Transcription and Trancriptional Regulation (Lac Operon)

Question 1

In what direction does RNA polymerase read and in what direction does transcription occur?

Answer 1

Reads 3’ to 5’ BUT Transcribes 5’ to 3’

Question 2

5’ -1-2-4-6-8-9PROMOTER125689 3’
In this text, where is upstream and where is downstream.

Answer 2

Upstream is towards the 5’ end AND downstream of the promoter is towards the 3’ end

Question 3

What are trans-acting element vs cis-acting element and mention 2 key examples of them in transcription along with their function.

Answer 3

Trans actin factors: bind to Cis to transcribe genes, like Activators or Repressors, transcription gactors

Cis actin elements: promoter, operator, enhacer

WRONG

Question 4

Is the promoter considered upstream or downstream of the transcription start site? WHY

Answer 4

Transcription starts after RNA pol binds to the promoter so the promoter is upstream

Question 5

What is the basic mechanism of transcription in prokaryotes

Answer 5

RNA pol binds to promoter in dsDNA»pol separates the dsDNA forming a transcription bubble»pol adds new rna nucleotides to the 3’ end»pol continues to move and elongate»termination signal»pol dissociates»dna ressociates

Question 6

Why is one side of DNA labelled in DNA finger printing?

Answer 6

It simplifies the analysis by labelling only one side of the DNA fragment (which lies within the protein binding site) we can can see exactly where the protein binds and determine its exact binding site

Question 7

What can I use to confirm the initial results of an EMSA

Answer 7

A supershift can be used to bind antibody to the bound protein of interest to confirm its presence

Question 8

I want to confirm that a protein does bind to DNA, what techniques could I use? And how are they done

Answer 8

DNA footprinting: label one part of DNA fragment»incubate it with protein»the digest with DNAse»if protein binds (DNA-protein complex will be indigested). Then we know protein bound and the exact binding site.

EMSA: we label DNA»incubate with protein&raquo_space;run on gel»if protein bind the band will be larger and slower on gel. Confirm result using supershift (check antibody can bind to protein as a control to confirm the protein bound)

Question 9

If you were going to use ChIP for checking if a protein bound to DNA, how would you do it?

Answer 9

ChIP-chromatin immunoprecipitation assay
Hint: chromatin = DNA plus protein

Cross link dna+protein~>lyse cells to release cheomatin~>break into smaller fragments ~>do immunoprecipitation by adding antibody specific to protein~> reverse crosslink to separate protein from DNA~>do sequencing or microarray to see results

Question 10

What is the basic gene arrangement of the lac operon and the function of the genes

Answer 10

I-repressor that binds to O to inhibit rna pol
P-promoter where rna pol binds to begin transcription
O-the operator where I binds
Z-bgalactosidase that cleaves lactose to glucose and galactose
Y-permeate that transports lactose
A-transacetylase

Question 11

What happens to cAMP when glucose is low in bacteria cell but there is lactose available?

Answer 11

cAMP level increases in low glucose condition as it enhances the binding of rna pol to the lac promoter which then leads to transcription of the lac genes