Unit 8: DNA Replication and Gene Expression

The Role of DNA

DNA Replication

Transcription/Translation

The effects of mutations

How do we know that DNA is the hereditary molecule

The process by which cells use hereditary information stored in DNA is very elegant

DNA must be retained intact, yet copied to make new cells (DNA replication)

It must be turned into working copies in the form of mRNA (Transcription)

mRNA molecules must be read and decoded to form the enzymes/structural proteins of the cell (Translation)

System must have ability to deal with damage (DNA repair)

The Griffiths Experiment

Some genetic factor can be transformed between S and R bacteria

Took heat killed infectious bacteria that were lysing and mixed it with live noninfectious bacteria and inserted it into the mouse. The mouse then died

Avery, MacLeod, McCarthy Experiment 

Attempted to determine if it was DNA, RNA, or proteins that were transferrable from the Griffith’s experiment

Used mixtures obtained from S strains (infectious) but digested away the other components (proteases for proteins, RNAase for RNA, DNAase for DNA)

Only the mixture with DNA left could still transform R cells into S cells indicating that was the molecule responsible 

Hershey-Chase Experiment

Used Radioactive labeling of either proteins or DNA in bacteriophages

They let the labeled phages infect bacterial cells, then determined where the tag ended up for each set up 

Only the labeled phage DNA went into the bacterial cells further proving that DNA was the hereditary molecule in these experiments 

DNA Replication is Semiconservative

Each time the dsDNA is copied, each copy carries one strand of the original molecule and newly made strand 

Structure of DNA

Each nucleotide building block of DNA consists of a five carbon sugar (2-deoxy/ribose), a phosphate group attached to carbon 5 of the sugar, a nitrogenous base attached to carbon 1

Complementary

Phosphodiester covalent bonds form the sugar/phosphate backbone of each strand

Higher GC content more stable the DNA, organisms with low GC content have mechanisms to deal with stability 

How DNA is packed

Bacteria: A single circular chromosome, has plasmids, histone like proteins but not homologous

Archaea: A single circular chromosome packaged around histone proteins, has plasmids 

Eukarya: Multiple linear chromosomes packaged around histone proteins, rarely plasmids 

Histones 

The wrapping of dsDNA 

Helps compact the very large chromosome structures of eukaryotic cells 

Bacterial Exception

D. radiodurans may possess 4 to 10 copies of its genome

The stacked genome copies may help give this bacterium its strong radiation-damage-resistance trait

If one of the copies is mutated the others will take over  

Bacteria DNA Replication: Initiation 

DNAa protein binds to 9-bp repeats of oriC and subsequently w/13-bp repeat this separates the strands (particular part where DNA replication starts)

DNAb (helicase) is recruited with DNAc (helicase loader) for further unwinding

DNAg (primase) is recruited to lay initial complementary RNA primers needed for DNA polymerase to work (ESSENTIAL STEP FOR DNA REPLICATION)

Single stranded DNA binding proteins are recruited to help keep the DNA unwound

This all forms the replication fork/bubble

Eukaryotic DNA Replication: Initiation 

Multiple origins of replication on each chromosome

Needs multiple enzymes to initiate and create the replication fork 

Chromosomes are much larger this causes multiple origins of replication (every 10-100,000 bp)

ARS: Autonomously replicating sequence forms binding site for initiator ORC 

ORC recruits additional proteins to unwind DNA 

Archaea are very similar to Eukaryotes 

Bacteria DNA Replication: Elongation

Occurs bidirectionally (helicases continue unwinding DNA)

Production of new DNA strands complementary to old strands catalyzed by DNA polymerase

Primase RNA polymerase synthesizes short single stranded primer to start the strand (This must be removed later)

Once replication fork forms DNA polymerase III adds nucleotides to the 3’ OH group of initial RNA primers (5’→3’) using DNA as a template

A continuous leading strand and a discontinuous lagging strand both synthesized at the same time

One molecule of double stranded DNA leads to 2 daughter dsDNA

Okazaki Fragments

Formed on the lagging stand

Strand can only be synthesized 5’ to 3’ so opposite strand must synthesize in fragments

DNA pol I removes the RNA primers by endonuclease activity and fills gaps with new nucleotides 

Fragments are later joined together by DNA ligase (5’ phosphate and 3’ OH sugar backbone)  

Eukaryotes DNA Replication: Elongation

Replication is highly conserved

It is largely the same in bacteria and Eukarya 

Bacteria DNA Replication: Termination

Termination of DNA replication mediated by ter sites on opposite side of oriC (DNA polymerase makes its way bidirectional to ter sites) 

Tus proteins bind to tern sites and stops the forward process of one of the replication forks 

Two halves of newly synthesized DNA are joined when other replication for arrives causing two identical circular dsDNA molecules 

Topoisomerase II forms transient double strand break then disentanglement (then pushed towards middle by parM)

DNA Replication Overview

DNA structure clearly determines the mechanism of replication

Allows the production of two new molecules identical to original 

Transcription

The conversion of dsDNA into ssRNA

Has specific initiation, elongation, and termination processes

Gene

A segment of DNA that gets transcribed into +ssRNA

Associated DNA regions necessary for transcription 

Differences between RNA and DNA

Ribose in RNA, Deoxyribose in DNA

Uracil in RNA, Thymine in DNA

The RNAs

Different forms of RNA and they each serve a different purpose

mRNA: 500-10,000 nucleotides, coding molecule translated into proteins (template for translation)

tRNAs: 75-100 nucleotides, Involved in translation charged with amino acids (not translated but participate in translation)

rRNA: 1500-1900 (small unit) 2900-4700 (large) 

miRNA: less than 100 nucleotides functions to regulate gene expression (not translated but participate in translation)

Transcription Initiation Bacteria

Must identify the beginning and end of the segment

Starts at a promoter where DNA dependent RNA polymerase binds to promotor and separates the DNA and synthesizes complementary strand 

RNA Polymerase reads the template DNA 5’ to 3’ but does not need a primer

RNA is the same sequence but there is U not T

RNA Polymerase Bacteria

Consists of 5 different polypeptide chains, alpha, beta, beta’, omega, and sigma

Sigma factor bound to RNA polymerase core enzyme and directs the combined holoenzyme to a promoter (this is very important in order for RNA polymerase to bind and the disassociation causes the DNA to unwind) 

The better the binding the better the transcription and the more mRNA produced

How does Transcription begin Bacteria 

At a consensus sequence at positions -10 (Pribow-Box or TATA box) and -35 in bacteria

Differences in the sequence dictates the affinity of the sigma factor 

Eukarya Transcription: Initiation and elongation

Resembles bacteria

However…

Eukarya possesses 3 different RNA polymerases (I, II, III)

RNA pol II transcribes all protein encoding genes (similar to bacteria)

RNA pol I: rRNA genes

RNA pol III: tRNA and miRNA

There are individual transcription factor proteins that must associate with the promoter regions first

RNA pol is recruited to the transcription factor/DNA complex

Binding initiates the unwinding of DNA and the start of transcription 

Archaea Transcription: Initiation and elongation

Not well understood

Possess only one DNA dependent RNA polymerase like bacteria but it looks more like eukaryotic RNA polymerase II

RNA pol does not bind to DNA directly

Transcription factors are homologous to eukaryotes and it directs RNA pol to promotor regions like Eukarya 

Identifying DNA Binding Proteins

Can use EMSA (Electrophoretic Mobility Shift Assay)

Many DNA binding proteins like histones, DNAa, Orc, Sigma factors

Band shift indicates DNA protein interactions 

Smaller migrates further, larger migrates shorter 

How can this be used: You can test if protein binds with DNA after incubation, this would show if there is binding or not 

Bacteria Transcription Termination

Rho dependent, a rho factor follows RNA pol and pops it off the DNA when it reaches a termination sequence

Rho independent, genes and GC rich repeat followed by string of adenines forms an RNA hairpin loop structure that causes RNA polymerase to dissociate 

Eukarya Transcription Termination

More complex

RNA pol I resembles rho dependent

RNA pol III resembles rho independent

RNA pol II: cleaved by specific endonucleases 

Post Translational Modifications

Eukaryotic pre-mRNA (product of RNA polymerase II) modified after transcription in 3 ways

Modified nucleotide (7-methylguanosine) added to 5’ end transcript and 5’ cap added

Poly A tail added by polyA polymerase

Introns (non-protein coding regions in eukaryal pre-mRNA) spliced out and exons are joined 

5’ cap and poly A protect against degradation (RNA need to be protected because transcription and translation are separate) 

Translation

Information contained in the mRNA molecule is decoded to form proteins 

Extremely energy intensive so it is tightly controlled 

Ribosomes

Interact with three nucleotides (codons) on mRNA and tRNAs which are charged with amino acids

Each nucleotide triplet matches complementary anticodon on each tRNA, amino acid is determined by the anticodon sequence

Ribosomes read the mRNA sequence 5’ to 3’ and recruits the tRNA

Peptide bonds are then formed between amino acids that are delivered

Structure of tRNA

Cloverleaf structure

Undergo intra molecular base pairing

High degree of hairpin structure which allows for stability

Anticodon must remain unpaired to match mRNA

Highly specific aminoacyl-tRNA-synthetase enzymes achieve charge this charge must be before translation 

One tRNA for each amino acid (20 different tRNAs) so there are 20 different genes for tRNA 

The Genetic Code

4 bases made in mRNA creates 64 codons

61 codons, 1 Start codon (AUG), 3 Stop codons (UAA, UAG, UGA)

Identical in all organisms 

The codon on the mRNA does not need to be exact to match the anticodon

This is known as a wobble (3rd base is not essential) 

How does Ribosome know where to start

fMET-tRNA (initiator tRNA) binds to AUG after the Shine-Dalgarno sequence mediated by initiation factor 2 and modified to N-formyl-methionine 

First AUG after Shine-Dalgarno box will be the start 

Bacteria Translation: Initiation

Initiation depends on interaction between small ribosome subunit (16s) and the Shine-Dalgarno sequence on mRNA

This aligns all the machinery to start 

Bacteria Translation: Elongation

Charged tRNA with complementary anticodon enters aminoacyl A site of ribosome

Peptide bonds form in peptide P site

Empty tRNA exits ribosome from exit site (uncharged)

When ribosome reaches a stop codon, release factors cause complex to come apart, releasing new protein for folding and modification 

Bacteria and Shine Dalgarno sequences

mRNA is polycistronic (monocistronic in eukaryotes)

Which means it can code for more than one protein 

Contain the genetic information for more than one gene however, every single gene has its own Shine-Dalgarno sequence this leads to faster translation as a ribosome can bind to each one 

Role of Nuclear Membrane 

Because of the lack of a nuclear membrane in bacteria transcription and translation are coupled

Also the lack of introns and PTMs of mRNA make coupling easier

Mutations

All of the following only refer to a mutation in a coding region

Silent: Usually the 3rd AA results in no change

Missense: A change in a codon that results in coding for a different AA

Nonsense: A change that forms a stop codon where is should not be 

Frameshifts: insertions or deletions can change how ribosome reads and can drastically alter

Inversion: DNA becomes inverted at the same location

Translocation: DNA segment breaks off and reattaches at different chromosomes

Mutations can be good or bad

If a mutation was in a non-coding region (depending on what it is) would affect the protein

Mutations From Envionment

Chemicals (nitrous acid) can induce mutations by removing amino groups

Ultraviolet light can cause higher than expected mutation rate in DNA by forming thymine dimers 

Ionizing Radiation can cause double strand to break in phosphate sugar backbone 

Repair of Mutations

DNA pol can proof read

Mismatch repair systems

Photolyase cleaves covalently linked T-T (thymine dimer)

Alkyl transferase can remove methyl groups adding to bases by DNA modifying agents

DNA glycosylases recognize damaged bases and initiates excisions