M2 L4

Question 1

Define what a core promotor is

Answer 1

Where RNAPII is recruited and overlaps TSS

Question 2

2 experiments that suggested events at the core promotor regulated transcription

Answer 2

RNA-seq
combination of RNA-seq and ChIP-seq experiments

Question 3

Outline RNA seq experiment

Answer 3

1. RNA is extracted and converted to cDNA using random hexamer of primers so that all RNA is converted to cDNA not just the mRNA
   - first depleted the cells of rRNA tho
2. cDNA is then sequenced using high-throughput sequencing – produces lots of short reads
3. short reads are mapped back to the genome -> amount of reads correlates to the level of gene expression

Question 4

What did RNA-seq experiment show

Answer 4

transcription was pervasive, happened even in regions without a core promotor => suggests core promotor isn’t necessary for transcription to happen

Question 5

What did further RNA-seq and chIP seq experiments show

Answer 5

there were 3 diff types of promotors each transcribed a particular type of gene -> there would be no need to have different types if it were machinery

Question 6

Name the 3 types of core promotors

Answer 6

1. Core promotor that transcribed genes involved in differentiated cells
2. Core promotor that transcribed housekeeping genes
3. Core promotor that transcribed developmental transcription factors

Question 7

describe the Core promotor that transcribed developmental transcription factors

Answer 7

• Broad TSS (but a bit smaller)
• Have specific motifs it binds to in the core promotor region, some are the same as the core promotor for housekeeping genes and some are different
• Histones each side tend to have trxG marks but can also have PcG marks on them at the same time i.e. have bivalent marks/are poised

Question 8

Describe the core promotor that transcribed genes involved in differentiated cells

Answer 8

• Tight/focused TSS (~1-2 bp)
• Nucleosomes can vary around it
• Has specific motifs in the core promotor region
• Tends to be marked by TrxG proteins

Question 9

Describe the core promotor that transcribed housekeeping genes

Answer 9

• Broad TSS (~100 kb) where transcription can start
• Has set nucleosome positions around it, which tend to have trxG histone marks
• Have specific motifs in the core promotor region

Question 10

Role of core promotor and PIC

Answer 10

The role of the core promotor is to recruit and help RNAPII recognise the TSS. it does this by recruiting the PIC complex. PIC is composed of RNAPII and GTFs which help stabilise RNAPII

Question 11

Example GTF in PIC complex

Answer 11

TFIID.
helicase activity - opens up DNA and feeds template into the active site
kinase activity - stabilises RNAPII and P ser5 on CTD which recruits capping factors that add 5’ cap