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Transcription Results in an Unprocessed Message 5 ´ Cap added immediately to 5 ´ sequence, in this case ACATTTG Poly(A) tail added when sequence (5 AATAAA 3 ) is transcribed Heterogeneous nuclear RNA (hnRNA) also known as (aka) pre-mRNA or the primary transcript
Modular Nature of Upstream Region for Tissue-Specific Gene Expression Note that many different transcription factors can bind to one gene It is the set of proteins bound to a gene that determine the level and location of gene expression
Transcription can also be arrested at promoter escape, potentially by TFII F binding to promoter ahead of transcriptional start site
Can be suppressed by TFII E & TFII H-XPB DNA helicase (ATP-dependent) activity which act to disrupt TFII F's interaction with the promoter
TFII H also recognizes damaged template DNA and recruits proteins for DNA excision-repair
Mutations in TFII H can result in diseases with sensitivity to light and increased risk of cancer such as xeroderma pigmentosum, trichthiodystrophy, or Cockayne syndrome (depending on mutation severity)
Trans-Splicing joins exons from two different RNAs
Self-excising group I & 2 introns 2 nucleophilic transesterification reactions 3 -OH guanosine on right side of intron is transferred to nucleotide at 5 end of intron Guanosine acts as cofactor "New" 3 -OH on left side of intron and phosphate group on 3 end of right side of intron interact leaving phosphate for ligation of exons 1 and 2
Alternative splicing results in families of proteins (splicing isoforms)
Types of Alternative Splicing (a) Alternative 5 ´ splice site (b) Alternative 3 ´ splice site (c) Skipping the variable alternative splice exon (d) Mutual exclusion of exons (e) Gender-specific splicing Alternative Poly(A) site Found in prostate cancer
Combinatorial Control Sex lethal binding results in stop codon being spliced out Functional transformer binding causes doublesex to be spliced in a female-specific fashion Stop codon remains Transformer not functional Male-specific doublesex
Lac Operon Has 3 Operators All 3 operators must be bound for maximal repression . Repressor binding to 2 operators causes a DNA conformational change DNA bends away from the repressor forming a repression loop, preventing RNA polymerase access to the promoter
Affinity of eIF4E for cap increased with phosphorylation
MAP kinase interacting protein 1 PKC Preiss and Hentze, 1999 Current Opinion in Genetics & Development
eIF4E Binding Proteins Compete for Binding with eIF4G eIF4E, A and G = eIF4F complex Sonenberg & Gingras, 1998
Control at initiation by numerous signal transduction pathways Many signal transduction pathways converge on eIF4E and phosphorylate it as well as other IF factors & ribosomal proteins Sonenberg & Gingras, 1998 eIF, Devers, 1999
The ribosome is a ribozyme – the 23S rRNA (large subunit) catalyzes peptide bond formation
Ribosome very accurate – uses 3 mechanisms in addition to codon-anticodon interactions to select against incorrect codon-anticodon pairings:
Two adenine residues in 16s rRNA form tight interaction with minor groove of correct base pair. Don’t recognize non-Watson-Crick base pairs b/c form a minor groove they don’t recognize, significantly reducing affinity for mismatches.
Proofreading - One mismatch dramatically reduces GTPase activity of EF-Tu. Mechanism very similar to #1.
Accomodation – a form of proofreading that occurs after EF-Tu is released. tRNA is moved closer to peptidyl transferase center by rotation. Incorrectly paired tRNA will usually dissociate here.
A chimeric molecule called a tmRNA mimics tRNA and mRNA
For example, SsrA (charged with an alanine) can bind to EF-Tu-GDP, enter the A site and cause translocation and release of mRNA
In addition, a part of SsrA enters the mRNA channel of the ribosome and extends the ORF by 10 codons and a stop codon
This results in a protein with 10 extra AA that tag the protein as incomplete, causing cellular proteases to digest it
What does the cell do if there is an early stop codon?
Normally, exon junction complexes (from splicing) are removed during translation
If a premature stop codon (a nonsense mutation) is encountered then the complexes still exist downstream from the mutation b/c translation stops before whole protein is made
This activates the nonsense mediated decay process where the remaining exon junction complexes recruit Upf proteins to the ribosome. Upf proteins activate the decapping enzyme that removes the 5 cap resulting in degradation of the mutated mRNA by 5 3 exonuclease.