molec chap 13 transcription in bacteria

RNA to DNA viruses

retroviruses - reverse transcription by rna-dep dna pol

RNA to RNA viruses

coronaviruses, reoviruses, many eukaryotes - rna genome replication, rna amplification, rna-dep rna pol

open reading fram (ORF)

Rna composed of consecutive, non overlapping codons (nulceotides triplets)

these codons can be translated in protein during translation by ribosomes

polycistronic mRNA

multi - ORF. in prokaryotes, rare in euk

monocistronic mRNA

eukaryotic, one ORF

DNA encoding polycistronic mRNA is called an

operon

Operons are under the control of….

a single promoter in transcription

what is the main objective of prokaryotes

to quickly adapt to the env (temp, nutrients, pH, O2, stresses)

What is the half life of mRNA in prokaryotes

short (abt 10 min)

mRNA’s are quickly/slowly synthesized and degraded in prokaryotes.

quickly

Regulation in prokaryotes is

simple and achieved at level of transcription

how are genes often organized in prokaryotes

in operons

regulation in eukaryotes is…

highly refined and intricate, enabling adapt. to env changes, response to stimuli

intricate regulation in eukaryotes is essentral for…

cell differentiation and dev

Operons are rare/common in eukaryotes

rare

mRNA half life in eukaryotes

long half life (days)

how is mRNA expression controlled in eukaryotes

at multiple levels, including transcription, rna processing, rna stability, and translation

spatiotemporal regulation of genes

In eukaryotes, where specific genes are activated at precise times and locations → key for cell dif and specialization

what are required for RNA synthesis by RNAPol

ATP, CTP, GTP, UTP

in RNA synthesis, RNA chain grows

5’ to 3’ direction

in RNA synthesis, DNA template sequence determines…

RNA sequence. There is complementary pairing. one DNA strand is template, other is sense strand that has same sequence as RNA

where is the transcription start site/start point

At the +1 nucleotide, the fourth one.

What causes transcription to start

when RNA polymerase binds to the promoter at the beginning of the gene

What does the promoter surround

the first base pair that’s transcribed into RNA, the start point

How long does RNA polymerase move along the template after binding to the promoter

until it reaches a terminator sequence, which defines a transcription unit that extends from the promoter to the terminator.

what are the various proteins that interact w DNA at/near promoter to regulate transcription intitiation

RNA pol, transcrip factors = activators, repressors

How is synthesizing the new complementary strand different in transcription, compared to replication

complementary strand is composed of ribonucleotides (NTPs) bc there is a 2’ OH on the sugar meaning it is RNA not DNA

does RNA pol need a primer

No!

is RNA or DNA pol more error prone

RNA pol

in transcription are there many RNA pol or little

many

Replication of part of/entire once/multiple times

entire genome once

transcription of entire/part of genome once/multiple times

part of genome multiple times

error rate of replication

10^-9 - 10^-10

error rate of transcription

10^-4-10^-6

error rate of translation

10^-4

Base selection and proofreading by DNA Pol are _____ more/less accurate than those of RNA Pol

10-100x more

DNA Pol has _____ and ______

proofreading capability and post synthesis mismatch repair mechanisms

RNAPol has ______ but there are no _____ after RNA or protein synthesis

proofreading capabilities, but no repair mechanisms

RNA and proteins can/can’t be prod with lower accuracy without long term consequences

can

DNA replication requires ____ to maintain inheritance of function, but _____ allow genome evolution

high accuracy, occasional errors

mRNAs

code for proteins

rRNAs

form basic struc of ribosome and catalyze protein synthesis

tRNAs

central to protein synthesis as adaptors between mRNA and amino acids

snRNAs

small nuclear rnas, func in variety of nuclear process, including splicing of pre-mRNA

snoRNAs

small nucleolar RNAs, used to process and chemically modify rRNAs

other noncoding RNAs

func in diverse cellular processes, including telomere synthesis, X chrom inactivation, and transport of proteins into ER

coding strand (sense strand)

has same sequence as mRNA and is related, by genetic code, to protein sequence that it represents

antisense strand

template strand, complementary to sense strand, and is one that acts as template for synthesis of mRNA

RNAPol

enzymes that synthesize RNA using DNA template(formally described as DNA-dep RNA Pol)

promoter

region of DNA where RNA Pol binds to initiate transcription

start point/site

position on DNA corresponding to first base incorporated into RNA

terminator

sequence of DNA that causes RNAPol to terminate transcription

transcription unit

seq between a site of initiation and termination by RNA pol; may include more than one gene

upstream

identifies seq in opposite direction from expression; for ex, bacterial promoter is upstream of transcription unit, initiation codon is upstream of coding region

downstream

seq proceeding farther in direc of expression; for ex coding region is downstream of initiation codon

primary transcript

original unmodified RNA product corresponding to transcription unit

what are the 3 steps of transcription

initiation, elongation, termination

The RNA pol core is made of

subunits!

Core enzyme

minimal set of subunits for activity in rna pol

what can core enzyme do

initiate and synthesize rna from ssDNA and nicked DNA templates

be reassembled invitro from subunits

what can’t the core enzyme do

initiate transcrip w intact dsDNA as template

5 subunits in core enzyme

a2B B’w

a2w

enzyme assembly, interac w regulators

BB’

interface forms active site

initiation

promoter recognition by the RNAPol holoenzyme

holoenzyme

a’a’’B B’wσ

what does the sigma factor do in the RNApol holoenzyme

confers RNAPol w promoter specificity and promotes isomerization (DNA melting)

initiation steps:

R + P → KB-< RPc → (Kf) RPo

what is the promoter recognized by in initiation

RNAPol holoenzyme

where is promoter located at initiation of transcrip

beginning of transcrip unit

what is the promoter constituted by

elements -10, -35, options UP-element

in initiation, core promoter is made of

-10 and -35 motifs

sigma factors are essential for

promoter recognition in initiation

what are sigma factors comprised of

4 domains: σ1-4

sigma factors recognize……. and cannot…….

2 seq of 6 nucleotides )-10,-35) and cannot bind to DNA on own

what is promoter strength

not well conserved.

strength of RNApol interac w promoter is set by

seq of -35, -10 regions and type of sigma factor, length of spacing region(16-19, optimum 17 bp), and presence of UP element

transcription specificity

dif sigma factors recognize dif consensus sequences for expression of regulons

a regulon is

genes (bacteria) under coordinated control of a single regulatory mechanism

structure of sigma factor

helix-turn-helix

dna melting in initiation is promoted by

sigma 2 domain

Initiation: in certain bcateria some sigma factors recognize…

dif elements of promoter

initiation: initial transcription

aboritve initiation. no need for a primer and 3 hypotheses for abortive initiation (synth of short RNAs < 10nt)

3 hypotheses for abortive initiation

transiet excursions, inchworming, scrunching

initiation: promoter escape

isomerization (slow - rate limiting, and irreversible) 10s - min

initial transcrip (abortive transcrip) can be slow too

promoter clearance- signam factor dissociates from core

what can inhibit initiation

Rifampicin

Rifampicin

inhibits bacterial RNA pol but not eukaryotic enzyme and medically useful as a potent bacteriocidal

how does Rifampicin inhibit initiation

blocks RNA exit tunnel, doesn’t inhibit elongation complex, RNApol-rifampicin complex remains stuck at the promoter

steps of transcription initiation

closed complex, intermediate stage, open complex, scrunched complex, promoter escape, elongation complex

sigma 54 RNAPol req

an activator protein (enhancing binding proteins - EBPs aka AAA+ protein)

-req for promoter melting, ATP dep

sigma 54

nitrogen metabolism regulon

elongation

RNA + rNTP → (favors forward) ← RNA+1 + PPi

whats required in elongation

2 Mg2+ req for addition of a new nucleotide to RNA strand. one Mg2+ bound in RNAPol active site. each nucleotide comes w additional Mg2+

Translocation in elongation follow a

brownian ratchet mechanism

how many channels does rna pol have in elongation complex

3 - duplex binding, rna exit, ntp

Duplex-binding channel

formed by B’ subunit, strand separation begins around +2, DNA makes sharp 90 deg bend

RNA exit channel

interacts w ssRNA that has separated from hybrid

NTP channel

NTPs bound to one Mg2+

dna supercoiling can occur during

elongation of transcription

elongation: processivity

ability to perform consecutive rxns