Transcription I Flashcards
RNA polymerase, nature of the chemical rxn?
RNA polymerase is processive (starts and finishes job alone). Stable complex between RNA pol and DNA.
Unidirectional, only add at 3’ OH. It catalyzes phosphodiester bond formation b/w ribonucleosides.
Attack high energy phosphoanhydride bonds, hydrolyze 2 of them. Nucleophilic attach of OH group on gamma phosphate!! Need triphosphate nucleoside to provide enough energy. Break 1st then 2nd anhydride bond.
Very first base of any RNA chain at 5’ end is special. It maintains 3 P’s (5’ triphosphate).
Catalysis means rx is done one way.
5 steps common to bacterial and eukaryotic RNA polymerases?
INITIATION
1) RNA pol binds to promoter sequence on DNA. CLOSED COMPLEX.
2.) Polymerase melts DNA strands apart near trx start site and forms OPEN COMPLEX (trx bubble)
3.) Phosphodiester bond catalyzed by polymerase b/w 2 initial rNTP’s (RNA bases) (1st base is triphos)
ELONGATION
4.) RNA pol moves away from promoter 3’ to 5’, as it moves so does trx bubble. As it moves it melts the DNA, links rNTP (spit out growing nascent through channel)
TERMINATION
5.) Pol releases completed RNA, dissociates form DNA
What makes RNA pol II different?
It has a C-terminal domain. Makes mRNA and structural/noncoding (snRNA and miRNA)
What is promoter?
Sequence upstream of start site that positively affects the expression of the gene
TATA box
frequently conserved promoter. 30 bases upstream. Mutations in TATA box lead to decreased gene expression (beta thal and beta hemoglobin)
Tata binding protein helps ASSEMBLE PRE-INITIATION COMPLEX of general trx. factors at the promoter
Alpha-amanitin
Death cap mushroom.
Inhibits the movement of RNA pol II (binds its bridge substructure) so it can’t translocate down DNA chain.
Blocks transcription elongation!
Rifampicin
Also blocks trx. elongation.
Antibiotic (used to be for TB) binds beta subunit of bacterial RNA pol and plugs up the exit chamber of the growing RNA, thus blocking elongation.
4 components of the RNA pol II pre-initiation cmplx?
Transcription factor II (TFIIH and TFIID), general trx factors
TBP (TFIID) binds TATA box on DNA at the MINOR groove (less sequence specificity) and causes structural distortion, looks like saddle with stirrups.
Mediator: involved in DNA melting. helps direct assembly of the complex.
RNA Polymerase II
TFIIH: facilitates NER, also fx in DNA repair. XPB, XPD, and p44 subunits. XPB helicase opens up DNA at promoter to permit initiation.
CKD7 phosphorylates RNA pol II’s CTD domain during PROMOTER clearance.
TBP is a shared general trx factor used by RNA pol I, II, III
TFIIH subunit mutations
Xeroderma pigmentosum, cockaynes,..
pigmentation issues, UV damage repair, cancer susceptibility.
because TBP works in trx and DNA repair.
TATA element: imp inherited mutations in this, defects in HB chains. one base change in beta-chain of Hb.
