Eukaryotic promoters, RNA polymerases + the assembly of PIC

Question 1

TATA box

Answer 1

• In highly transcribed genes, a conserved sequence called the TATA box is found 26-30 bps upstream of transcription start site
• A single base change in TATA sequence drastically reduces transcription

Question 2

Initiator sequence (Inr)

Answer 2

• Instead of TATA box, some eukaryotic genes contain alternative promoter (Inr)
• Most initiators have a C at -1 position and an A residue at the transcription start site (+1)

Question 3

CpG Islands

Answer 3

• In mammals #60-70% of protein coding genes lack obvious TATA or Inr
• Txn initiation occurs at lower rate + at several alternative sites within regions of 100-1000 bps that have unusually high frequencies of GC sequences (CpG islands). These genes code for proteins that aren’t required in large quantities
• In mammals, Cs followed by Gs that are not associated with CpG Islands are methylated at position 5 of the pyrimidine ring, but C residues in CpG islands escape methylation (hypomethylation). Methylation of CpG islands is associated with silencing genes.
• CG rich sequences are bound by histone octamers more weakly because more energy is needed to wrap them into small diameter loops, so CpG islands coincide with nucleosome free regions

Question 4

UAS/Enhancer

Answer 4

• activator binding sites
• proximal <1Kb
• distal >1Kb

Question 5

URS/Silencer

Answer 5

repressor binding site

Question 6

Sequence Comparison

Answer 6

Consesus sequence was determined by looking at frequency of bases at a certain location in the promoter region

Question 7

Reporter Analysis

Answer 7

• Reporter genes encode proteins whose levels can easily be measured (e.g. GFP, luciferase, LacZ)
• Amount of reporter protein provides a measure of gene expression
• Replace promoter sequence with the sequence you’re interested in, and see how expression of gene is affected.
• Reporters can also be used to identify: when a gene is expressed, where it is expressed, what signals it responds to and what factors and sequences control it’s expression

Question 8

Identification of promoter elements using 5’ -deletion series of reporter constructs

Answer 8

• Using recombinant DNA techniques, a 5’ prime deletion series is made from the suspected promoter sequence
• each set of sequences from the deletion series is ligated into a plasmid containing a reporter gene
• Each type of plasmid is separately transfected into cultured cells
• the amount of reporter protein is measured and compared.
• The amount of protein transcribed helps to determine which sequences are most critical for txn initiation

Question 9

RNA pol I

a) target genes
b) location

Answer 9

a) rRNA (28S, 18S, 5.8S)

b) Nucleolus

Question 10

RNA pol II

a) target genes
b) location

Answer 10

a) mRNA (encodes protein), snRNAa (RNA splicing), miRNAs (Translational control), siRNA (chromatin-mediated repression, translational control)
b) Nucleus

Question 11

RNA pol III

a) target genes
b) location

Answer 11

a) tRNA (protein synthesis), 5S RNA (ribosomes), U6 snRNA (splicing), 7S RNA (signal recognition particle for insertion of polypeptide into endoplasmic reticulum)
b) Nucleus

Question 12

How many subuntis does RNA pol II have?

Answer 12

12

Question 13

Gene Transcription Factor characteristics

Answer 13

• RNA pol specific (pol II)
• Multi component factors
• Form a complex on TATA box
• Recruit RNA pol II to promoter
• Direct initiation at start site

Question 14

PIC assembly

Answer 14

1) TATA binding protein (TBP- subunit of TDII) binds to the TATA box promoter. TBP folds in a saddle shape and interacts with the minor groove of DNA, bending the helix
2) Once TBP is bound TFIIA and TFIIB can bind, making contact with both TBP and the DNA. )TFIIB assists RNA pol II in melting the DNA strand at the start site, and interacts with the template strand near RNA pol II active site).
3) A preformed complex of TFIIF and Pol II binds, positioning the polymerase over the start site
4) TFIIE and TFIIH then bind before transcription can begin. TFIIE provides a docking site for TFIIH, and the helicase activity of TFIIH separates the template strand at the start site (open-complex) - requires ATP hydrolysis
5) As Pol II begins transcribing (Mg2+ bound to active site to assist catalysis of phosphodiester bond synthesis) TFIIH phosphorylates Pol II on the C terminal domain (CTD) so that transcribed RNA can be capped
6) TFIID and TFIIA stay behind
7) TFIIB, TFIIE and TFIIH are released
8) TFIIF moves down the template with Pol II

Question 15

TFIID

a) subunits
b) property

Answer 15

a) 13
b) - binds to TATA box
- recruits TFIIB
- Trilobular structure
- TFIID = TBP + TBP Associated Factors (TAFs)
- TBP can direct assembly of PIC on a TATA-containing promoter (in vitro) without any TAFs, but alone, it can’t direct PIC assemblt on a TATA-less promoter, and can’t support “activated” transcription
- TAFs promote interaction of TFIID with basal promoters (other than TATA, e.g. Inr), and interact with other activators

Question 16

TFIIA

a) subunits
b) properties

Answer 16

a) 3
b) - Stabilises TFIID binding
- Anti-repression function

Question 17

TFIIB

a) subunits
b) properties

Answer 17

a) 1
b) - recruits RNA pol II-TFIIF
- important for start site selection

Question 18

TFIIF

a) subunits
b) property

Answer 18

a) 2
b) - interacts with TFIIB
- destabilises non-specific RNA pol II DNA interaction

Question 19

TFIIE

a) subunits
b) Properties

Answer 19

a) 2

b) recruits TFIIH and modulates TFIIH activity

Question 20

TFIIH

a) subunits
b) properties

Answer 20

a) 9
b) Promoter melting and clearance
CTD kinase activity
DNA repair
- Can be divided into CORE + CAK
- CAK module contains a kinase for CTD, and can dissociate away from TFIIH + has other roles in cell cycle regulation
- TFIIH contains 2 helicases (XPD and XPB)
- XPB play key role in promoter melting