Molecular Genetics Lecture 18

Bacterial Transcription 2

what do sigmas do in RNAP?

promoter recognition

Sigma 1.1 Domain function

in the groove before DNA is seated

negatively charged

in the closed promoter complex

in N terminus, what does 1.1 sigma domain do?

folds back and covers up domain 4.2 so it can’t see the -35 or -10 regions, masks the DNA recognition binding sites

When RNAP binds to the core, what opens up?

N terminus so the holoenyzme can search for a promoter

Holoenzyme

Core RNAP (a2BB’w) + sigma

recognized promoters and initiates transcriptionn

core RNAP

a2BB’w, inactive RNAP

if domain 1.1 is deleted then…

the free sigma will bind to a promoter without a core

sigma domain 1.2 function

recognized the discriminator element, binds the few nts in front of the discriminator

sigma domain 2.1 and 2.2 function

bind sigmas to the core

are very conserved

sigma domain 2.2 function

interact with core beta’

recognizes the core

sigma domain 2.3 function

melt the DNA strand at the -10 promoter region with tryptophan and tyrosine

sigma domain 2.4 function

recognizes the first two bases of TATA

sigma domain 3.0 function

binds to the extended -10 (bases before the TATA region)

sigma domain 3.2 function

snakes into the active site through the exit pore bumping into the transcript

makes contact with the first two bases, involved in abortive cycling

sigma domain 4.2 function

recgonizes the -35 promoter region, a helix turn helix

sometimes bumps against alphaCTD

what does helix turn helix mean?

the dominant DNA fold that interacts sepcifically with DNA

can recognize promoter elements

recognizes the major groove, fits perfectly into the major groove

how does helix turn helix read DNA bases?

by reading the side of the bases

what does TBP stand for?

TATA binding protein

mutations in -35 promoter region affect…

affinity of the initial binding in the formation of the closed promoter complex

why is the binding low affinity on purpose?

so the promoters need another element, like a cap for stability

mutations at the -10 promoter region effect…

the rates of forming both open and closed promoter complexes

when does true elongation begin?

when the polymerase leaves the promoter

to leave the promoter what must push domain 3.2 back out of the exit pore?

the nascent transcript

when 3.2 is removed from the exit pore…

this reduces affinity for sigma to the core (by 30%), now the polymerase can leave the promoter

what does the C terminal domain of the alpha subunit make contact with?

the UP element

C terminal means

carboxyl terminal

the ribosomal cintron synthesizes…

ribosomal genes, most active promoter in E. Coli, structural ribosomes

what is rifampicin?

an antibiotic that interacts with the nascent transcript

rifampicin occupies the same domain as…

sigma domain 3.2

what is the difference between sigma domain 3.2 and rifampicin?

it cannot be pushed out of the way so it blocks transcription

sigma 70 is coded by; function

gene rpoD; recognizes most of the promoters (1000)

What is the -10 and -35 region of sigma 70?

TTGACA-16 and 18 bp-TATAAT

which is written first, the -10 or -35 element?

-10 element

how is the cap regulated?

glucose levels

changing the spacing between promoters lowers…

affinity

what gene codes for Sigma S?

rpoS

what does Sigma S do?

stationary phase/some stress responses

what is the stationary phase?

when the glucose in a petri dish is all digested, this is the stationary phase. must slow metabolism because there is no more nutrition

Sigma S recognizes which promoters?

same as Sigma 70

TTGACA-16 to 19 bp-TATAAT

Sigma S has a mutation that prevents it from binding to…

the C terminal domain of Alpha

so it prevents the promoter from becoming active

gene that codes for sigma 32 is…

rpoH

sigma 32 does what?

heat shock response (activated by heat shock)

promoter region for sigma 32…

CCCTTGAA-13 to 15 bp-CCCGATNT

what does sigma 54 do?

nitrogen assimilation, in nitrogen starvation conditions

what gene codes for sigma 54?

rpoN

what is the promoter sequence for sigma 54?

CTGGNA-6 bp TTGCA

sigma 54 is missing which domain?

1.1 so it binds to the promoter before it binds to the core

sigma 54 cannot melt the promoter until…

it has a helper protein and aTP

at the open promoter complex, DNA is seated…

in the groove

at closed promoter complex where is DNA?

on top of the complex

things that RNAP can do that DNAP cannot, DNA Duplex

can melt the DNA duplex

things that RNAP can do that DNAP cannot; initiation

can initiate synthesis independently

things that RNAP can do that DNAP cannot; 2’-OH

can make multiple contacts with the 2’-OH of the incoming NTP

things that RNAP can do that DNAP cannot; abortive cycling

abortive cycling can occur while still in contact with the promoter

what is DNAse I footprinting used for?

to map where a protein sits on DNA

when the core binds to random DNA, it has a half life of…

60 min

when the core binds to DNA it is what kind of binding

loose binding, it is reversible

when the sigma joins to the core enzyme it has what fold reduction in the affinity for random DNA?

10k

when the sigma attaches to the core RNAP, it gains BLANK fold affinity for promoter DNA

1000

half life for non-specific DNA is

less than a second

fastest rate of initiation is

once a second

slowest rate of initiation isis

once every half hour (lac repressor)

when the polymerase changes from initiation to elongation it also changes

confirmation smaller footprint 75-35

when do stalls in the RNAP happen?

at strings of As or Gs

when a stall happens the transcript…

protrudes out of the front of the enzyme

to recover from a stall you must…

reverse the catalysis so it cleaves a nt in the active site so the end will float away

which proteins bind to the polymerase for it to cut when a stall happens?

GreA and GreB

in eukaryotes, transcription factor II S does what?

what GreA and GreB do

which promoter is the strongest promoter?

the rrnB operon; makes ribosomal RNAs

what makes the rrnB operon so high affinity?

the UP element increases affinity 60 fold

FIS sites 5 fold increase

what does UP stand for in UP element

upstread promoter

NtrC needs to touch which two elements to work?

sigma and needs ATP