MBB 331 Lecture 11

Which DNA strand does RNA polymerase read

The template strand

Which strand has the same sequence as the RNA transcript (except T→U)

coding strand

Why is it called the coding strand

Because its sequence matches the RNA sequence (except T is replaced by U)

Which direction does RNA polymerase read DNA

3' → 5'

Which direction is RNA synthesized

5' → 3'

RNA polymerase moves along

template strand.

RNA polymerase builds

complementary RNA

What are the component

2 beta, 2 alpha, and 1 omega

Has a cleft which is the

active site and hydrolysis/addition of new nucleotides

What is the role of α subunit

Assembly and interactions with regulatory proteins

What is the role of ω

Stabilizes assembly of the enzyme

What is the holoenzyme

Core enzyme + sigma factor.

Core enzyme can recognize

promoters and start transcription

In eukaryotes

3 RNA pol

What does sigma factor do

Recognizes promoter sequences and directs RNAP to promoters

Is sigma required for elongation

No

What often happens to sigma during elongation

It falls off the complex

Why is sigma essential

Without sigma, RNAP cannot efficiently recognize promoters

Missing sigma can cause what

RNAP can't properly start transcription

Sigma recoganizes what

TATA box and -35 box

Why are UP elements benefical

genes are highly expressed because they allow for tighter binding

What is the transcription bubble

short melted region of DNA maintained by RNA polymerase

What happens ahead of the bubble

DNA unwinds

What happens behind the bubble

DNA reanneals

Why are topoisomerases needed

To relieve strain generated during transcription

Bubble moves with

RNAP

What does it lack

3-5' exonuclease proofreading

Why is exonuclease not required

because chances all transcripts have mistake is super low

What does upstream mean

DNA before the transcription start site

What does downstream mean

DNA after the transcription start site

What is proximal

Close to the transcription start site

What is distal

Far from the transcription start site

What is a promoter

DNA sequence where RNAP binds to initiate transcription

What are the three promoter elements

UP element, -35 element, and -10 element

Which promoter element is recognized first by sigma

-35 element

Which promoter element melts most easily

-10 element (AT rich).

What is down mutation

divergence from -10 and -35 consequence or insertions between the two reducing transcription

What is up mutation

deletions that bring distance closer to 17 bp and optimal match between region increasing transcription

What makes a promoter strong

Close match to consensus sequences

Why is the lac promoter weak

The -35 box is unlike the consensus sequence

Stronger promoter means what

RNAP binds more easily and more transcription

lac promoter activation requires

CAP-cAMP.

What proofreading mechanisms does RNAP use

Kinetic proofreading and nucleolytic proofreading.

Does RNAP have 3'→5' proofreading

NO 3' to 5' proofreading

What is kinetic proofreading

RNAP stalls after incorporating a mismatched base, allowing pyrophosphorolysis to remove it

What is nucleolytic proofreading

RNAP backs up and cleaves the rNTPs upstream of the mismatch

bascially “redoes” the stretch of sequence

RNAP proofreading Option 1

stall- Pyrophosphorolysis removes base

RNAP proofreading Option 2

Backtrack- Cut RNA

What are the major stages of transcription

Recognition & binding → Closed to open promoter melting → abortive initiation/promoter clearance → Elongation → Termination

RNA polymerase/promoter binding

the core is next to the transcription site and sigma factor helps bind RNA polymerase to promoter

What is Closed to open complex

DNA opens up and we melt promoter region and can read nucleotides

What is abortative initaiation

couple nucleotides added and then transcription stops couple times until promoter clears

What is promoter clearance

RNA polymerase is able to move away from promoter and begin transcription

What direction is RNA synthesized

5'→3'.

What does processive mean

RNAP is not released until the transcription is finished

How many nucleotides remain paired with template DNA

8-9 nucleotides

What enzyme relieves torsional strain

Topoisomerase

Ensures transcription throuhg

kinetic/nucleolytic proofreading no 3-5 proofreading

What creates intrinsic termination

DNA sequences transcribed into RNA hairpins

What follows the termination hairpin

Stretch of U's less than 10 nt

Why does RNA fall off in rho independent termination

Weak A-U bond between DNA-RNA easily detaches transcript from DNA

Rho-dependent what protein is required

Rho factor

What type of protein is Rho

Hexameric ring.hydrolyzes ATP and has RNA-DNA helicase activity

Where does Rho bind

Rut site

What genes are in the lac operon

lacZ, lacY, lacA

What does lacZ encode

β-galactosidase

What is the inducer of the lac operon

Allolactose

What happens when lactose is absent

Repressor binds the operator and blocks RNAP

What happens when lactose is present

Allolactose binds LacI causing it to dissociate from operator

What activates high transcription

CRP-cAMP

When does cAMP rise

Low glucose

What is required for high lac operon expression

Lactose present AND low glucose

What does a gel shift assay detect

DNA-protein binding

What indicates binding in a gel shift assay

Slower migration (shifted band)

What does DNase footprinting identify

Exact DNA sequence bound by a protein

Why does a footprint appear

Bound protein protects DNA from DNase digestion