chem 114c mt 2

TFIIA

stabilized TBP and TAF binding

TFIIB

Stabilizes TBP binding, recruits RNAP, influences start site selection

TFIID and TBP

recognizes TATA box, recruits TFIIA and TFIIB, has positive and negative regulatory functions

TFIIE

an a2b2 heterotetramer, recruits TFIIH and stimulates its helicase activity, enhances promoter melting

TFIIF

facilitates promoter targeting, stimulates elongation

TFIIH

contains SsI2, an ATP dependent helicase that functions in promoter melting and clearance, and a CTD kinase

step 1 of the assembly of the pre initiation complex

the TBP component of TFIID binds to the data box of the promoter

step 2 of the assembly of the pre initiation complex

TFII and TFIIB bind

step 3 of the assembly of the pre initiation complex

TFIIF binds to RNAP II and escorts it to the complex

step 4 of the assembly of the pre initiation complex

TFIIE and TFIIH are sequentially recruited

what are the traits of tRNA

5’ terminal phosphate, 7 bp acceptor stem, D arm, 5 bp stem with anticodon loop, TpsiC arm, amino acid attached to 3’acceptor stem

Describe the two sieve mechanism

first only Ile and val bind at rossman then val also binds at editing/hydrolysis site

Step 1 in initiation in eukaryotes

initiation factors assemble with mRNA, elF-2a activated with gtp, and tRNAmet on small ribosomal subunit to form pre initiation complex

Step 2 in initiation in eukaryotes

other initiation factors bind the 5’ cap of the mRNA

Step 3 in initiation in eukaryotes

pre initiation complex binds mRNA

Step 4 in initiation in eukaryotes

small subunit scans mRNA for AUG start codon

Step 5 in initiation in eukaryotes

initiation tRNA binds start codon, elF2a hydrolyzes bound gtp

Step 6 in initiation in eukaryotes

phosphate, gdp, elF2a, and other initiation factors dissociate and recycle

Step 7 in initiation in eukaryotes

small subunit binds to larger subunit

Step 8 in initiation in eukaryotes

elongation begins

Initiation Factor-1

assists IF-3 binding

Initiation Factor-2

binds initiator tRNA and GTP

Initiation Factor-3

releases mRNA and tRNA from recycled 30s subunit and aids new mRNA binding

elongation factor -Tu

binds aminoacyl-tRNA and GTP

elongation factor -Ts

displaces GDP from EF-Tu

elongation factor -G

promotes translocation through GTP binding and hydrolysis

release factor - 3

binds to stalled ribosome and stimulates RF-1/RF-2 release via GTP hydrolysis

Ribosomal recycling factor

binds to a site followed by EF-G-GTP, induces ribosomal dissociation to small and large subunits

Decoding

aminoacyl tRNA binds in A site, if anticodon matches codon, EF-TU hydrolyzes GDP and dissociates

transpeptidation

peptide bond formation between peptidyl group in P site and aminoacyl tRNA in A site

translocation

A site tRNA is transferred tp p site p site trna is transferred to e

When does RRF enter

once there is a stop codon

Trigger factor/NAC

helps proteins fold as they emerge from the ribosome

GroEL/GroES complex

sequester unfolded proteins until they can fold

tRNA synthetase

aminoacyltates tRNA activation of the aa followed by transfer of the aminoacyl group to the tRNA

how doe the tRNA synthetase verify the tRNA

by reading the anticodon loop and acceptor stem

Ribosome composed of more rna than protein

use catalytic rna to do peptide transferase

What controls protein abundance

transcription

RNA polymerase

binds to temple/non coding strand and adds 5’ triphosphate to 3’OH

promoters

specific site where RNA synthesis is initiated

why have an operon controlled by lactose

when lactose is present lacl won’t bind to lac operator and stop RNA polymerase, need lactose metabolizing enzymes

what happens when lactose in not present

E. coli don’t need need lactose metabolizing gens so operon is kept OFF by lacl control of transcription

When do you add IPTG why

when E. coli are grown, causes e. coli to make t7 rna polymerase which binds to t7 promote and makes lots of RNA fro translation

where are all the genes we want to express in pET system located

downstream of t7 promoter

what happens when we add lactose

transcription occurs and we can express protein of interest

intrinsic termination

RNA with a self complementary GC rich segment forms a hairpin followed by a string of Us that base pair with the template As in transcription bubble which bocks the exit channel and stalls elongation

rho dependent termination

rho factor attaches to rna and moves 5’ to 3’ until it encounters RNAP at termination site and pushes it forward so that ir rewinds dsDNA helix and unwinds rna dna hybrid helix

RNA pol 1

in nucleoli where ribosomes are assembled

RNA pol ii

main producer of mRNA in nucleus can fix errors

RNA pol iii

in nucleus responsible for specific rnas

What role does the trigger loop play in RNA Polymerase II?

Its movement helps the nucleotide pivot from the E site into the A site

How does the bridge helix function in RNA Polymerase II?

Acts like a brownian ratchet =, pushes nucleotides along during transcription

How are the trigger loop and bridge helix coordinated in RNA Polymerase II?

are in physical contact and therefore have coordinated movements

How does RNA Polymerase II select nucleotides?

2 steps: nucleotide entry and pivoting from e site to a site, trigger loop and bridge helix movement that promotes correct nucleotide incorporation

How are pol ii promoter elements identified

by mutating and seeing if gene transcription levels decrease

tata binding protein

kinks and unwinds dna and recruits the rest of pre initiation complex

What happens on promoters that don’t have a TATA box

the conserved 7 nt Inr sequence is sufficient to recruit TFIID

How do eukaryotes put the 5’ end cap on mrnas

remove terminal P from 5’ end, add gtp, methylation of g

what do eukaryotes use to add the tail

polymerase a which adds poly a tail and protects the 3’ end of the mrna from nucleases

splicing step 2

U6-RNA coordinates a metal ion that helps the 3’OH attack

what does alternative splicing do

magnifies eukaryotic protein from a single gene

what helps determine splicing specificity

exonic and intronic splicing enhancers

PlacI

lac I promoter makes inhibitor bind to Olac and prevents RNA pol from moving along DNA

lac I

inhibitor or repressor protein

Plac

lac operon promoter

O lac

operatore sequence where I protein binds and prevents RNA pol from copying

lac z

codes for b galactosidase

lac y

codes for lactose permease

lac A

codes for thiogalactoside transactylase

In Eukaryotes, the _______, positions the kinase responsible for phosphorylating the RNA Pol II CTD and

the __________, which contains the kinase subunit

mediator, TFIIH

In Eukaryotes but not in Prokaryotes, there is a region between -38 and -32 that contains methylated C’

allows inheritance of gene expression patterns