Molecular Genetics Final

transcription

RNA synthesis; 5’ to 3’ polarity; uracil instead of thymine; sugar-phosphate backbone (ribose); phosphodiester bonds; 2’ OH

eukaryotic gene organization

exons; introns; pre-mRNA/primary transcript; splicing; 5’ cap; 3’ poly A tail

DNA template

complementary and anti-parallel to RNA; antisense to RNA

DNA non-template

same sequence and orientation as RNA; sense to RNA

coding RNA

mRNA

non-coding RNA

rRNA; tRNA; snRNA; miRNA

RNAP

RNA polymerase; does not require a primer

RNA synthesis

antiparallel orientation of strands; template strand; NTP; 5’ to 3’ direction

initation

RNA polymerase binds to promoter, forms closed complex, RNA pol “melts” DNA forming open complex, covalent linkage of first two nucleotides

elongation

synthesis of RNA in 5’ to 3’ direction

termination

polymerase releases RNA and dissociates from template

Euk RNAPs

have many more polypeptides (12-17); homologous to 5 core subunits

bacterial RNAPs

5 core subunits

RNA pol II

products: mRNA; lncRNA; snRNA; miRNA

CTD

carboxy terminal domain; consists of heptapeptide repeat and 5 phosphate targets

core promoters

short sequences near a transcription start site that serve as assembly points for Pol II and general txn factors

dispersed (broad) promoters

have several txn start sites spread over 50-100 bps; more common in mammals; characterized by a CpG island, a 300-3000 bp region enriched for CpG dinucleotides

CpG islands

common on constitutively expressed genes; also found at many developmentally regulated and tissue-specific gene promoters

active dispersed promoters

CpG islands are hypomethylated; maintaining an open, nucleosome-free regoin over the txn start site that permits PIC assembly

repression of gene activity

1) methyl CpG binding proteins (MeCP2) recruit HDACs, producing repressed chromatin

2) repressive histone marks accumulate, particularly H3K9me2/3 and H3K27me3

Together create a stable, silenced state

focused (single peak) promoters

a single transcription start site ± one or two bp and one or more identifiable sequence element (cis-acting element); typically found in developmentally regulated and tissue-specific genes;

cis-acting element

DNA sequences involved in transcription of a gene

trans-acting factor

proteins that bind to cis-acting elements

TATA box

first core promoter element identified; sequence found centered between -35 and -25 with initiation at +1; only a small proportion of eukaryotic promoters have; sufficient by itself

Initiator

most widely used core promter motif; weak consensus seq around +1; sufficient by itself

BRE

TFIIB recognition element; located at -35 position; cannot function alone

DPE

downstream promoter element; common in Drosophila, rare in humans; located at +30 position; cannot function alone

general txn factors (GTFs)

assist RNA Pol II in promoter recognition and transcription initiation at most Pol II promoters

GTF functions

core promoter recognition; recruit RNA pol; stabilize binding of RNA pol; integrate regulatory information from gene-specific transcriptional activator and repressor proteins; promoter clearance (RNA pol activation)

How do Pol II and GTFs assemble on a core promoter with a TATA box

two non-mutually exclusive mechanisms: sequential assembly and holoenzyme

sequential assembly

TFIID; TFIIA/TFIIB; TFIIF; TFIIE/TFIIH

TFIID

recognizes core promoter elements; consists of TATA binding protein (TBP) and TBP associated factors (TAFs)

TBP

TATA binding protein; binds directly to TATA; binds to DNA causing an 80 degree bend in DNA, facilitating interactions between DNA and other PIC components; required for txn of most Pol II promoters

PIC

pre-initation complex

TAFs

TBP associated factors; bind to other core promoter elements other than TATA box; recognize TATA-less promoters; interact with transcriptional activator and repressor proteins; chromatin modification activity

TFIIA/TFIIB

stabilize binding of TFIID to DNA; TFIIB can recognize and bind to BRE

TFIIF

escorts RNA pol II with hypophosphorylated (low phosphorylation) CTD of largest subunit to promoter

TFIIE/TFIIH

bind to complete formation of PIC for in vitro transcription; initially a closed PIC

closed PIC

promoter region is not unwound

TFIIH

10 subunits including DNA helicase activity to form open PIC, creating a transcription bubble; possess kinase activity which phosphorylates pol II CTD allowing the release of pol II from PIC and formation of elongating complex

EMSA

electrophoretic mobility shift assay; discovered sequential assembly; standard method to test for the presence of a protein that binds to a particular cis-acting element

EMSA method

incubate labeled dsDNA containing a cis-acting element with protein fractions; run on non-denaturing gel; protein bound DNA migrates more slowly than the free probe

mediator

large multi-subunit complex; required for initiation of transcription by pol II in vivo and thus a part of the PIC; provides many potential targets for interaction with activator proteis and other proteins that regulate transcription

How does RNA pol initiate RNA synthesis after PIC formation

after assembly of PIC, open complex forms, abortive initiation occurs, extensive phosphorylation of CTD tail; Pol II dissociates from GTFs and from mediator and begins elongating transcript

promoter-proximal pausing

RNA pol II paused 20-60 nt downstream of the txn start site; allows rapid induction in response to signals

uaRNAs

upstream antisense RNAs; typically short and rapidly degraded

RNA modifications

removal, addition, and chemical modifications of nucleotides

Euk pre-mRNA processing

involves capping, poly A addition and splicing of exons; co-transcriptional with key coordinating role played by CTD of large subunit of RNA pol II

CTD phosphorylation

reversible and dynamic, altering function of RNA pol II and changing which Rna processing factors can interact with CTD

CTD function

an assembly point where capping, splicing, and polyadenylation factors assemble

5’ capping

7-meG, 5’ to 5’ linkage with 3 phosphates, methylation of 2’ OH on the next 1 or 2 nts; recognition of first exon for splicing, recognition of mRNA for translation; protection from degradation; export to cytoplasm

cleavage/polyadenylation

form a large multiprotein complex associated with the RNA pol II CTD; CPSF; CStF; CF I/II

CPSF

cleavage and polyadenylation specificity factor; binds to poly A signal in RNA; cleaves RNA downstream of polyadenylation signal

CStF

cleavage stimulation factor; binds to G/U-rich region and recruits additional cleavage factors (CF I/II)

PAP

poly A polymerase; binds to 3’ end and uses ATP to add As to 3’ end

PABP

poly A binding protein; binds the growing tail and controls its final length

poly A tail function

protection from degradation; transport to cytoplasm; translation initiation

pre-mRNA splicing

involves removal of introns from primary transcripts and ligation of exons; catalyzed by spliceosomes

consensus sequences

GU at 5’ splice site; AG at 3’ splice site; polypyrimidine tract; A at branch point

exonic splicing enhancers

different sequences within exons that facilitate splicing

trans-splicing

exons located on different pre-mRNA molecules can be spliced together

cis-splicing

splicing that occurs between exons on the same pre-mRNA molecule

snRNPs

small nuclear riboproteins; catalyze splicing; recognize 5’ splice site and branch point; bring splice sites and branch point into correct configuration; catalysis of transesterifications

major steps of pre-mRNA splicing

recognition of branch point and 5’ and 3’ splice sites; exchange of SF1 for U2; binding of tri-snRNP and spliceosome forms; dissociation of U1, U4, and U2AF; first transesterification; second transesterification

recognition of branch point and 5’ and 3’ splice sites

U1 binds to 5’ splice site; U2AF (auxiliary factor) binds to pyrimidine rich region and AG at 3’ splice site which facilitates binding of SF1 (splicing factor 1); SF1 binds to branch point

exchange of SF1 for U2

U2AF recruits U2 to branch site which displaces SF1; U2 recognizes branch point consensus sequence by RNA-RNA base pairing

binding of tri-snRNP and spliceosome forms

U4/U5/U6 tri-snRNP binds, bringing splice sites and branch point in close proximity; spliceosome formed but not catalytically active

dissociation of U1, U4, and U2AF

U6 is exchanged for U1 at the 5’ splice site; U1, U4, and U2AF leave spliceosome, active spilceosome forms; U6 recognizes 5’ splice site by RNA-RNA base pairing

first transesterification

U2-U6 interaction mediated by RNA-RNA base pairing; U2 and U6 RNAs carry out catalysis; 5’ splice site placed in active site with branch point and forms lariat

second transesterification

U2 and U6 catalyze second transesterification linking 5’ and 3’ exons (aided by U5); spliced exons released; snRNPs release lariat which is usually degraded

ESEs

exonic splicing enhancers; short sequences recognized by SR proteins

SR proteins

rich in serine and arginine; interact with U1 and U2AF to define exon boundaries

exon definition complex

assembly of exonic splicing enhancers with SR proteins and U1/U2AF

alternative splicing

genes spliced in different ways and polyadenylated at multiple sites yielding more than one kind of mRNA from a single gene

translation

process of protein synthesis; occurs in cytoplasm

genetic code

a list of which codons specify which amino acids; codons are arranged with 5’ to let and 3’ to the right

how are mRNAs decoded and used to synthesize proteins

need tRNAs, aminoacyl-tRNA synthetases, and ribosomes

anticodons

complementary and anti-parallel to codons

aminoacyl-tRNA synthetases

adds amino acids to tRNAs; must add amino acid to corresponding tRNA with high accuracy

wobble pairing

some tRNAs must recognize multiple codons; unusual base pairing

r-proteins

ribosomal proteins; hold rRNA in configurations necessary for function and protect rRNA from degradation

ribosome tRNA binding sites

A (aminoacyl); P (peptidyl); E (exit)

ribosome small subunit

interacts with tRNA anticodons and mRNA

ribosome large subunit

interacts with amino acid end of tRNA and contains peptidyl transferase activity

protein synthesis

initiation; elongation; termination

initation

mRNA binds to ribosome, charged tRNA enters P site, start codon identified

types of initiation

cap-dependent initiation and cap-independent initiation

cap-dependent initiation

5’ cap on an mRNA is recognized and translation initates at a start codon

cap-independent initiation

occurs for some mRNAs where start codons are nmot near a 5’ cap or for mRNAs that lack a 5’ cap

IRES

internal ribosome entry sites; cap-independent translation initiation sites

key steps of cap-dependent initiation

formation of 43S pre-initiation complex; mRNA activation

43S pre-initiation complex

40S (small) ribosomal subunit, binding of 5 initiation factors, GTP, and initiator tRNA-met

mRNA activation

subunits of eIF4F complex bind to mRNA

eIF4F complex

eIF4E; eIF4G; eIF4A; PABP

eIF4E

cap-binding protein

eIF4G

interacts with poly A binding protein

eIF4A

RNA helicase

recognition of initiation codon (Kozak consensus sequence)

ribosome scans using eIF4A to unwind secondary structure in UTR to first AUG found in a good sequence context

completion of initiation

binding of 60S large subunit with eIF5b-GTP, displacement of other eIFs, formation of 80S initiation complex

elongation

aminoacyl tRNA binds to A site; peptide bond formation; translocation