Transcription II

Question 1

3 major ways to process pre-mRNA?

Answer 1

Capping
Splicing: excise introns, desegment exons
Cleavage/polyadenylation

TAKE place WHILE RNA is being made

Question 2

Capping

Answer 2

Replace 5’ triphosphate w/ a backwards 7-methylguanosine (no phosphate) .
3 steps:
1.) Triphosphatase removes Pi
2.) Guanylyl transferase (removes PPi), to produce gaunosine cap
3.) Guanine 7-methyltransferase (gives methyl group to guanine cap)

Question 3

Cleavage/polyadenylation

Answer 3

cleavage of RNA at 3’ end past the consensus sequence, polyadenylation (>200 A’s ) of the cleaved site (at 3’ OH exposed by cleavage) (partly responsible for Pol II being released from DNA)

Question 4

Pre-mRNA vs. mature mRNA

Answer 4

pre-mRNA is considerably longer (has introns), no cap… has triphosphate group at 5’, no poly-A tail.

Question 5

Function of 5’ cap?

Answer 5

Makes 5’ end resistant to exonucleases that target “long ends” of single D/RNA strands
Helps with splicing & processing through cap-binding complex that recognizes cap (primers for splicing, 3’ tail, translation)
In cytoplasms the cap-binding complex is replaced by eIF4E… vital facilitator of translation! for transport to ribos.

WHEN CAP REMOVED, signals mRNA to be degraded

Question 6

3 rxns to add 5’ cap to pre-mRNA

Answer 6

1. ) Cut off last Po4 from triphos group at 5’ end, triphosphatase
2. ) Add GTP backwards via guanylyl transferase (it loses 2 Po4 groups of its own, making GMP) and forms a 5’ to 5’ triphosphate bond (2 PO4 from mRNA, 1 Po4 from GMP) to the end of the mRNA.
3. ) Guanine 7-methyl transferase: methylate the 7-position of the guanosine cap via S-adenosyl-methionine (SAM)… to form 7-methylguanosine at cap

Question 7

Important sequences

Answer 7

GU marks beginning of every intron at 5’ end, AG at 3’ end.

Consensus seq at poly A site: AAUAAA

Question 8

Splicing defects?

Answer 8

Cause genetic disorders of CD44 (cancer metastasis) and spinal muscular atrophy

Question 9

Function of U1 and U2 snRNA’s?

Answer 9

Located in nucleus (where splicing happens)
U1snRA binds the GU 5’ splice site
U2snRNA binds to the branch point (A) on the pre-mRNA seq b/w 5’ and 5’ splice sites
U2AF binds 3’ splice site (AG)

Question 10

Lariat splicing

Answer 10

U1snRNP brings 5’ splice site near to branch point
U2snRP activates 2’ OH group at branch point, which attacks the phosphodiester just past the GU 5’ splice site

Now there is a free 3’ OH on the 5’ exon end- this attacks phosphodiester bond at AG 3’ splice site- linking 2 exons and excising the snRNP/intron complex to be degraded.

OH on branch point attacks 5’ end, revealing OH group there, which then attacks the 3’ end. This links the exons.

Question 11

Ways to get multiple proteins from splicing?

Answer 11

Include or skip exons
Mutually exclusive exons
Alt 3’ splice sites (extension or truncation)
Alt 5’ splice sites (extension or truncation)
Intron retained (acts like an exon)

Question 12

5’ splice site

Answer 12

Is vulnerable. Is part of DNA seq.
Vulnerable to being corrupted by mutation and being unrecognizable as an intron and then mRNA has an extra sequence (final protein is sig different).

HIV.

Question 13

Stop codons are…

Answer 13

UAG, UGA, UAA

Question 14

Tail

Answer 14

3’ end formation of cleavage-polyadenylation is coupled to termination of trx by RNA pol II

Question 15

Fx of poly-A tail

Answer 15

Enhances stability & translation of mRNA

PolyA tail is bound by poly A binding protein which fx. to remove poly A tail… RNA degradation key step

Question 16

Alt poly-A tail sites

Answer 16

Most can be poly-A’ed in more than one way
IgM, poly A tail added at 2 different sites. HEavy chain or light chian. IgM.

WHERE THEY ARE ADDED MATTERS NOT HOW MANY

frequently altered in cancer cells (shorter mRNA and escape normal regulatory mechanisms).