lecture 5

Question 1

what forms RNApol?

Answer 1

RNAP and sigma factor

Question 2

what happens during transcription?

Answer 2

• the RNA holoenzyme forms (RNAP and sigma factor)
• recognizes the promoter in a closed complex
• covers approximately ~from -55bp to +15 bp
• transcription bubble
• open complex
• RNA synthesis
• elongation (sigma factors not required)

Question 3

what is the structure of bacterial RNAP?

Answer 3

• highly conserved structure
• composed of 5 subunits: alpha, alpha, beta, beta’, omega (helps B’)
• RNAP is capable of random transcription (no promoter)
• a sigma factor is necessary for recognizing a promoter
• lineage specific inserts- specific partners

Question 4

what is the role of RNAP inhibitos?

Answer 4

RNAP inhibitors can block transcription at different stages

Question 5

what are the features of sigma factors?

Answer 5

lead RNAP to the transcription start site (TSS)
-initiate strand separation of dsDNA for the transcription bubble
- incapable to recognize DNA alone, only with RNAP
- evolutionary related

Question 6

what is sigma 70?

Answer 6

housekeeping factor- responsible for recognizing most promoters

• each specific domain recognizes specific promoter elements

Question 7

what is sigma2?

Answer 7

sigma two is a domain of sigma70
- can remain attached to the elongating RNAP; continues to recognize -10 and -35 like sequences

• this is transcriptional pausing

Question 8

what are sigma ECF?

Answer 8

Extra Cytoplasmic Function sigma factors

Question 9

what are the features of Extra Cytoplasmic Function sigma factors?

Answer 9

• homology with sigma70
• minimal size sigma (only two domain- 2&4)
• a bacteria has on average six sigmaECF
• recognized by anti-sigma factors

*released in response to stress

Question 10

what is the function of anti-sigma factors?

Answer 10

sigma factors sequestered by anti-sigma factors and released when needed

they arrest the sigma factors in a conformation so they are unable to associate with RNAP

Question 11

what are anti-anti-sigma factors?

Answer 11

sequester anti-sigma factors

Question 12

what are the features of sigma factor 54?

Answer 12

• has 3 domains
• recognizes -24 and -12 promoter sites
• uncapable to allow the formation of the open complex and transcription bubble
• sigma54 requires an ATP-dependent activator
• activators ATPase is regulated by stress-related signals
• activator binds upstream of the promoter- DNA looping is required

Question 13

what is the structure of sigma factor 54?

Answer 13

Region I- inhibits polymerase isomerization and initiation
Region II- implicated in DNA melting
Region III- the primary DNA-binding region and recognizes the -12 and - 24 elements

Question 14

what is the relation between sigma70 and sigma 54?

Answer 14

two structurally and evolutionari;ly distinct families of sigma factors

Question 15

what is PRE binding?

Answer 15

the cII protein binding site overlaps with the -35 elements

Question 16

what is the function of the PRE promoter?

Answer 16

does not have the autonomous capability of binding RNAP holoenzyme

Question 17

what are the features of bacterial 6S RNA?

Answer 17

non-coding RNA that binds sigma70 holoenzymes (not other sigma)
- promotes the increase activity of other sigma factors
- 6S RNA has a central bubble- mimics promoter structure

Question 18

what is sequestering RNAP?

Answer 18

transcriptional reprogramming during the transitions between exponential and stationary growth phases

Question 19

what are the environmental cues that can effect promoter activity?

Answer 19

ligands, proteins, covalent modification
Abundance, availability

Question 20

what is the role of transcription factors?

Answer 20

• control promoter activity
  Most TF regulate many proteins
  Most promoters are regulated by more than one transcription factor

Transcription factors are expressed from promoters regulated by other transcription factors

The compaction of the DNA impede the access to promoters (further control of transcription)

Question 21

what are class I activators?

Answer 21

• activators bind to an upstream operator that is located upstream -> recruits RNAP
• RNAP recruited by protein-protein interaction

Question 22

what are class II activators?

Answer 22

• activator binds to operator at the -35 element (interaction with domain 4 of sigma factor)
  Conformation change
• operator between the -35 and -10 elements
• activator realigns the DNA to bind RNAP

Question 23

what is steric hindrance?

Answer 23

• block RNAP access to promoter
• promoter might have several operators: this increases the stength of repression

Question 24

what are the mechanisms of transcriptional repression?

Answer 24

Steric hindrance
Looping
Anti-activators

Question 25

what is repression through looping?

Answer 25

operators binding outside of promoter

Question 26

what is repression through anti-activators?

Answer 26

prevent the recruitment of RNAP

Question 27

what are the features of the Lac Repressor?

Answer 27

• part of the first regulatory network discovered
• binds tightly to a specific region before three lactose-metabolizing proteins
• when bound to lactose and similar sugars, it changes shape and no longer can bind to the DNA

Question 28

what is the structure of the Lac Repressor?

Answer 28

Lac repressor has 3 domains:
- the C-terminal tetramerization domain
- the core domain that binds to LacI sugar ligands in a cleft between two subdomains
- the helix-turn-helix motif and hinge- bind operator DNA

*functional as a dimer or dimers