COPY Module 5: DNA and RNA (copy)

Describe conservative, dispersive and semiconservative processes of DNA replication and state which one DNA uses

• conservative: one of the two daughter duplexes is conserved parental duplex while the other is synthesized de novo

• dispersive: parental material is scattered through the structures of the daughter duplexes

• semiconservative: one strand is conserved in each progeny

how is DNA extended?

DNA polymerase (DNAP) uses free nucelotides and adds them to the 3’ OH group of a growing polynucleotide chain (5 → 3)

DNA is replicated in what direction?

bidirectionally

first step of replication: helicase

separates the DNA double helix into 2 antiparallel strands

second step: SSB proteins

• SSB proteins bind to the single stranded DNA strands

• coat each DNA single strand to prevent them from further reannealing before replication can occur

• interact with the ss-DNA (single stranded) very loosely and can be easily knocked off the strands

3rd step of replication: Primase

• synthesizes short RNA primer: 5 → 3 direction

• this primer acts as a free 3’ -OH for the DNA poly to initiate DNA replication on

4th step replication: DNA poly 3

(DNAP)

• binds to the 3’ end of the RNA primer and begin replicating the DNA, using the original parent strand as a template in a 5’ → 3’ direction (towards the movement of the replication fork)

5th step of replication: RNase H

• small RNA primers cannot be left in the seq

• RNase H recognizes the RNA nucleotides and hydrolyzes them

• RNA primer is removed from the daughter DNA strand

6th step in replication: DNA poly 1

• fills the gap of the RNA primer that was removed by replicating a DNA primer to put in its place

7th step of replication: DNA ligase

binds to and rejoins the covalent phosphodiester bonds (backbone) between the newly replicated DNA primer and the strand

difference in the lagging strand vs leading strand

same way except one exception:

• the ss-DNA is in the wrong orientation with respect to how DNAP works

  • have to add them to the growing 3’ OH, which is moving away from the replication fork

  • the lagging strand moves towards the replication fork

• primase makes short RNA primers and dna poly 3 binds to these primers until it reaches the next RNA primer

  • called okazaki fragments

• covalently linked together by DNA ligase once the RNA primers have been hydrolyzed and replaced with DNA

what is the fidelity of DNA polymerase in an E. coli cell

DNA poly makes mistake in DNA rep once every 10^9 to 10^10 nucleotides added

• thus makes an error every 1000 - 10 000 replications

two major factors that play into the fidelity (making mistake) of DNA poly 3

1. active site constraints

2. proofreading activity of DNA polymerase

Explain how active site constraints play into the lack of mistakes from DNA poly

• active site of DNA poly 1 only accommodates the correct base pairs

• if a nucleotide incorrectly h-bonds with a base in the template dna, it most likely wont fit correctly in the active site

• exception: purines nad pyrimidines can exist in two or more tautomeric forms depending on pH → incorrect pairing and fitting in active sites

Explain how polymerase proofreading activity play into the lack of mistakes from DNA poly

• posses a 3 → 5 exonuclease activity that double checks work after adding a nucleotide

• once poly detects an incorrect base pair match, it is prohibited from moving on

  • polymerase repositions the mispaired 3’ terminus into the 3’ → 5” exonuclease site

  • exonuclease hydrolyzes the mispaired base

  • the 3’ terminus repositions back to the polymerase site

  • polymerase incorporates the correct nucleotide

How were the DNA polymerases discovered in E.coli

• 1995: DNA poly 1

  • too slow for DNA replication (but capable, just not primary polymerase)

  • performs number of clean up functions during replication, recombination, and repair

• 1970: poly 2 and 3

  • dna poly 3 is main replication enzyme in e.coli

• dna poly 4 and 5

  • involved in a specific type of DNA repair

What are the key attributes of dna poly 3 (found in E.coli)

• holoenzyme made up of 10 subunits

• Sits at each replication fork (ready to go)

• Works as a dimer, performing leading and lagging strand synthesis at the same time

• Uses an RNA primer, which is made by primase

• Has enormous processivity

  • Processivity is, in general terms, defined as an enzyme's ability to catalyze "consecutive reactions without releasing its substrate"

differences between rna and dna

• rna carries out its function in a cell as a single stranded moiety

  • can fold back on themsleves and obtain a broader range of structural conformations than dna

  • suited for a lot of cellular functions

• rna is a macromolecule known to have roles in storage and transmission of information and catalysis

3 major kinds of rna and roles

1. messenger (mrna) → encode for polypeptides

2. transfer (tRNA) → read the mRNA code and transfer the appropriate amino acid to a growing polypeptide chain in a process called translation

3. Ribosomal RNAs (rRNAs) → Ribosomes are composed of rRNA and proteins in a perfect molecule whose sole purpose is to translate the RNA message into proteins

what is a consensus seq and what are two regions that are these

refers to certain nucleotides that are particularly common at each position

• -10: 5’ TATAAT 3’

• -35: 5’ TTGACA 3’

• up (upstream promoters)

  • between -40 and -60 positions in promotors that have certain highly expressed genes

Main similarities between transcription and DNA replication

• Same fundamental chemical mechanism ( creation of phosphodiester bond)

• Same direction of synthesis (5’-->3’)

• Same 3 phases: initiation, elongation, termination

Main differences between transcription and DNA replication

• Transcription does NOT require a primer

• Transcription utilizes limited segments of the DNA molecule

• Transcription uses only one of the two DNA strands as a template

features of RNA polymerase

• haloenzyme (catalytically active form of the enzyme)

• 5 subunits

  • has a 6th sigma subunit that binds transiently to the core rna haloenzyme and directs it to specific binding sites on dna

• does not have 3’ → 5’ proofreading exonuclease activity

  • high error rate for transcription

  • not as detrimental compared to dna error

what strand of dna is used by the RNAP as a template

• 3’ → 5’ (antisense) is used as the template strand for RNA polymerase

• mRNA is thus made in a 5' → 3’ orientation

steps of initiations and elongation in transcription

1. RNA poly binds to the DNA promoter

• the rna poly is directed by its bound sigma factor to the promoter

  • there are many sigma subunits but most predominant is sigma 70

2. rna poly-sigma 70 bound → creates a closed complex

• promoter dna is stably bound not unwound

• immediately followed by the open complex

  • difference: 12-15 bp region of dna from the -10 to +3 region is unwound

3. sigma 20 dissociates and is replaced by the protein NusA

• NusA: facilities transcription termination

4. dissociation of NusA → marks termination of RNA transcript

what are the 2 mechanisms termination of transcription proceeds through

1. Rho factor-independent:

• 2 distinct features:

  • a region that produces an rna transcript with self-complementary seq which folds in on itself

    • the harpin structure is found approx 15-20 nucleotides before the rna strand

  • downstream run of 3-8 adenine (AAA…) residues in the template strand that are transcribed to a poly-Uracil run at the 3’ end of the harpin

• when rna poly arrives at the harpin structure it pauses transcription

• harpin also causes disruption between the RNA-polymerase and the RNA-DNA hybrid → more dissociation of the complex and termination of transcription

2. Rho factor dependent

• less frequent

• req additional protein called Rho (atp depdendent rna-dna helicase)

  • binds to the 3’ end of the nascent transcript (at a CA-rich site called the rut)

  • RNAP pauses transcription → Rho proceeds down the transcript towards the 3’ end unwinding the 3’ end of the transcript from the DNA template → RNAP and other protein factors are released along w the transcript

how are amino acids compiled

3 nucleotides (called a codon) encode for a specific amino acid

3 major discoveries that led to the current understanding of protein synthesis (translation)

1. The discovery of where in the cell proteins are synthesized, namely the ribosomes and the ER

2. The discovery of transfer RNAs and aminoacyl-tRNA synthetases

3. adaptor hypothesis:

• proposed by Francis Crick.

• puts together many research ideas and describes one way in which the genetic information encoded by the 4-letter language of DNA could be translated to the 20 letter language of proteins

• was later shown to be correct with the tRNA serving the role of adaptor

features of the prokaryotic mRNA

• has a start and stop codon denoting the beginning and end of the Open Reading Frame, which typically encompasses the coding region for the protein being translated

• prokaryotic mRNA also has other untranslated regions (UTRs) that are important for proper translation of

  the mRNA

  • Shine-Dalgarno sequence: allows for proper positioning of the start codon (AUG) on the mRNA relative to the ribosome, which allows for initiation of translation

Aminoacyl-tRNA synthetases (aaRS)

enzymes whose function is to covalently attach amino acids to their respective tRNA molecules.

• This occurs between the carboxylate of the amino acid and the ribose 3’ OH of the invariant 3’ terminal adenosine residue on the tRNA

ribosome

• bacteria: 70s ribosome (two subunits: 50s and 30s)

• ribosomes binds to the mRNA and the tRNAs + other soluble factors

• has 3 tRNA binding sites:

  • E (exit), P (peptidyl site), A (aminoacyl site)

step 1 of translation

activation of amino acids

• carboxyl group of each amino acid has to be activated → in order to facilitate the formation of the peptide bond AND a link needs to be established to ensure the correct amino acid is present

• role of aminoacyl-tRNA synthetases: prep or “charge” each tRNA ready for protein synthesis

tRNA

recognize specific nucleotides on the mRNA and bind to these nucleotides

• also carry amino arm, hence they bring amino acids to the site of translation

• some tRNA can recognize more than one codon when the difference is in the third/wobble position

  • one amino acid is encoded by multiple codons that just have the last letter difference

• features of note:

  • anticodon triplet (bottom of structure) complementary to the mRNA codon and will base-pair with it in an antiparallel direction

  • acceptor stem (top of structure) is the amino acid attachment site (using 3’OH)

step 2 of transcription

initiation

• begins on the mRNA with the codon AUG

  • encodes methionine

  • in bacteria: modified methionine called tRNA-fMet

    • f: N-formyl group attached to methionine

  • only charged tRNA molecule (that encodes for met) that can bind to the 30s subunit on its own in the absence of the fully assembled ribosome

• 30s subunit of a dissociated ribosome binds two initiation factors

  • IF1

  • IF3 → prevents the 30s and 50s from interacting too early

• mRNA binds next

  • shine-dalgarno seq on mRNA ensures it is placed optimally

  • allows for the binding of IF2-GTP: serves to recruit the initiation tRNA (f-Met-tRNA)

• anticodon of tRNA pairs correctly with the mRNA initiation codon

  • allows for the ribsosome to be assembled through the addition of the 50s ribosomal subunit

• gtp → gdp and pi: dissociation of all initiation factors from complex → translation can proceed

3rd step of translation: elongation

• req ribosomal complex + 3 soluble cytosolic proteins called elongation factors + GTP

3 steps:

1. Codon recognition (tRNA entry)

   • A charged tRNA enters the A-site of the ribosome.

   • It is brought in by EF-Tu + GTP.

   • GTP is hydrolyzed → EF-Tu leaves.

2. Peptide bond formation

• The amino acid in the P-site is transferred to the amino acid in the A-site.

• This forms a peptide bond.

• Catalyzed by 23S rRNA (a ribozyme).

3. Translocation

• The ribosome moves forward along the mRNA.

• The tRNA in the A-site shifts to the P-site.

• The empty tRNA exits.

• This step uses EF-G + GTP.

These 3 steps repeat over and over to build the full protein.

step 4 of translation: termination

signaled by one of 3 codons: UAA, UAG, UGA

→ immediately followed by the final coded amino acid

• Stop codon enters the A-site of the ribosome

• A release factor (RF) binds to the A-site

• This triggers hydrolysis of the bond between the polypeptide and tRNA in the P-site

• The completed polypeptide is released

• The uncharged tRNA exits

• The ribosome dissociates into 30S and 50S subunits

• All translation components separate and are recycled