Lac Operon

Question 1

What is LacZ responsible for?

Answer 1

B-galactosidase synthesis (z +/-)
z^1 is inactive enzyme so no activity

Question 2

What is LacY responsible for?

Answer 2

Permease (y +/-)

Question 3

What is LacA responsible for?

Answer 3

Acetylase

Question 4

What are constitutive mutations and how are they represented on the Lac Operon?

Answer 4

Get transcription w/ or w/o inducer (o^c)
Active in cis position

Question 5

What is the role of the repress, where does it bind, and how is it represented in the Lac Operon

Answer 5

Acts at the operator (i +/-)
Active in trans position
i+ is inducible, i- is constitutive
i+ is dominant

Question 6

What are the 4 pieces of evidence for RNA?

Answer 6

1. Fast enzyme synthesis when gene is expressed
2. Constant and max rate of expression (rapidly forms and doesn’t accumulate)
3. Short 1/2 life of expression
4. Ribonucleotide analogs inhibit enzyme production

Question 7

Describe the lac operon and how it is regulated

Answer 7

LacI binds operator and acts as repressor

Lactose (or IPTG) binds LacI and removes from operator to allow basal transcription

cAMP binds CAP protein which then binds upstream of RNAP binding site to enhance transcription

Question 8

Subunits and functions of RNAP

Answer 8

6 subunits
B & B’: polymerize
2 alpha: determine frequency of initiation
w: stabilizes enzyme
Sigma: recognizes promoter then dissociates during elongation

Reads 3’-5’ to synthesize 5’-3’

Question 9

5 steps of transcription by RNAP

Answer 9

1. sigma and core recognize promoter and bind
   (~35 & 10 bp upstream of initiator)
2. Core opens DNA
3. Transcribe short “testing fragments” and sigma begins to dissociate
4. sigma fully dissociates –> elongation while core pries RNA and DNA apart
5. Termination by AU rich hairpin loop

Question 10

What is Rho-independent termination?

Answer 10

Hairpin loop secondary structure disrupts elongation complex at ends of operons

Question 11

What is Rho-dependent termination?

Answer 11

Rho binds mRNA = helicase activity at RNAP active site (binds at C rich mRNA)

Results in termination by disrupting elongation complex

Question 12

What are the 4 types of transcription control in prokaryotes?

Answer 12

1. Alternative sigma factors
2. Promoter-proximal elements
3. Catabolite repression
4. Attenuation

Question 13

Describe alternative sigma factor transcription control

Answer 13

Various sigma factors that bind various promoters (Hsp, motility, stress response, etc)

Question 14

Describe promoter-proximal elements in terms of transcription control

Answer 14

Interact w/ transcription factors to modulate transcription at promoter site

Question 15

Describe catabolite repression in terms of transcription control (example of lac operon)

Answer 15

Metabolites can influence transcription
Ex: Lac Operon
Lactose binds repressor (LacI) which allows expression
cAMP binds CAP which enhances expression

Question 16

Describe attenuation in terms of transcription control

Answer 16

Site to prematurely stop transcription

Ex: Trp binding
Low Trp = transcription (pause)
hairpin forms close to ribosome rather than RNAP
High Trp = no transcription (no pause)
hairpin forms close to RNAP, get short mRNA