Prokaryotic Transcription (Lec 6 + 7)

Question 1

RNA Polymerase

+ Two components

Answer 1

• de novo synthesis 5’ -> 3’ direction
• no exonuclease activity

Holoenzyme (whole enzyme)

• five subunits
• two components: sigma factor and core enzyme

sigma factors
- initiation, exchangeable, shortens binding half-life

core enzyme

• elongation
• basic (positively charged)
• long binding half-life

Question 2

RNA Polymerase subunits + position and function

Answer 2

alpha (a): core enzyme
- promoter binding

B: core enzyme
- nucleotide binding

B’: Core enzyme
- template binding

sigma: Sigma factor
- initiation

Question 3

sigma factors give holoenzyme the ability to

Answer 3

recognize and bind specific DNA nucleotide sequences (promoters)

holoenzyme- binds very strongly to promoters

Question 4

Promoter + conserved areas

Answer 4

DNA nucleotide sequences where RNA Pol holoenzyme associate with DNA

SEQUENCES DIFFER FROM ONE ANOTHER

except two short conserved areas

1. -10 DNA n. sequence [Pribnow Box]
   - 5’ TATAAT3’
2. -35 DNA n. sequence
   - 5’TTGACA3’

Question 5

Function of the two conserved DNA nucleotide sequences + UP element

Answer 5

• 35
• necessary signal for RNA Pol Holoenzyme to bind to promoter - form a binary closed complex
• 10 [Pribnow box]
• consists mainly of As and Ts
• allow complex to change to an open complex
• DNA melts to form single strand

UP elements

• additional Upstream Promoter (UP) element
• binds C-terminal domain (a-CTD) of RNA Pol
• allows tight biniding between promoter and RNA pol
• facilitate high transcription levels - eg highly expressed rRNA gene (rrn loci) that encode for ribosomal RNAs

Question 6

Models how RNA Pol finds promoters

Answer 6

1. Random diffusion to target DNA
2. Random displacement between DNA helix (strands)
3. Glide along DNA helix

Question 7

How many RNA Pol molecules are freely available to associate with the sigma factor and bind to promoter

Answer 7

almost no free RNA pol

• ~50% of core enzyme busy with elongation
• ~30% holoenzyme are in loose binding or binary closed complexes
• ~20% of core enzymes are in loose binding

Question 8

Transcription Process - write it (Lec 7)

Answer 8

Look at notes

Question 9

Attenuation

Answer 9

termination before end of transcription units is reached

Question 10

E. coli two types of termination at the end of transcription units

Answer 10

1. Rho independent Termination (Simple termination)
   core enzyme terminates in absence of any other factors

properties:

• requires reverse nucleotide sequences
• forms hairpinloop
• string Us 3’ to hairpinloop
• mechanism

2. Rho dependent termination
   - requires rho-factor
   - mechanism

Question 11

Rho dependent terminators

Answer 11

rho factor anchors as hexamer to cytosine-rich area (rut site) on 5’ end of mRNA

• glides on mRNA towards RNA pol
• anchor and gliding motion is associated with hydrolysis of ATP

when RNA Pol reaches rho dependent terminator it moves slower

rho factor factor catches up to RNA Pol
- termination is brought about

rho factor has ATPase activity

• causes unwinding and dissociation of RNA:DNA hybrid in absence of RNA Pol
• ATP IS USED TO DRIVE RXN