that can be joined

    • Blunt ends can also be joined

DNA ligase

• Joins the two fragments forming a stable DNA molecule

• Catalyzes formation of a phosphodiester bond between adjacent phosphate and hydroxyl groups of DNA nucleotides

• Same enzyme joins Okazaki fragments on lagging strand in replication

Transformation

• Introduction of foreign DNA from an outside source into a cell

• Natural processes in many bacterial species

  • E. coli does not

• Temperature shifts can induce artificial transformation in E. coli

• Transgenic organisms are all or part transformed cells (more later...)

Molecular cloning

• Clone: genetically identical copy

• Molecular cloning- isolation of a specific DNA sequence (usually protein-encoding)

  • Sometimes called gene cloning

• The most flexible and common host for cloning is E. coli

  • Vector: carries DNA in host and can replicate in the host

Plasmids

• Small, circular chromosomes

• Used for cloning small pieces of DNA

• 3 components

  • Origin of replication allows independent replication

  • Selectable marker: allows the presence of plasmid to be easily identified

  • Multiple cloning site (MCS)

Artificial chromosomes

• Plasmids have limited insert size

• Yeast artificial chromosomes (YACs)

• Allow for larger insert for large-scale analysis of genomes

Polymerase chain reaction (PCR)

• Developed by Kary Mullis (1985)

  • Awarded Nobel Prize

• Allows the amplification of a small DNA fragment using primers that flank the region

• Each PCR cycle involves three steps:

  • Denaturation (high temperature)

  • Annealing of primers (low temperature)

  • DNA synthesis (intermediate temperature)

    • Taq polymerase

• Applications of PCR

  • Allows the investigation of minute samples of DNA

  • Forensics: drop of blood, cells at base of a hair

  • Detection of genetic defects in embryos by analyzing a single cell

  • Analysis of mitochondrial DNA from early human species

Gel Electrophoresis

• Separate DNA fragments by size

• Gel made of agarose or polyacrylamide

• Submersed in buffer that can carry current

• Subjected to an electrical field

• Negatively-charged DNA migrates towards the positive pole

• Larger fragments move slower, smaller move faster

• DNA is visualized using fluorescent dyes

DNA libraries

• A collection of DNAs in a vector that taken together represent the complex mixture of DNA

• Genomic library: representation of the entire genome in a vector

  • Genome is randomly fragmented w/restriction enzymes

  • Inserted into a vector

  • Introduced into host cells (e.g., E. coli)

  • Usually constructed in BACs

• Complementary DNA (cDNA)

  • DNA copies of mRNA

  • MRNA isolated

    • Represents only actively used genes

    • No introns

• Use reverse transcriptase to make cDNA

• CDNA used to make library

• All genomic libraries from a cell will be the same but cDNA libraries can be different

Molecular hybridization

• Technique used to identify specific DNAs in complex mixtures such as libraries

• Also termed annealing

• Known single-stranded DNA or RNA is labeled

• Used as a probe to identify its complement via specific base-pairing

• The most common way of identifying a clone in a DNA library of interest

Southern blotting

• Sample DNA is digested by restriction enzymes and separated by gel electrophoresis

• Double-stranded DNA denatured into single-strands

• Gel “blotted” with filter paper to transfer DNA

• Filter is incubated with a labeled probe consisting of purified, single-stranded DNA corresponding to a specific gene

Northern blotting

RNA is separated by electrophoresis and then blotted onto the filter

Western blotting

• Proteins are separated by electrophoresis and then blotted onto the filter

• Detection requires an antibody that can bind to one protein

DNA fingerprinting

• Restriction fragment length polymorphisms

• Generated by point mutations or sequence duplications

• Restriction enzyme fragments are often not identical in different individuals

• Can be detected by Southern blotting

• STRs

  • Identification technique used to detect differences in the DNA of individuals

  • Short tandem repeats (STRs)

    • Typically 2-4 nt long

    • Not part of coding or regulatory regions

  • Population is polymorphic for these markers

  • Using several probes, probability of identity can be calculated, or identity can be ruled out

  • Also used to identify remains

Methods of sequencing

• All modern-day methods utilize the chain termination method (the dideoxy method) of sequencing developed by Fred Sanger (1977)

• Types:

  • Shotgun sequencing

  • Pairwise-end sequencing

  • Next generation sequencing

Dideoxynucleosides and deoxynucleotides

• A dideoxynucleoside is similar in structure to a deoxynucleotide, but is missing the 3’ hydroxyl group (indicated by the box)

• When a dideoxynucleoside is incorporated into a DNA strand, DNA synthesis stops

Genetic Engineering: “Knockout” mice

• Can replace a wildtype gene with mutant copy to determine function

• “knockout” mice: known gene is inactivated

• Effect is then assessed in adult mouse (or if lethal- the stage of development at which function fails can be determined)

• Cloned gene interrupted by replacement with a marker gene

• Marker gene codes for resistance to the antibiotic neomycin

• Interrupted gene is introduced into embryonic stem cells (ES cells)

• ES cells injected into embryo early in development

How does regulation benefit organisms?

• Each somatic cell contains the full genome of an organism

• All genes do not need to be expressed simultaneously in each cell (in fact, that would be detrimental to the organism)

• Regulation maintains efficiency

  • Energy: expressing all gene would require a massive amount of energy

  • Space: cells are kept to a manageable size

  • Time: genes can be expressed as needed and more rapidly

General model of transcription and translation in prokaryotic organisms

• Transcription and translation occur simultaneously in the cytoplasm

• Regulation occurs at the transcriptional level

• Eukaryotic transcription and RNA processing occurs in the nucleus

• Translation takes place in the cytoplasm

• Gene expression is regulated in following ways:

  • Epigenetic

  • Transcription

  • Post-transcription

  • Translation

  • Post-translational modification of proteins

Prokaryotic gene regulation

• Prokaryotic DNA is organized into a circular chromosome located in nucleoid region of cytoplasm

• Proteins with similar function or in the same biochemical pathway are organized in blocks called operons

• Operons are regulated by three molecule types

  • Repressors: suppress transcription

  • Activators: increase transcription

  • Inducers: may suppress or activate transcription depending upon the needs of the cell

The trp Operon: A repressor operon

• When tryptophan is plentiful

  • Two tryptophan molecules bind the repressor protein at the operator sequence

  • The complex physically blocks the RNA polymerase from transcribing the tryptophan genes by binding to the operator

• When tryptophan is absent

  • The repressor protein does not bind to the operator

  • The RNA polymerase can access the operator and the genes are transcribed

Negative regulators

proteins that bind to the operator silence trp expression

Glucose supplies become limited in cell

• CAMP levels increase

• CAMP binds to the CAP protein (a positive regulator)

• CAMP/CAP protein complex binds to an operator region upstream of the genes required to use other sugar sources

Positive Regulators

Proteins that bind the promoter in order to activate gene expression

The lac Operon: An inducer operon

• In the absence of lactose, the lac repressor binds the operator, and transcription is blocked

• In the presence of lactose, the lac repressor is released from the operator, and transcription proceeds at a slow rate

• CAMP-CAP complex stimulates RNA Polymerase activity and increases RNA synthesis

• However, even in the presence of cAMP-CAP complex, RNA synthesis in blocked when repressor is bond to the operator

Inducible operons

Proteins that activate or repress transcription. Activation/repression depends on the local environment and the needs of the cell

Eukaryotic Epigenetic gene regulation

• Eukaryotic gene expression is more complex than in prokaryotes:

  • Transcription and translation are physically separated

  • Regulation can occur at many levels

  • 1st level begins with control of access to the DNA-epigenetic regulation-and occurs before transcription

  • Transcription factors are proteins that control the transcription of genetic information from DNA to RNA

Epigenetic control: regulating access to genes within the chromosome

• Organization of human genome

  • 20,000 genes

  • 23 chromosomes (thousands of genes per chromosome)

  • DNA is wound/compacted tightly with proteins (histones)

  • Expressed genes must be unwound and made available to polymerases

Organization of DNA and Proteins

• DNA is folded around histone proteins to create nucleosome complexes

• These nucleosomes control the access of proteins to the underlying DNA

• When viewed through an electron microscope, the nucleosomes look like beads on a string.

Nucleosomes control access to DNA

• When nucleosomes are spaced closely together (top), transcription factors cannot bind, and gene expression is turned off

• When nucleosomes are spaced far apart (bottom), the DNA is exposed

• With the DNA exposed, transcription factors can bind to it, allowing gene expression to occur

Chemical modifications of histones and DNA affect gene expression

• Chemical tags are added to histones and DNA

  • Phosphate, methyl, acetyl groups serve as tags

  • Tags are not permanent-can be added or removed

• Acts as signals to tell histones if region of chromosome should be open or closed

• Epigenetic regulation: “around genetics” temporary changes to nuclear proteins and DNA that do not alter nucleotide sequence but do not alter gene expression

Histone proteins and DNA nucleotides can be modified chemically

• Modifications affect nucleosome spacing and gene expression

• Unwinding and opening of DNA allows transcription factors to bind promoters and other upstream regions and initiate transcription

Eukaryotic Transcription Gene Regulation

• Gene transcription in Eukaryotes requires RNA polymerases

• These RNA polymerases require transcription factors (specialized proteins) to initiate transcription

• These factors bind the promoter sequence and other DNA regulatory sequences

• Eukaryotic RNA polymerases require transcription factors in order to initiate transcription

Promoter

• Region of DNA upstream of coping sequence (a few nucleotide to 100’s of nucleotides long)

• Purpose is to bind transcription factors that control the initiation of transcription

TATA Box

A series of thymine and adenine dinucleotides within the promoter 25-36 bp upstream of the transcriptional start site

Transcription factor TFIID binds the TATA box

• This recruits additional transcription factors to form a complex at TATA box

• RNA polymerase can bind to upstream sequence

• RNA polymerase is then phosphorylated, and part of protein is released from DNA

• RNA polymerase is in proper orientation for transcription

Enhancer: a DNA sequence that promotes transcription

• Each enhancer is made up of short DNA sequences called distal control elements

• Activators bind to the distal control elements and interact with mediator proteins and transcription factors

• Two different genes may have the same promoter but different distal control elements, enabling differential gene expression

Eukaryotic Post Transcriptional gene regulation

• RNA transcripts must be processes into final form before translation can begin-post transcriptional modification

• This step can be regulated to control gene expression

RNA splicing, the first stage of post-transcriptional control

Pre-mRNA can be alternatively spliced to create different proteins. This process occurs in the nucleus

How could alternative splicing have evolved?

• Splicing requires proper identification of introns

• Errors in this process could lead to splicing out of an intervening exon

• Usually, would be deleterious to organism

• But it could produce a protein variant without loss of original protein

• New variant might have had an adaptive advantage

Control of RNA stability

• The protein-coding region of mRNA is flanked by 5’ and 3’ untranslated regions (UTRs)

• RNA-binding proteins at these UTRs influences the RNA stability:

  • Can increase or decrease the length of time mRNA is present in the cytoplasm

  • They also regulate mRNA localization and protein translation

RNA stability and microRNAs

• MicroRNAs (miRNAs): short RNA molecules (21-24 nucleotides) that recognize specific sequence of mRNA

• They associate with ribonucleoprotein complex called RNA-induced silencing complex (RISC)

• RISC/miRNA bind to and degrade the mRNA

The initiation complex and translation rate

1. Translation controlled by proteins that bind and initiate process (formation of initiation complex)

2. Eukaryotic initiation factor-2 (eIF-2)- first protein to bind and form complex

3. GTP binds to eIF-2 and this complex binds to 40S ribosomal subunit

4. Methionine initiator tRNA brings mRNA and binds the eIF-2/GTP/40S complex

5. GTP is converted to GDP and energy is released

6. Phosphate and eIF-2 are released and 60S binds and translation occurs