Prokaryotic transcription

Question 1

RNA polymerase properties
(3)

Answer 1

1. builds up RNA chain by attaching 5’-P of an NTP to the 3’OH of the nucleotide preceding it
2. does not need a pre-existing primer to initiate synthesis of a new chain
3. Composed of a sigma subunit that recognizes promoter and the core RNA pol complex is made up of 5 subunits

Question 2

Desribe the bacterial RNA polymerase holoenzyme

Answer 2

6 subunits, 5 core subunits and 6th sigma unit is needed for initiation and cycles in and out of complex

Question 3

Mechanism of RNA synthesis

Answer 3

addition of an rNTP in a Mg2+ dependent reaction that produces 5-3 phosphodiester linkages

Question 4

What is the other strand called that is not the template

Answer 4

coding strand or non-template strand

Question 5

direction of RNA synthesis

Answer 5

5’-3’

Question 6

what is the transcription bubble?

Answer 6

the RNAP maintains a bubble of melted DNA to reveal the template strand
DNA duplex is unwound for about 17 bp, forming a bubble, which enables RNA polymerase to access the template strand.

Question 7

What are promoters

Answer 7

regions of DNA where the RNAP holoenzyme can bind the dsDNA and begin transcription at a transcriptional start site on the template strand

Question 8

Promoters recognized by the same sigma factor share a particular ______ _______

Answer 8

consensus sequence

Question 9

Which promoters do RNAP bind to

Answer 9

sigma70
o-70

Question 10

How can you predict the strength of a promoter

Answer 10

Promoters with a close match to the sigma 70 consensus and have an UP element are especially strong promoters

Question 11

three distinct phases of RNA synthesis

Answer 11

initiation: RNA polymerase binds to promoters
Elongation: process of adding nucleotides to the growing RNA strand
Termination: release of the product RNA when the polymerase reaches the end of a gene
formation of transcription bubble happens before all

Question 12

T/F RNA polymerase requires a primer like DNA polymerase

Answer 12

F

Question 13

what is the -10 region

Answer 13

site of strand melting that reveals the template strand
binds sigma 2

Question 14

describe the conversion of the closed complex to the open complex by sigma factors
(2)

Answer 14

1. the DNA is melted around the -10 element to reveal the template strand
2. The sigma factor repositions away to open the channel through which NTPs must enter the polymerase active site

Question 15

Describe the transcription initiation

Answer 15

After open complex formation reveals the template strand the RNAP holoenzyme starts transcribing from the +1 position

Question 16

How can extension of the RNA chain be halted

Answer 16

can be halted by a steric clash of the growing RNA chain with the s subunit, this sometimes causes transcription to abort after extension of the RNA chain to 8-9 nt, otherwise s is released and the RNAP core escapes the promoter

Question 17

What is promoter clearance

Answer 17

movement of the transcription complex away from the promoter, which marks the beginning of the elongation stage of transcription

Question 18

What is abortive initiation

Answer 18

release of an 8 to 10 base pair RNA transcript from the bacterial RNA polymerase initiation complex before it clears the promoter and enters the elongation stage.

Question 19

what is the sigma facotr

Answer 19

directs the enzyme to the promoter

Question 20

What is a consensus sequence

Answer 20

DNA or AA sequence consisting of residues that commonly occur

Question 21

what is the transcription elongation complex

Answer 21

composed only of the RNAP core without the sigma factor. Initiates the processive function of elongation until termination signal is encountered

Question 22

What is the transcription termination

Answer 22

signals for RNA extension to stop
2 types
1) rho-independent terminators
2)Rho-dependent terminators

Question 23

Rho-independent terminators

Answer 23

composed of 2 elements
a) an inverted repeat that can form a hairpin structure in the RNA transcript
b) a run of A’s in the DNA that promote pausing and RNAP release (U’s in transcript)

mRNA forms a hairpin followed by three U residues, stalling and separating the polymerase from the mRNA

Question 24

Rho-dependent terminators

Answer 24

1) A rut site where the Rho helicase loads
2) a string of A’s in the template strand shortly after the rut site. This slows down the RNAP core to allow Rho to catch up to the RNAP core complex.

Question 25

Describe the regulation of gene expression in bacteria

Answer 25

Bacteria need some proteins most of the time, like the proteins involved in DNA replication so they are expressed at most times. However, many other genes are only expressed when their products are needed by the cell

Question 26

When is it most efficient to regulate gene expression

Answer 26

Transcription initiation step or shortly after because gene expression is very expensive

Question 27

Describe the regulation of the gal operon (mechanism, repressors, operators)

Answer 27

repressed when galactose is absent. When present, GalR binds galactose and falls off the operator removing the repression
2 repressors: GalR and GalS
2 operators: Oe(external), Oi(internal)

GalR is bound to Oe and Oi that represses Gal

Question 28

What is catabolite repression? what is it mediated by

Answer 28

Bacterial cells prefer to use glucose as an energy and carbon source and repress expression of operons for utilizing other sugar sources until the glucose is used up

this process is mediated by cAMP and cAMP receptor protein (CRP)

Question 29

Describe the cAMP-CRP mechanism

Answer 29

Binds to operator sites near the promoters of sugar utilization operons and stimulate transcription of these neurons when glucose is absent
Acts like an accelerator

Question 30

what is a corepressor

Answer 30

a protein or small molecule that inhibits transcription by binding a repressor or repressors, without binding the DNA directly

Question 31

What is the trp operon in E. coli? Describe the regulation

Answer 31

produces enzymes needed to form the amino acid tryptophan

In the absence of tryptophan the Trp repressor cannot bind the operator and transcription is initiated, tryptophan serves as an effector molecule for the repressor to bind the operator

Question 32

What is transcriptional attenuation

Answer 32

a process for the regulation of expression of a bacterial operon in which transcription constitutively initiated but is halted in the leader region.
Termination occurs if products are not needed in the cell

Question 33

What is the leader sequence

Answer 33

a short sequence near the 5′ end of an RNA that has a specialized targeting or regulatory function

Question 34

what is a leader peptide

Answer 34

a short sequence near the amino terminus of a protein that has a specialized targeting or regulatory function

Question 35

What is the SOS response

Answer 35

a coordinated induction of a variety of genes in response to high levels of DNA damage
LexA interaction with RecA-ssDNA causes LexA to inactivate itself. Lack of LexA allows SOS to be expressed

Question 36

What is a riboswitch

Answer 36

Structure within an mRNA that binds to a specific ligand and undergo a conformational change to regulate gene expression affecting the translation or processing of the mRNA.

Question 37

Describe the expression of the B. subtilis trp operon

Answer 37

responsive to cellular tryptophan
uses a tryptophan sensing RNA-binding repressor

Question 38

What is the TRAP

Answer 38

Binds to mRNA if it is itself bound to tryptophan
Binding of TRAP to leader RNA sequesters the RNA at an essential point (antiterminator point)

Question 39

What is the glmS ribozyme

Answer 39

enzyme needed for the biosynthesis of a cell wall component in B. subtilis
the glmS riboswitch is a ribozyme that binds to glucosamine 6-phosphate
upon binding, the glmS cleaves itself and promotes its own degradation