Lecture 02 - Post Translational Modification Flashcards
Why is prokaryotic mRNA not usually modified
since there are no compartments separating it, it doesn’t need to last as long
Where are eukaryotic mRNA modified
nucleus
What types of RNA are modified in both eukaryotes and prokaryotes
tRNA and rRNA
What are the coding tracts called
exons
What are the non-coding tracts called
introns
How much of a primary transcript is usually exons
10%
What removes introns from the primary transcript
splicing
What occurs during eukaryotic mRNA processing
addition of a 5’ cap, poly A tail, splicing and editing
What is the role of the C terminal domain (CTD) within transcription
it becomes phosphorylated because of the serine residues which removes TF2A and forces the transition from initiation to elongation
What is an exonuclease
an enzyme that cleaves the 5’ end of the mRNA
When is the 5’ cap added
early in transcription (20-30 nucleotides are synthesized before the cap is added)
What is the function of the 5’ cap
protect mRNA from ribonucleases, binds to specific cap binding complex which directs the mRNA to stay associated with RNAP, participates in binding to ribosome to initiate transcription
What is the multistep process of the 5’ cap
- removal of 5’ phosphate
- GTP addition
- methylation of G- generating the mature cap
Where do the capping enzymes bind to
RNAPII phosphorylated CTD
How is 7-methyguanosine attached to the 5’ nucleotide
triphosphate linkage
What binds the 5’ cap to the CTD
the cap binding complex
What is the role of the 5’ cap
protects RNA from degradation, involved in transport
What is the poly A tail
~80-250 A residues at the 3’ end of the transcript
What is the purpose of the poly A tail
protects RNA from degradation
Is the poly A tail encoded in the genome
no
What is the multistep process of the poly A tail
- recogniton of the sequences
- cleavage
- addition of A
What is the sequence seen 10-30 nucleotides before the poly A tail
AAUAAA
What binds to the AAUAAA region
an enzyme complex containing endonuclease, polyadenylate polymerase
What does the endonuclease do to add the poly A tail
cleaves it past the AAUAAA region
What does the polyadenylate polymerase do to add the poly A tail
add A residues to the free 3’ -OH
What are the only known vertebrate structural genes to lack introns
histone and interferon genes
What are the self-splicing (autocatalytic) introns
Group I and Group II
What are spliceosomal introns
splicing is mediated via a large protein complex
What occurs in the unnamed class of introns
they require ATP and an endonuclease to be spliced
Where are group I introns found
nuclear, mitochondrial and chloroplast genes of diverse eukaryotes and in some bacteria
Where are group II introns found
primary transcripts of organelles in fungi, algae and plants
Where do spliceosomal introns occur
in nuclear mRNA primary transcripts
Where do unnamed introns occur
in certain tRNA
Why do group I and II introns not need ATP
they undergo transesterification (break a bond to make a new bond)
What is the splicing mechanism of group I introns
the 3’OH of guanosine acts as a nucleophile and attacks the phosphate at the 5’ splice site, then the 3’OH of the 5’ exon becomes the nucleophile completing the reaction and joining the two exons togther
What is the splicing mechanism of group II introns
the 2’OH of an internal nucleotide acts as a nucleophile attacking the 5’ splice site to form a lariat structure, the 3’ OH of the 5’ exon acts as a nucleophile completing the reaction
What is the basis of spliceosomal introns
lariat forming mechanism but uses large splicing complex
What are spliceosomes made out of
RNA-protein complexes (5 snRNAs U1, U2, U4, U5, U6), ~50 proteins
What does the spliceosome do
recognizes and brings the two exons together
What is the mechanism of spliceosomal introns
U1 and U2 recognize the exon-intron junction, base pairing forms a bulge around a nucleotide that activates it into a nucleophile, U4/U6 complex and U5 bind to form the inactive spliceosome, U1 and U4 are expelled and U6 binds with both 5’ splice site and U2 to form an active spliceosome, the rest if the mechanism carries out like Group II to form a lariat intron that is released alongside the spliceosome
What is the directionality of intron splicing
5’ to 3’
Do all primary transcripts produce the same mRNAs
no, there can be different cleavage and polyadenylation sites and/or alternative splice patterns
