Molec Bio II Exam I (Ch 13-14)

open reading frame (ORF)

- Nucleic acid, composed of consecutive, non-overlapping triplets.

- These triplets are translated in proteins during translation by ribosomes.

- Start and stop codons

- Encodes for polypeptide

polycistronic mRNA

- Has more than one orf's

- Often found in Prokaryotes

- Located in 5'UTR and 3'UTR

- 2+ polypeptides

monocistronic mRNA

- Only one orf

- Found in Euks

- 5'cap and poly-A tail

- encodes for 1 polypeptide

operon

- Cluster of genes under the control of a single promoter

- Produces polycistronic mRNA

- Often encodes enzymes for consecutive steps of a metabolic pathway

coding strand

- also called Sense Strand

- the strand of DNA has the same sequence as the mRNA, and is related, by the genetic code, to the proteins sequence that it represents

template strand

- also called the antisense strand

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

apoenzyme

- also called core enzyme

- the minimal set of subunits for activity

- alpha2 Beta Beta' omega

- synthesizes RNA from ssRNA and nicked DNA samples

- cannot use intact circular dsDNA as template

- can be reassembled in vitro from subunits

Bacterial RNA Polymerase subunits and their functions (core enzyme)

- alpha2 Beta Beta' omega

- a2w: enzyme assembly, interaction with regulators

- BB': interface forms the active site (pincers of crab claw)

holoenzyme

- alpha2 Beta Beta' omega sigma factor

- apoenzyme + sigma factor

direction of transcription

5' to 3'

Steps of Transcription

initiation, elongation, termination

what are the components of bacterial promoters?

-10, -35, optional UP-element

Where are promoters located?

beginning of transcription unit

what does the optional UP-element do?

inc binding affinity

What sigma factor binds to -35?

Sigma factor 4

What sigma factor binds to -10?

Sigma factor 2

-10 element is also called what?

pribnow box

What binds to the promoter region during transcription initiation?

Upstream RNA Pol --> forms closed complex

What occurs during the melting/isomerization step?

The DNA near the transcription site starts to unwind to form an open complex. Once open, the RNA polymerase will add enzymes (rNTP's) on the template, DNA

Unlike DNA polymerase RNA does not need what for initiation of transcription?

a primer

What happens after melting/isomerization of transcription initiation? What is it?

- once short stretches of RNA are formed the open complex undergoes abortive initiation

- a lot of short RNA will be produced from the previous step and RNA is too short to bind well, so they dissociate

Why does RNA dissociate in abortive initiation?

Because the sigma factor blocks the RNA exit channel of RNA polymerase

What are the three models for abortive initiation?

Transient excursions, inch worming, scrunching

What happens in transient excursions?

RNAP 'leaps'

What happens in inch worming?

RNAP moves

What happens in scrunching?

DNA moves, RNAP stays in place

How many nucleotides must be added to an RNA to escape the promoter?

About 10

What is the last step in initiation of transcription?

promoter escape

What occurs in promoter escape?

The release of the sigma factor from the core

What is the rate limiting step of initiation of transcription?

isomerization

During elongation, the active site is made up of regions from what two subunits?

B and B'

Where is the polymerase active site found during elongation?

At the base of the pinchers within a region called the active center cleft (B and B')

What does the active site bind to during elongation?

Two magnesium ions

What are the three channels that RNA polymerase has?

Duplex binding channel, RNA exit channel, NTP channel

duplex binding channel

- formed by B' subunit

- encircles ~9 bp of downstream dsDNA

- strand separation begins around +2

RNA-DNA hybrid binding site

- formed by the B and B' subunits

- surrounds 8-9 bp of RNA-DNA hybrid

RNA exit channel

Interacts with ssRNA that has separated from the hybrid

NTP channel

NTPs are bound to one Mg2+

What happens when elongation goes wrong?

Recruitment of transcription coupled DNA repair machinery:

Pyrophosphorolytic editing and Hydrolytic editing

Pyrophosphorolytic editing

Remove single misincorporated nucleotide using pyrophos

Hydrolytic editing

- Backtracking and hydrolysis of several nucleotides

- Happens when there's a big error

What are the two mechanisms of termination of transcription?

Rho independent, and rho dependent

Rho independent

- does not require external proteins for termination to occur

- intrinsic RNA terminator: inverted sequence, hairpin structure that halts RNAP, and A:U base paris are formed (weak)

Rho dependent

- depends on rho factor (requires ATP)

- Rho protein binds ssRNA rich in cytosine (in rut site) and pulls

- recruited to loading site

- catches up with RNAP (pause)

What role do sigma factors have on transcription specificity?

different sigma factors recognize different consensus sequences for the expression of regulons

What are regulons?

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

During promoter clearance, what disassociates from the core?

sigma factor

What does Rifampicin inhibit?

bacterial RNA pol but not euk enzyme

What does rifampicin do to inhibit initiation?

Blocks addition of the second nucleotide during abortive transcription

What does rifampin not inhibit?

the elongation complex

Where does RNAPol-rifampicin get stuck?

the promoter

What is ChIP sequencing? What does it do?

- chromatin immunoprecipitation and seq

-Helps find where transcription factors are bound to DNA.

What does transcription assays do?

Measure RNA synthesis

What does NusA do?

Inc RNAP pausing--> inc termination

What is EMSA?

Electrophoretic mobility shift assay

What does EMSA do?

determine protein binding affinity, binding order stoichiometry

What does footprinting do?

identify protein-DNA interactions

T7 of RNA Pol is _________

monomeric

Does T7 RNA Pol need a sigma factor to initiate transcription?

No

What type of promoter does T7 RNAP have?

Simple

Where do T7 RNAP specificity beta sheets interact?

in the groove -7 to -11

Many plasimids in _____ exhibit a T7 promoter.

E coli

What does T7 RNAP allow?

better control of expression -- advantageous for toxic proteins

In euks, what recognizes the promotors?

transcription factors NOT RNAP

In euks, what are transcription factors needed for?

initiation

How many general transcription factors (GTFs) does RNA Pol II have?

6

What are the 6 GTF's in RNA Pol II?

TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH

In euks, promotors span across _____

the start site

What does RNA Pol I and III do?

Synthesize non-coding RNA (rRNA, tRNA, snRNA)

What does RNA Pol II do?

synthesizes coding RNA (mRNA and hnRNA) and snRNA

What is hnRNA?

heterogenous nuclear RNA is the unprocessed mRNA in the nucleus

In what order are the GTFs added in transcription initiation?

D, A, B, F, Pol II, E, and H

TFIID binds to what in the promotor?

TATA element

TATA box details

- TATAXAX consensus sequence

- Located 30 bp upstream of transcription start site

- In highly regulated genes

_________ binding protein binds TATA sequence

TBP-TATA

What does the TBP-TATA do?

- bends DNA sequence by 80 degrees

- allows for better binding of TFIIA and TFIIB

What does TFIIA do?

helps in stabilizing of TFIID with the promoter

What does TFIIB do?

reacts with TBP and the promoter region downstream to the data sequence

What does TFIIB recruit?

Pol II and TFIIF

What does TFIIE bind to?

preinitiation complex

What does TFIIE do?

creates a docking site for TFIIH

What are the roles of the TFIIH subunits?

- 2 subunits have ATPase activity

- 7 subunits have kinase activity

The subunits with ATPase activity do what in TFIIH?

Act as helicase and melts promotor causing the transition from preinitiation to open complex

What do the subunits with kinase activity do in TFIIH?

Phosphorylates the C terminal domain (the tail of polymerase 2) leading to promoter escape and transcription elongation

What GTFs are positioning factors?

TFII D, A, and B

What transcription factors help in enlongation?

TFEb and TFIIS

What recruits TFEb to polymerase?

transcription activators

What is TFEb?

a kinase protein and phosphorylates serine residues in the C terminal domain of the polymerase to stimulate elongation

What does TFIIS do?

helps increase rate of transcription at the region where the rate of transcription becomes slow

What does TFIIS not allow?

RNA Pol to pause and encourages it to move on

What does TFIIS contribute to?

proofreading

mRNA forms in what direction?

5' to 3'

What is the first RNA processing event to occur during elongation?

5' capping

RNA triphosphatase removes what?

Terminal gamma phosphate of the nucleotide

Guanylyl transferase enzyme carries out the reaction between ____ phosphate of the first nucleotide and ____ phosphate of GTP

beta; alpha

After a guanine is attached and a methyl group transfers, what is formed?

5' cap

What does the 5' cap help in?

the recruitment of mRNA for the initiation of translation

What are the main functions of 5' capping?

- necessary for nuclear export

- prevents degradation

- promotes translation

How does termination of transcription begin?

RNA Pol reaches end of gene -> C terminal domain (CTD) of RNAP interacts with CstF and CPSF

What is CstF and what does it do?

- cleavage stimulation factor

- Dissociates once the mRNA is cleaved