chapter 15 transcription

transcription

doesnt require a primer

dna to RNA in the dogma

synthesize RNA 5’ → 3’

can occur on either strand of DNA as long as its 5→ 3

mrna encoded protein

transcription NEVER beings at a start codon

extra RNA sequences (5’ Untranslated Regions-UTRs) are required upstream of the start codon to

1. Load ribosomes

2. regulate translation efficiency

3. regulate RNA stability

reason for UTRs

1. load ribosomes

2. regulate translation efficiency

3. regulate RNA stability

how much DNA is wound up from RNA polymerase

~15bp

RNAP

what does RNA pol require

Mg+2 and NTP

mg+2 (active site) and generally neutralizes the neg charge of the phosphates

number of RNA pol in bacteria

only one

number of RNA polymerase in eukaryotes

three

• Pol I (rRNA)

• Pol II (mRNA and microRNA)

  • other non-coding RNA

• Pol III (tRNA, 5S rRNA)

  • 7SL RNA (involved in membrane protiens)

active site for RNA pol

between the two beta pincers? overall structure of RNA pol is conserved

actinomycin D

inhibits RNA and DNA synthesis, mushroom toxins

inhibits the RNA elongation

it is used in the laboratory to identify cell processes that depend on RNA synthesis

alpha amanitin

selectively inhibits RNA pol II

disrupts eukaryotic mRNA synthesis by blocking Pol II and, at higher concentrations, Pol III.

“useful in the laboratory as a specific inhibitor of eukaryotic Pol II, or to determine the polymerase responsible for transcribing a particular gene“

Indeed, Pol I, Pol III and bacterial RNA polymerase are insensitive to alpha-amanitin

transcription cycle

1. binding of RNA polymerase core to DNA promoter

   • reversible, closed complex

2. formation of transcription bubble

   • isomerization

   • open complex: irreversible

3. initiation

   • lots of abortive initiations

4. elongation (promoter clearance)

   • 9+ nucleotides Promoter ESCAPE

transcription cycle written out

The polymerase binds the promoter (step 1), forming first a closed complex, in which the bound DNA is intact, and then an open complex (step 2), in which the bound DNA is partially unwound near a region 10 bp ahead of (upstream of) the transcription start site.

Transcription is initiated within the complex (step 3), leading to a conformational change that converts the complex to the form required for elongation.

Promoter clearance, involving movement of the transcription complex down the DNA template and away from the promoter, leads to the formation of a tightly bound elongation complex (step 4).

Once elongation begins, RNA polymerase becomes a highly efficient enzyme, completing synthesis of the transcript before dissociating from the DNA template (step 5)

supercoils from RNA polymerase

underwound dna negative right handed supercoils go in

overwound DNA positive left handed supercoils go out

bacterial RNA pol

4 core subunits

• basic machines to make mRNA

sigma factor

• where to start

• which direction to go

single rna polymerase enzyme

promoters in bacteria

• -10 binding/spec

  • all promoters have some kind of -10

  • those without the -35 are generally weaker

• some very strong promoters e.g. for ribosomal RNA genes contain “Up-element“

  • -60 to -40 region upstream

  • binds RNAP alpha SU

consensus sequence at -10 region

5' -TATAAT-3';

all promoters have some kind of -10 region

consensus sequence at -35 region

is 5'-TTGACA-3'

those without the -35 region are generally weaker

upstream promote r (UP) element

• occurs between positions -40 and - 60 in the promoters of certain highly expressed genes

• bound by one of the alpha subunits of RNA polymerase.

divergence from consensus

determines basal transcription rate

sigma 70 factor

occupies both DNA entrance channel and RNA exit channel

Nterminus is displaced during open complex formation

typically multiple rounds or abortive initiation before C terminal end is displaced

• growing mrna chain is very not stable intil 10 nts long

disociates after promoter clearance

NusA binds RNA pol when sigma 70 leaves

closed complex?

the holoenzyme binds to its promoter to form what is initially a closed complex, with the DNA maintaining its double-stranded structure; formation of this closed complex is readily reversible. In complexes with the cr70-class factor, the closed complex can spontaneously convert to a transcription-competent open complex, in a process of isomerization

RNA pol proofreading

rna pol error rate 10^-4 to 10^-5

DNA pol error rate 10^-6 to 10^-8