Molecular Biology (7) Flashcards
What does cotranscriptional mean?
RNA processing occurs at the same time as transcription (in eukaryotes)
What is the effect of TBP binding to the TATA box in eukaryotes?
Forms TBP-DNA complex
Bends DNA - due to weaker A-T hydrogen bonds
Aids DNA melting
Describe eukaryotic transcription initiation at the RNA polymerase II promoter
General transcription factors position RNA polymerase at start site (TFIIA, TFIIB, TFIID, TFIIF)
TFIID first - then TFIIA - then TFIIA bound to RNA polymerase
TFIIH and TFIIE bind to RNA polymerase - forms pre-initiation complex (PIC) - TFIIE assists TFIIH
TFIIH is helicase and kinase - assists DNA unwinding and phosphorylates C-terminal domain (CTD) of RNA polymerase - CTD bound over TFIID and TFIIA
Phosphorylation of CTD triggers transcription starts
DNA melts - forms trancription bubble
RNA polymerase escapes promoter - general transcription factors dissociate - TFIIF remains bound
What is found at the 5’ end of an RNA primary transcript immediately after synthesis?
Triphosphate group - α, β, γ
What is the structure of the 5’ methylated cap and how is it joined to eukaryotic mRNA?
7-methylguanylate - methyl group of 2’ hydroxyl of ribose of first mRNA nucleotide
5’-5’ phosphodiester linkage with 5’ end of primary transcript
Describe the process of the addition of the 5’ methyl cap to pre-mRNA
Phosphatase removes γ-phosphate from 5’ end
5’ end receives GMP from GTP - using guanyltransferase
Base of guanylate group methylated at N-7
2’ hydroxyl groups of terminal and penultimate riboses may also be methylated
What are the roles of the 5’ methyl cap?
Prevents RNA degradation by exonucleases
Marks RNA as substrate for splicing
Marks ribosomal recognition site for start of translation
What is the tole of the CTD of RNA polymerase?
Carries enzymes for capping and other factors - transferred to RNA
What allows RNA polymerase escape from transcription factors?
Phosphorylation of serine 2 and 5 in heptad repeats of CTD sequence
What is the direct result of CTD phosphorylation?
Exchange of capping enzymes for factors involved in elongation and RNA processing
Which types of RNA receive methyl cap?
Only mRNA
What is the effect of reaching the termination signal sequence at the end of a gene?
Transfer of polyadenylation enzymes to RNA
Describe the process of RNA polyadenylation
CstF (cleavage stimulation factor) and CPSF (cleavage and polyadenylation specificity factor) transferred from CTD to RNA poly-A signal sequence
Cleavage of RNA transcript - forms 3’ end
CstF diffuses away
CPSF remains bound - recruits PAP (poly-A polymerase) - binds to 3’ end - prevents degradation and adds poly-A tail - prevents base pairing to poly-T sequences
CPSF and PAP diffuse away
Which types of RNA have a poly-A tail?
Only mRNA
Where are RNA splicing signals located?
Exon-intron boundaries
What is the consensus sequence of the RNA 5’ splice site?
GU
What is the consensus sequence of the RNA 3’ splice site?
AG
What forms the branch site?
Adenine residue in middle of intron
Describe the process of pre-mRNA splicing
2 trans-esterification reactions
Nucleophilic attack of branch site adenine on 5’ splice site - cleaves 5’ end
5’ end loops and binds to branch site - forms lariat
Nucleophilic attack of free 3’ OH of exon on 3’ splice site - cleaves 3’ end
Lariat cut away
Phosphodiester bond forms between cleaved exon ends
What does the spliceosome consist of?
~150 proteins and 5snRNA complexed with proteins to form snRNPs
What are the roles of the spliceosome?
Protects free ends of exons from exonuclease degradation
snRNA acts as catalytic enzyme in splicing reaction
What is the action of the spliceosome in splicing?
U1 (snRNP) binds to exon 5’ site - gives specificity
U2AF and branch point binding protein bind to branch site - recruit U2 - binds to branch point
Interaction between snRNPs- causes splicing
What is the role of alternative splicing?
Expands informational content of genome
Give an example of tissue-specific RNA processing
Calcitonin gene - 6 exons
Exons 1-3 constitutive - transcribed in brain and thyroid cells
Exon 4 transcribed in thyroid cells - forms calcitonin
Exons 5 and 6 transcribed in brain - forms calcitonin gene-related peptide (CGRP)
What is the role of calcitonin?
Protects skeleton during calcium stress
What is the role of CGRP?
Endogenous vasodilatory peptide
