Prokaryotic Transcription 2

Question 1

what are the two subunits that show specific DA recognition in binding?

Answer 1

• alpha subunits
• sigma subunit

Question 2

what are the two elements most promoters have?

Answer 2

-10 and -35

Question 3

what is the UP element?

Answer 3

• stimulates upward transcriptional activity
• upstream promoter element
• found in ribosomal structural genes
• alpha subunits makes contact with it and recognizes the sequence from the closed promoter
• does this by having two hairpin motifs with the two hairpins contacts both sides of the groove

Question 4

domain 1.2

Answer 4

• in the sigma element
• discriminator element
• some bacteria has additional info upstream of -10, so it recognizes this info

Question 5

domain 3

Answer 5

recognizes the extended TATA
- binds to the extended -10

Question 6

domain 3.2

Answer 6

in the RNA exit pores and 5’ end of transcription
- contacts first nt of transcript
- region 3.2 of the sigma subunit contributes to the binding of the 5’ initiating nucleotide in the RNA polymerase active center and facilitates promoter clearance during initiation

Question 7

domain 4.2

Answer 7

• uses the major groove of the sequence that is found at -35
• also called the -35 site
• helix turn helix
• activator contacts and alpha CTD

Question 8

sigma 70

Answer 8

most required functions

Question 9

sigma S

Answer 9

stationary phase/some stress responses
- cannot interact with alpha CTD, specificity involves UP motif

Question 10

Sigma 32

Answer 10

heat shock

Question 11

sigma E

Answer 11

periplasmic/extracellular proteins

Question 12

sigma 54

Answer 12

nitrogen assimilation
- It is unusual in that it can bind to the promoter in the absence of the Core. It has no inhibitory domain. This makes it more like a eukaryotic transcription factor.
- no 1.1 domain

Question 13

sigma F

Answer 13

flagellar synthesis/chemotaxis

Question 14

sigma fecl

Answer 14

iron metabolism/transport

Question 15

what happens when a mutation introduces more spacing in the binding domains?

Answer 15

• decrease binding and efficiency of promoter
• this is because the sigma is not very flexible and has set -10 and -35 domains

Question 16

domain 2.2

Answer 16

• makes contact with core beta prime unit
• connection to the core, recognizing tata and melting the promoter

Question 17

domain 2

Answer 17

• domain 2 is the most conserved domain

Question 18

domain 2.3

Answer 18

• melting: aromatic and basic aa
• aromatic acids trp433 and tyr430 make contact and promote melting

Question 19

domain 2.4

Answer 19

base specific interactions with -10
- recognizes tata
- domain 2.4 helix recognizes TA of TATAAT on non-template strand

Question 20

domain 2.1

Answer 20

most conserved-contact with core

Question 21

domain 1.1

Answer 21

initially binds in the active site in place of DNA

Question 22

what happens if you mutate away from the consensus?

Answer 22

• decreases the rate of closed promoter complex formation (promoter recognition)
• if you mutate at the -35, you decrease initial contact

Question 23

what happens if you mutate the -10 site?

Answer 23

• mutations away from the consensus may decrease the rate of open and closed promoter complex formation
• decreased promoter recognition and melting

Question 24

what is the role of rifampicin?

Answer 24

blocks bacterial RNA polymerase and is the major drug against tuberculosis. It binds close to the active site and blocks the formation of RNA beyond 2-3 nt. Blocks exit of nascent RNA. Mimics activity of sigma, but can not be dislodged.

Question 25

what is an additional role for domain 1.1?

Answer 25

- prevents sigma from binding promoter without first binding to the RNA polymerase core - when sigma is floating with no attachment, domain 1.1 folds back onto the body of sigma and covers domain 4.2 - sigma 54 lacks a 1.1 domain

Question 26

which subunit of RNA polymerase holoenzyme partially blocks the entry of DNA into the channel?

Answer 26

sigma region 1.1

Question 27

how does the holoenzyme transition from abortive cycling to elongation?

Answer 27

Sigma loses affinity for the promoter DNA and RNA polymerase, thus allowing the enzyme to move away from the promoter as elongation proceeds (70% sigma still loosely attached). Sigma then slowly disassociates from RNA polymerase. The growing RNA chain dislodges bound sigma (3.2) from the RNA exit pore.

Question 28

what are the seven differences between RNA polymerase and DNA polymerase?

Answer 28

1) RNAPs can initiate synthesis which involves promoter recognition 2) RNAPs can melt the DNA duplex 3) RNAPs initiation is primed by a single nucleotide, not an oligo as is the case for DNAPs 4) RNAPs make multiple contacts with the 2’-OH of the incoming NTP 5) DNA scrunching occurs for RNAPs allowing abortive cycling while still retaining contact with the promoter 6) For RNAPs, the transcript is peeled away from the template; not so for DNAPs where the open cleft allows the duplex to extend out of the enzyme 7) Initiation of synthesis is regulated by many proteins for RNAPs, but not for DNAPs

Question 29

what are the steps of footprinting?

Answer 29

Step 1: end-label the DNA at a specific site Step 2: incubate DNA with Protein (RNA polymerase in this example) Step 3: incubate DNA:Protein complex with dilute DNase I enzyme Step 4: remove protein and run polyacrylamide gel electrophoresis (PAGE)

Question 30

What are the two major changes in RNA polymerase as it transitions from promoter recognition phase to elongation phase of transcription?

Answer 30

- release of sigma - smaller footprint (changing of conformation)

Question 31

what is the process of RNA polymerase recovering from a stall?

Answer 31

Sometimes RNA polymerase pauses due to a temporary shortage of the complementary nucleotide. Thermal agitation, RNAP backtracks & 3’end out of active site. When this occurs, restarting synthesis requires the GreA and GreB proteins to release the pause. Acidic portion of the factors GreA & GreB repositions the Mg2+ ion at the active site. This makes RNAP act as a nuclease. RNP cleaves the 3’ end so that it is properly aligned within the catalytic site of the polymerase again. - then resumes elongation

Question 32

which eukaryotic factor performs that same function as GreA and GreB?

Answer 32

TF2S

Question 33

the most heavily transcribed genes in e. coli fire once per second

Answer 33

- true - ribosomal genes are the most actively transcribed of any in e. coli

Question 34

how much does the UP element increase the activity of the promoter?

Answer 34

60 fold

Question 35

core promoters

Answer 35

-10 and -35 elements

Question 36

-10 element

Answer 36

TATAAT

Question 37

-35 element

Answer 37

TTGACA

Question 38

upstream activating sequences

Answer 38

1, 2, and 3 - FIS sites that are positively acting - stabilizes UP - increases activity of the promoter 5 fold

Question 39

why is the distance between the -10 and -35 sites are so conserved?

Answer 39

the single protein sigma simultaneously makes contact

Question 40

what types of base changes would lead to increases or decreases in promoter activities?

Answer 40

- anything that makes it more like the consensus and anything that makes the distance more like the consensus - there are not many consensus promoters

Question 41

What is a promoter to an operon regulated by sigma 54?

Answer 41

- glutamine synthase in cyanobacteria - nif in e, coli

Question 42

dual promoters

Answer 42

: the vegetative promoter is read by sigma 70 and is weak. The nif-like promoter is much stronger and is read by sigma 54. This allows the gene to be active under normal conditions and when NH4 is in short supply.

Question 43

what are the limitations to sigma 54?

Answer 43

- it is defective and unable to melt the promoter without added ATP and the NtrC protein