Lec 4- RNA processing

Question 1

RNA capping

Answer 1

1) The 5’ phosphate of the first nucleotide is removed by guanylyl transferase
2) The alpha and beta phosphates are removed from the guanine triphosphate that is to be added
3) Guanine monophosphate is joined to the 5’ mRNA end by a 5’-5’ triphosphate linkage. Additional nucleotide methylation occurs.

Question 2

Post transcriptional processing

Answer 2

5’ capping
3’ polyadenylation
intron splicing

They’re tied to the end of transcription so once fully complete the mRNA is released from the nucleus and makes it way to ribosomes where translation takes place

Question 3

5’ capping

Answer 3

the addition of a modified nucleotide at the 5’ end of mRNA, 7-methylguanosine

Question 4

3’ polyadenylation

Answer 4

cleavage at the 3’ end of mRNA & addition of a tail of multiple adenines to form the poly-A tail. Occurs when RNA is still being transcribed.

Question 5

Intron splicing

Answer 5

RNA splicing to remove introns and ligate exons

Question 6

Polyadenylation process

Answer 6

1) Polyadenylation begins w/ the binding factor, cleavage and polyadenylation specificity factor (CPSF) near a 6 nucleotide mRNA sequence that is downstream of the stop codon. The binding of cleavage stimulating factor (CStF) to a uracil-rich sequence several dozen nucleotides downstream of the polyadenylation sequence quickly follows and the binding of two other cleavage factors, CF1 & CFII & polyadenylate polymerase (PAP) enlarges the complex.
2) the pre-mRNA is then cleaved 15-30 nucleotide down stream of the polyadenylation signal sequence
3) the cleavage releases a transcript fragment bound by CFI, CFII,CstF which is later degraded
4) through the action of CPSF and PAP, the 3’ end of the cut pre-mRNA then undergoes the enzymatic addition of 20-200 adenine nucleotides
5) After addition of the first 10 adenines, molecules of poly-A-binding protein II join the elongating poly-A tail and increase the rate of adenine addition

Question 7

5’ capping process

Answer 7

1) phosphatase removes the 5’ phosphate of the first nucleotide from growing mrna
2) Guanylyl transferase adds a guanine to the 5; end of the mrna creating a 5’-5’ triphosphate linkage
3) methyl transferase adds a methyl group making the 7-methylguanosine cap

Question 8

3’ Poly-A tail functions

Answer 8

1) facilitating transport of mRNA across nuclear membrane
2) protecting mRNA from degradation
3) enhancing translation by enabling ribosomal recognition of mRNA

Question 9

Torpedo RNase (highly processive enzyme)

Answer 9

is a 5’-3’ nuclease attacking the uncapped 5’ end following 3’ cleavage by CF I and CFII. once RNase destroys the residual mRNA and catches up to RNA pol II, it triggers dissociation of the polymerase from template strand of DNA to terminate transcript.

Question 10

are coding sequencing in our DNA continuous?

Answer 10

No due to introns

Question 11

what did the detection of R-loops evidence?

Answer 11

sequences in the DNA that do not hybridize and loop out, INTRONS

Question 12

5’ splice site (DONOR)

Answer 12

located at the 5’ intron end, where it abuts an exon. This site contains a consensus seq. with a nearly invariant GU dinucleotide forming the 5’ most end of the intron.

Question 13

3’ splice site (ACCEPTOR)

Answer 13

on the opposite end of the intron, a consensus sequence containing a pyrimidine-rich region and nearly invariant AG dinucleotide at the 3’ most end of the intron

Question 14

Branch site

Answer 14

located 20-40 nucleo. upstream of the 3’ splice site. This sequence is pyrimidine rich and contains an invariant adenine adenine, called the branch point adenine near the 3’ end.

Question 15

Spliceosome

Answer 15

1) SnRNP U1 binds 5’ splice site and U2 binds branch site
2) snRNPs U4,U5,U6 bind to complex and form the inactive spliceosome. A lariant intron structure forms.
3) U4 dissociates to form active spliceosome, followed by 5; cleavage and formation of a 2’-3’ phosphodiester bond to stabilize lariat intron
4) Lariat intron forms by a 2’-5’ phosphodiester bond between the 5’ guanine and the branch point adenine.
5) The 3’ end of the intron cleaved, leaving a 5’ monophosphate at the 5; exon end.

Question 16

Splice sites

Answer 16

donor (exon) GU

acceptor (exon) AG

Question 17

snRNA-protein subunits

Answer 17

U1, U2, U4, U5, U6

Question 18

Coupling transcription & mRNA processing in the nucleus

Answer 18

1) At the initiation of transcription the carboxyl terminal domain (CTD) of RNA polymerase II affiliates with capping (CAP), polyadenylation (pA), and splicing factors (SF), and torpedo RNase (RNase)
2) RNA pol II initiates transcription after dissociation of the general transcription factors (GTFs). Multiple aa in the CTD are phosphorylated. The pre-mRNA processing proteins on the CTD begin their work. starting with the CAP proteins carrying out 5’ capping
3) CAP proteins dissociates, leaving part of the capping complex behind, including SF. The pre-mRNA continues to elongate.
4) Spliceosome complexes affiliate with pre-mRNA with the aid of SF proteins. Intron splicing takes place ad RNA pol II continues elongation of mRNA.
5) Polyadenylation proteins identify the pA signal sequence and carry out polyadenylation. Transcription terminates. Splicing continues to completion. Torpedo RNase digests residual mRNA
6) Fully processed mature mRNA dissociates from pol II, is released through nuclear pores and is transported to cytoplasm for translation. RNA pol II dissociates from DNA.

Question 19

3 mechanisms associated for single DNA sequence to produce more than one polypeptide

Answer 19

1) Alternative mRNA splicing- a pre-mRNA can be spliced in alternative patterns
2) Alternative promoters- can initiate promoters at distinct start points
3) Alternative polyadenylation- uses different polyadenylation signal sequences

Question 20

group 1 introns

Answer 20

large, selfsplicing ribozymes (catalytically active RNAs) that catalyze their own excision from certain mRNAs

Question 21

pre-RNA parameters

Answer 21

30s Pre-RNA and cleavage releases 16S and 23S and tRNAs

in humans the precursors is 45S and cleaves releases 28S, 18S and 5.*S rRNAs

Question 22

tRNA processing

Answer 22

1) many tRNAs are cleaved from large precursor tRNA transcripts to produce several individual tRNA molecules
2) nucleotides are trimmed off at the 5’ and 3’ ends of tRNA transcripts to prepare the mature molecules
3) certain individual nucleotides in diff. tRNAs are chemically modified to produce a distinctive molecule
4) tRNAs fold into a precise 3D structure that includes 4 DS stems, 3 of which are capped by SS loops; each stem and loop constitutes an “arm” of the tRNA molecule
5) tRNAs undergo post-transcriptional addition of bases. The most common addition is 3 nucleotides, CAA, at the 3’ end of the molecules. This region is the binding site for the aa tRNA molecule transports to the ribosome

Question 23

Addition of U nucleotides

Answer 23

1) SS guide RNA pairs with a portion of mRNA. Adenine nucleotides are in unpaired loops
2) Nuclease enzyme cuts mRNA, and RNA polymerase uses unpaired adenine to guide RNA to add uracils to mRNA