Transcription II + regulation of gene expression Flashcards
What is the difference btw endo- and exonucleases?
- endonucleases: hydrolyze internal phosphodiester bond to produce additional 3’-/5’-terminals
-
exonucleases: hydrolyze terminal phosphodiester bonds
BUT: limited to one direction (5’→3’ or 3’→5’)
Which modifications does pre-mRNA receive?
Where?
first 3 in nucleus, later transported into cytoplasm
- 5’-cap added
- splicing to deleted introns, connect exons
- 3’-poly A tail added
- RNA editing (happens elsewhere in cell)
List some features that affect the stability of mRNA.
-
cap, tail → prevent exonuclease action
NOTE: histone mRNA is not capped/tailed, receive 3’ UTR stem-loop instead - AUUUA sequence at 3’ terminal → target mRNA for rapid degradation
Describe the final structure of mRNA.
- 5’ untranslated region (UTR) leader sequence w/ 5’-cap
- protein coding sequence, codons determine AA sequence of final protein (btw start + stop codon)
- 3’ untranslated region (UTR) trailer sequence w/ 3’-poly A tail
Which terminal of pre-/mRNA receives the poly A tail?
Function?
poly A tail at 3’ terminal
- stabilizes mRNA, preventing attack by 3’→5’-exonucleases
- important for cytoplasmic export
Describe the mechanism of tailing.
When and where does it happen?
happens post-transcriptional in nucleus
- proteins assemble at poly(A) signal recognition sequence on pre-mRNA
- cleaved off pre-mRNA at poly(A) site
- slow polyadenylation occurs
- rapid polyadenylation occurs
NOTE: no DNA template required for poly A tail synthesis
Which terminal of pre-/mRNA is capped?
Function?
7-methylguanosine cap at 5’ terminal
5’-5’ triphosphate bind
- recognized by cap-binding protein (CBC), required for translation
- helps to stabilize mRNA, preventing attack of 5’→3’ exonucleases
- enhances translation, splicing and cytoplasmic export
Describe the mechanism of capping.
When and where does it happen?
happens during transcription of primary transcript in nucleus
- γ-phosphate of nascent transcript lost at 5’ terminal
- GMP added, β- and γ-phosphates of cap are lost
- guanosine of cap methylated
What is splicing?
When and where does it happen?
co-transcriptional editing of pre-mRNA to remove introns and ligate exons, happens after capping
- carried out by the spliceosome, complex of snRNPs
- some self-splicing introns/ribozymes capable of catalyzing their own excision from their parent RNA molecules
How are exons recognized as such by the spliceosome?
mechanism called exon definition
-
SR (Ser-Arg) proteins bind to each exon sequence, mark off 3’, 5’ splicing sites
→ form exonic splicing enhancer (ESE) - begin at the CBC (cap-binding complex) at the 5ʹ
- U2 binds to downstream 3’ branch-point
- *U1** binds to upstream 5’ splice site
What are snRNPs?
small nuclear ribonucleoproteins
snRNAs (U1, U2, U4, U5, U6) form complexes w/ 6-10 proteins that bind to their Sm-binding sites
→ complex to form spliceosome
Describe how the spliceosome is exactly formed during splicing.
- U2AF (U2 auxiliary factor) helps U2 binding
- U1 and U2 form pre-spliceosome
- when U4/U5/U6 bind, spliceosome formed
- U1 and U4 detach, remaining 3 snRNPs rearranged → catalytic spliceosome formed
Explain the mechanism of splicing.
two step process of transesterification RXNs btw RNA nucleotides
- 2’OH of branch-point nucleotide within the intron performs nucleophilic attack on the first nucleotide of the intron at the 5’ splice site, forming the lariat intermediate
- 3’OH of the released 5’ exon then performs a nucleophilic attack at the last nucleotide of the intron at the 3’ splice site, joining the exons and releasing the intron lariat
What happens to the excised lariat after splicing?
brought back into typical linear state debranching enzyme, then quickly degraded by ribonucleases
What are self-splicing introns?
rare introns that form a ribozyme (no spliceosome required)
In what way is alternative splicing different from “normal” splicing?
How is it mediated?
mediated by interactions btw histone PTMs + alt. splicing factors
- selective inclusion/exclusion of exons
- alternative 5’-donor/3’-acceptor sites
- use of different polyadenylation sites
What is RNA editing?
Name one important example.
changes the AA sequence on mRNA (that coding the protein) → differs from transcribed one, predicted by DNA sequence
see image for example
What are the 2 types of RNA editing?
substitution editing
- chemical altering of individual nucleotides
- ex: deamination of C→U, or A→I (inosine, read as G by ribosome)
insertion/deletion editing
- deletion/insertion of nucleotides (mostly U)
- requires special guide RNAs (gRNAs)
What are housekeeping genes?
genes with constitutive gene expression in (nearly) all cell types
How can signals affect the duration of gene expression?
Explain the 3 types and give examples.
- type A: incr. gene expression as long as signal persists
- ex: after exposure of nutrients, hormones*
- type B: transiently incr. gene expression, even if signal persists
- ex: action of many pharmacologic agents*
- type C: permanently incr. gene expression in respsonse to single signal exposure, inheritable
- ex: organ development*
Explain the model of the operon in prokaryotes.
functional unit of genomic DNA in prokaryotes containing a cluster of genes under the control of a single promoter that regulates their gene transcription, therefore affecting gene expression
What is an operator?
How do they work?
DNA sequence in proximity to promotor in prokaryotes that is able to bind activator or repressor proteins
- when repressor bound → transcription blocked, RNA polymerase cannot bind to DNA
- when neither repressor/activator bound → basal transcription rate
- when activator bound → maximal transcription rate
How does an activator work?
simultaneously binds to operator on DNA and RNA polymerase for maximal transcription rate
What is the function of the lac operon in e. coli.
operon that controls transcription of 3 genes, important for the degradation of lactose when glucose not present
- β-galactosidase
- lactose permease
- lactose transacetylase
