Lecture 25 - Prokaryotic transcription

Question 1

Describe the difference between prokaryotic and eukaryotic transcription

Answer 1

Eukaryotic mRNA undergoes much more processing, transcription and translation are spatially separated. Prokaryotic transcription and translation can occur simultaneously

Question 2

How are prokaryotic genes regulated?

Answer 2

transcription initiation, RNA turnover (nuclease degradation), transcription termination

Question 3

Describe initiation of transcription

Answer 3

sigma factor binds to the promoter, RNA polymerase attaches to it with DNA closed, scans along promoter until it reaches the -10 Pribnow box where it opens up the DNA (open complex) and transcription starts at the +1 nucleotide. Sigma factor drops off after the polymerase clears the promoter

Question 4

Describe elongation of transcription

Answer 4

Doesn’t require the sigma factor, occurs slower than with DNA replication, RNA pol is highly processive, 3’OH acts as nucleophile to form phosphodiester bond, there is no editing/proof reading, so lots of mistakes are made, but since RNA doesn’t act as an info storage site, it doesn’t matter. Multiple RNA pols can attach to promoter one after another

Question 5

Describe the two different models of transcription termination

Answer 5

Rho independent: there is a GC rich sequence which causes the mRNA to form a stable hairpin turn which destabilizes the mRNA-DNA duplex. The GCs are followed by a strand of As which corresponds to a string of Us, these have only 2 h-bonds which are weaker to it further destabilizes complex, induces RNA to dissociate
Rho dependent: stop sequence in the template strand causes polymerase to stop. Rho protein binds at the rut in the mRNA, uses ATP to move 5’-3’ until it reaches the complex, where it forms a helicase-like interaction to dissociate the complex

Question 6

Describe gene regulation in prokaryotes

Answer 6

Initiation transcription regulated through use of repressors and activators. Some genes are constantly on (housekeeping genes), others are inducible (proteins increase under conditions) while others are repressible (protein concentration decreases under certain conditions). The repressors and activators bind to the operator site located at or in the promoter. Repressors perform negative regulation, where RNA pol transcription is blocked, while activators perform positive regulation, where RNA pol transcription is enhanced. These repressors and activators are then also controlled by effector molecules, which can increase or decrease their affinity for the operator (repressors: inducers or co-repressors).

Question 7

What is an operon?

Answer 7

Operons are groups of genes and a promoter for those genes and entire operons are transcribed into mRNA. However, during translation, each section of the mRNA can be expressed independently.

Question 8

Describe how the lac operon is regulated

Answer 8

Lac operon has lacZ (beta-galactosidase) lacY (glactosidase permease) and lacA (transacetylase). Converts lactose into galactose, glucose, and allolactose. Lac operon is usually repressed. Some small amounts of B-gal present, when lactose also present, some allolactose produced, which acts as an inducer and binds to the repressor (lacI) which causes it to dissociate from the operator. However, the lac promoter is weakly efficient, so needs an activator. When glucose levels are low, there is lots of cAMP which acts as an effector for CRP (activator) which binds and helps the RNA pol bind to the mRNA.

Question 9

How does the lac repressor bind?

Answer 9

helix-turn-helix motif: 2nd helix is recognition, lies in major groove, 1st helix interacts with the backbone, binds as a dimer with 2-fold symmetry