8_15_16 Hampsey I

Question 1

Template-product relationship

Answer 1

5’ to 3’ DNA coding/”non-template strand” CGCT
3’ to 5’ DNA template strand GCGA
5’ to 3’ RNA product CGCU

Conserve directionality bases except T to U

Question 2

Prokaryotic transcription

Answer 2

RNAP holoenzyme created when catalytic core enzyme (alpha2betabeta’omega) joins with a sigma subunit which binds to various promoters

Three stages: Initiation (regulation), elongation, termination RNAP needs Mg!!!

Initiation: sigma binds two consensus sequences on same side of helix before initiation site, the -35 sequence and the Pribnow box

Elongation: RNAP recruits nucleic acids and makes an RNA-DNA hybrid

Termination: rho-dependent, hexameric atp-ase winds until it reaches a termination sequence on new RNA OR rho-independent a loop and poly-U tail is transcribed signaling RNAP to leave

bacterial RNAP inhibited by Rifampicin

Question 3

Eukaryotic transcription

Answer 3

Needs Mg and Zn

Here we have different polymerases instead of different sigma subunits

Pol I for rRNA, Pol II for mRNA, and Pol III for tRNA and 5S rRNA

Regulation: Core promoter (-40, 40 (TATA Box)), proximal promoters (binds regulators for housekeeping proteins) and far away enhancers and repressors

Core promoters TATA box binds TATA-Box-Binding-Protein part of the TFIID Complex (TBP and 14 TAFs(TBP associated factors) TAFs 1 and 2 bind initiator seq. and 6/9 bind downstream promoter

-35 sequence binds the BRE (TFIIBRecognitionElement)

Pre-Initiation Complex: TFIID to TFIIB to TFIIA to TFIIF with RNAPII to TFIIE to TFIIH

First base is not coded but rather a 5’ to 5’ phospodiester bond caps the 5’ end with 7-methylguanosine

Poly A tail added to the 3’ end (non-exonic)

RNAPol-II inhibited by alpha-amantin in ‘shrooms

Question 4

Post-transcriptional processing

Answer 4

Splicing: recognize a 5’ GU and a 3’ AG
(think GULAG, with the L for lariat in the middle)

5’ splice site P-group attacks a 2’ -OH at the branch site A forming a lariat intermediate with 2’ to 5’ link (red flag)

newly freed 3’ end of Exon 1 (freed by first attack) attacks a P on the 5’ end of the 2nd exon forming the spliced mRNA and leaving lariat intron

Accomplished using spliceosome activity of proteins: SNURPs, U1, U2 etc. (malfunction causes diseases (beta thalassemia))

Alternative splicing generates protein diversity

Question 5

Regulation of gene expression

Answer 5

regulators include steroid hormones (carbohydrate based)

Agonists- bind receptors, stimulate expression e.g. anabolic steroids

antagonists- bind receptors to repress expression
e.g. tamoxifen binds estrogen receptor as a competitive inhibitor

steroid receptors bind DNA with a DNA binding domain (Zn) and a ligand (estrogen) binding pocket and an activation domain which binds a palindromic enhancer, which confers specificity

when the enhancers bind the conformation of the nucleosome changes loosening the histones allowing recruitment of coactivators to further remodel allowing RNAP-II to show up