Lecture 02 - Post Translational Modification

Question 1

Why is prokaryotic mRNA not usually modified

Answer 1

since there are no compartments separating it, it doesn’t need to last as long

Question 2

Where are eukaryotic mRNA modified

Answer 2

nucleus

Question 3

What types of RNA are modified in both eukaryotes and prokaryotes

Answer 3

tRNA and rRNA

Question 4

What are the coding tracts called

Answer 4

exons

Question 5

What are the non-coding tracts called

Answer 5

introns

Question 6

How much of a primary transcript is usually exons

Answer 6

10%

Question 7

What removes introns from the primary transcript

Answer 7

splicing

Question 8

What occurs during eukaryotic mRNA processing

Answer 8

addition of a 5’ cap, poly A tail, splicing and editing

Question 9

What is the role of the C terminal domain (CTD) within transcription

Answer 9

it becomes phosphorylated because of the serine residues which removes TF2A and forces the transition from initiation to elongation

Question 10

What is an exonuclease

Answer 10

an enzyme that cleaves the 5’ end of the mRNA

Question 11

When is the 5’ cap added

Answer 11

early in transcription (20-30 nucleotides are synthesized before the cap is added)

Question 12

What is the function of the 5’ cap

Answer 12

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

Question 13

What is the multistep process of the 5’ cap

Answer 13

• removal of 5’ phosphate
• GTP addition
• methylation of G- generating the mature cap

Question 14

Where do the capping enzymes bind to

Answer 14

RNAPII phosphorylated CTD

Question 15

How is 7-methyguanosine attached to the 5’ nucleotide

Answer 15

triphosphate linkage

Question 16

What binds the 5’ cap to the CTD

Answer 16

the cap binding complex

Question 17

What is the role of the 5’ cap

Answer 17

protects RNA from degradation, involved in transport

Question 18

What is the poly A tail

Answer 18

~80-250 A residues at the 3’ end of the transcript

Question 19

What is the purpose of the poly A tail

Answer 19

protects RNA from degradation

Question 20

Is the poly A tail encoded in the genome

Answer 20

no

Question 21

What is the multistep process of the poly A tail

Answer 21

• recogniton of the sequences
• cleavage
• addition of A

Question 22

What is the sequence seen 10-30 nucleotides before the poly A tail

Answer 22

AAUAAA

Question 23

What binds to the AAUAAA region

Answer 23

an enzyme complex containing endonuclease, polyadenylate polymerase

Question 24

What does the endonuclease do to add the poly A tail

Answer 24

cleaves it past the AAUAAA region

Question 25

What does the polyadenylate polymerase do to add the poly A tail

Answer 25

add A residues to the free 3’ -OH

Question 26

What are the only known vertebrate structural genes to lack introns

Answer 26

histone and interferon genes

Question 27

What are the self-splicing (autocatalytic) introns

Answer 27

Group I and Group II

Question 28

What are spliceosomal introns

Answer 28

splicing is mediated via a large protein complex

Question 29

What occurs in the unnamed class of introns

Answer 29

they require ATP and an endonuclease to be spliced

Question 30

Where are group I introns found

Answer 30

nuclear, mitochondrial and chloroplast genes of diverse eukaryotes and in some bacteria

Question 31

Where are group II introns found

Answer 31

primary transcripts of organelles in fungi, algae and plants

Question 32

Where do spliceosomal introns occur

Answer 32

in nuclear mRNA primary transcripts

Question 33

Where do unnamed introns occur

Answer 33

in certain tRNA

Question 34

Why do group I and II introns not need ATP

Answer 34

they undergo transesterification (break a bond to make a new bond)

Question 35

What is the splicing mechanism of group I introns

Answer 35

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

Question 36

What is the splicing mechanism of group II introns

Answer 36

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

Question 37

What is the basis of spliceosomal introns

Answer 37

lariat forming mechanism but uses large splicing complex

Question 38

What are spliceosomes made out of

Answer 38

RNA-protein complexes (5 snRNAs U1, U2, U4, U5, U6), ~50 proteins

Question 39

What does the spliceosome do

Answer 39

recognizes and brings the two exons together

Question 40

What is the mechanism of spliceosomal introns

Answer 40

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

Question 41

What is the directionality of intron splicing

Answer 41

5’ to 3’

Question 42

Do all primary transcripts produce the same mRNAs

Answer 42

no, there can be different cleavage and polyadenylation sites and/or alternative splice patterns