BCEM 393 - Gene expression, RNA processing, genetic code, tRNA charging and protein synthesis

requirements for transcription

template DNA, NTPs (ATPs, CTPs, GTPs, UTPs), RNA polymerase, Mg2+

RNA polymerase holoenzyme

a₁a₂BB’ow subunits

RNA polymerase core enzyme

a₁a₂BB’w subunits, active for transcription

no primer required for transcription

RNA pol a1a2

assembly of core enzyme, interacts with regulatory factors

RNA pol BB’

catalysis, interactions with DNA and RNA

RNA pol w

required for structure/folding

RNA pol o

decreases affinity of RNA pol for DNA and recognizes and binds the promoter sequence

falls off the core enzyme after several nucleotides of RNA are synthesized, and can bind to another core enzyme

promoter sequence

includes consensus sequence (-35) and pribnow box (-10) - both are upstream (towards the 5’ end) from the start site

elongation of transcription

begins after the formation of the first phosphodiester linkage, reaction is similar to DNA replication

3’OH of the terminal ribose of the growing chain attacks innermost/alpha phosphoryl group of the incoming ribonucleoside triphosphate

synthesis begins de novo (no primer needed)

termination of transcription

hairpin in RNA product causes RNA pol to pause, poly U region follows hairpin

rU-dA base pairs are wealk, so RNA dissociates from the DNA template and enzyme

protein dependent termination - Rho protein

rho protein

for terminating transcription, a helicase that pulls RNA through

transcription in prokaryotes

• transcription and translation can occur at the same time

• simple control elements

• termination signal is GC rich hairpin-poly(U) structure

• mRNA is not transported across a membrane

transcription in eukaryotes

• transcription and translation are separated in time and space

• more complex control elements

• termination signal is poly(A) tail

• mRNA is highly processed and transported across the nuclear envelope

eukaryotic promoter elements

for RNA polymerase II

elements define start site and involved in polymerase recruitment

TATA box (-25), Inr (+1), DPE (+30), enhancer (> 1 kb from start site), CAAT box and GC box (-40 to -150)

preinitiation complex

transcription factors bind cis-acting elements to recruit RNA pol II

TATA box recognized by TATA binding protein (TBP)

TFIID is a dynamic protein complex

TFIID

in eukaryotic transcription

binds DNA and the TBP domain moves between multiple conformation until it binds the TATA box

TFIIH

in eukaryotic transcription

helicase that unwinds DNA and phosphorylates the C-terminal domain of pol II (kinase activity)

this triggers transition to elongation and recruitment of RNA processing enzymes

combinatorial control

many TFs are needed to form a complex that stimulates or suppresses transcription

additional TFs that bind to other sites stimulate high levels of transcription

5’ cap

a 7-methylguanosine cap

begins very early during transcription (after 20-30 nt synthesized)

cap synthesizing complex is composed of 4 enzymes, associate with phosphorylated RNA pol II

protects mRNA from degradation by nucleases, interacts with ribosome to enhance translation

5’ cap processing

1) removal of the terminal (gamma) phosphate at the 5’ end by a phosphohydrolase

2) diphosphate 5’ end attacks to a-phosphate of a GTP to form a 5’ to 5’ triphosphate linkage

3) N7 of guanine is methylated

4) methylation of a 2’OH group of adjacent riboses

poly A tail

~250 adenylates at the 3’ end of the RNA

enzymes involved in building the poly A tail are associated with the phosphorylated C-terminal domain of RNA pol II

enhances stability and translation efficiency, also involved in nuclear export

poly A tail processing

CPSF (cleavage and polyadenylation specificity factor) recognizes the cleavage signal and an endonuclease cleaves the mRNA transcript

poly A polymerase adds ~250 adenylates to the 3’ end

splicing

removes introns (non-coding regions), and joins exons (coding regions) together

intron begins with GU and ends with AG so knows where to splice

spliceosome

complex of protein and RNA that excises introns and joins exons

catalyzes two transesterificaton reactions

1) branch site adenylate 2’ OH attacks the phosphoryl at 5’ splice site - exon 1 released, lariat intermediate formed

2) 3’ OH of exon 1 attacks phosphoryl group at the 3’ OH splice site - spliced produce exon 1-2 joined, lariat intron

transesterification reaction

reaction of an alcohol and ester to make a different alcohol and ester

spliceosome assembly

five critical small nuclear RNAs (snRNAs) - U1, U2, U4, U5, U6

U1

binds the 5’ splice site

U2

binds the branch site

U4-U5-U6 complex

assembles and displaces U1 and U4 - not involved in actual splicing

extensive interaction betweeen U2 and U6 - brings the 5’ splice site and branch site close together

U2-U5-U6

the catalytic core of the spliceosome, catalyzes the first transesterification

U5 - facilitates the second transesterification, Mg2+ required

forms spliced RNA product and lariat intron

genetic code

links nucleic acid and protein information

3 nucleotides encode an amino acid, read from 5’ to 3’ end of mRNA

degenerate - some codons are synonymous

start = AUG, stop = UAA, UAG, UGA

tRNA

adaptor molecules, 73-93 ribonucleotides, L shaped 3D structure

phosphorylated 5’ end

3’ CCA terminus - where amino acid will be attached

anticodon loop - base pairs with mRNA codon

inosine

is formed by deamination of adenine

tRNA contains modified bases like this

wobble base pairing

some tRNAs recognize more than one codon, eg. yeast alanyl-tRNA - anticodon IGC (I is inosine) which binds to all of GCC, GCU, GCA

when inosine is present at the 5’ end of the anticodon, the anticodon can recognize 3 bases

when U or G at the 5’ end of the anticodon, the anticodon can bind 2 codons

tRNA charging

creates an activated intermediate called amino acyl tRNA - more thermodynamically favourable

all catalyzed by aminoacyl tRNA synthetase - a ligase

1) activation by adenylation, releases PPi —> 2 Pi

2) transfer to tRNA, ester linkage to 3’ OH group of 3’ adenosine of the tRNA CCA arm

editing site

proofreading ability on aminoacyl tRNA synthetases

eg. threonine - cannot fit into threonyl-tRNA synthetase’s editing site because it is too bulky, but serine can to be proofread and removed

ribosome

50s subunit - has 34 proteins, 23S rRNA, 5S rRNA

30s subunit - has 21 proteins, and 16S rRNA

rRNA plays a catalytic role

3 binding sites - A (aminoacyl site), P (peptidyl site), E (exit site)

protein synthesis initiation

shine dalgarno sequence pairs with 16s rRNA

initiator codon on mRNA pairs with an anticodon on an initiator tRNA

bacterial protein synthesis begins with N-formylmethionine (fMet) - methionine is attached to an initiator tRNA (tRNA_f) and subsequently formylated —> fMet-tRNA_f

IF2

protein bound to GTP (a GTPase)

binds fMet-tRNA_f and mRNA - all bind the 30s subunit

30s initiation complex

complex of IF1, IF2(GTP), IF3 and fMet-tRNA_f and mRNA

subsequent binding of the 50s subunit results in hydrolysis of GTP by IF2 and release of IF2

EF-Tu(GTP)

binds to an aminoacyl tRNA and delivers the charged tRNA to the A site —> if correct bsae pairing between codon and anticodon, the EF-Tu(GTP) hydrolyzes GTP

protein synthesis elongation

peptidyl tRNA occupies the P site, aminoacyl tRNA occupies the A site - delivered by EF-Tu

peptide bond formation, peptide is attached to tRNA with its anticodon base paired to the codon at the A site

peptide grows in the N terminus to the C terminus

translocation

catalyzed by elongation factor G (a GTPase), moves the mRNA and tRNAs - shift by 1 codon

elongation factor G

GTPase that binds to the ribosome after peptide bond formation

hydrolysis of GTP pushes the mRNA and tRNAs through the ribosome by one codon

reaction happens 20 x per second so 20 peptides per sec

termination

RF1 or RF2 proteins recognize stop codons, bind at the A site and simulate the hydrolysis of the final ester linkage between peptide and the last tRNA resulting in release of the peptide

protein folds and may be post-translationally modified

disassembly of ribosome

RRF (ribosome recycling factor) and RF3(GTP) hydrolyze GTP and cause release of RF1 or RF2

posttranslational protein modifications

most commonly modified - hydroxy, amino, thiol side chains

influences chemical properties, results in changes of net charge, conformation, localization, binding and/or function

most are reversible and catalyzed by a specific enzyme

glycosylation

catalyzed by glycosyltransferases

can be N-linked or O-linked

transfer a carbohydrate from a nucleotide donor to a side chain of a residue in a protein

phosphorylation

catalyzed by protein kinases and reversed by protein phosphatases

eg. regulation of glycolysis

lipidation

attachment of a lipid to a residue in a protein - localizes proteins to membranes - eg. lipid-anchored proteins