pp 17

Question 1

What are the differences between mRNA processing between bacteria and eukaryotes?

Answer 1

Bacteria: translation and transcription happen in cytoplasm simultaneously
Eukaryotes: transcription occurs in nucleus, primary transcript that has coding regions cap 5’ and poly A tail, translation occurs in the cytoplasm

Question 2

What are exons

Answer 2

Blocking of coding sequences

Question 3

What are introns?

Answer 3

Blocks of non-coding sequences

Question 4

How and when are introns removed?

Answer 4

Before the coding sequence in the exons is to be translated in the cytoplasm, removed by splicing in the nucleus

Question 5

How have introns been evolved?

Answer 5

Involved in gene regulation, encode functional RNA resulting from further processing after splicing, can facilitate gene expression by recruiting proteins to bind mRNA, can result in many different protein variants from different combinations of exons from a single gene.

Question 6

What are the DNA names during the process of splicing?

Answer 6

Genomic DNA - promoter region, exons, introns
Pre-mRNA - after transcription of genomic DNA
Spliced mRNA - leaving of the introns, only exons

Question 7

What is the evidence for splitting of genes into exons and introns?

Answer 7

R-looping. This involves hybridization of RNA and DNA. You have DNA and mature mRNA, hydrize them, the mRNA will match up to the DNA, leaving out the introns into loops

Question 8

What is splicing?

Answer 8

post-transcriptional process that occurs in the nucleus, resulting in the removal of introns

Question 9

What chemicals are needed to remove introns from genes?

Answer 9

1. 2’-OH of adenosine wihin intron attacks 5, phosphate of guanosine at 5’ splice site
2. this results in free 3’-OH from the first exon attacks phosphate group 5’ of second exon

Question 10

What does the first step of splicing result in?

Answer 10

Formation of an intermediate called a lariat (lasso)

Question 11

What does the splicing sites require to get spliced?

Answer 11

specific sequences at 5’ and 3’ ends of the intron and also at the internal site in the intron

Question 12

What type of consensus sequences does the processing machinery require?

Answer 12

1. 5’ splice site (A/C, A, G)
2. Internal branch point (C, U, A/G, A, C/U
3. 3’-splice site (C, A, G)

Question 13

What 40S macromolecular structure splices introns?

Answer 13

spliceosome

Question 14

What does the spliceosome consists of?

Answer 14

Proteins and small nuclear RNA’s (snRNA’s)

Question 15

How does the spliceosome work?

Answer 15

snRNA associate with proteins to form small nuclear ribonucleoproteins (snRNP)
SnRNP recognizes consensus sequences of 5’ and 3’ splice site and the branch point through base-pairing to snRNA components

Question 16

What is the mechanism of spliceosome?

Answer 16

1. U1 binds to the 5’ splice site, U2 binds to the branch point
2. U4-U6 displaces U1 at the 5’ splice site, U4 dissociates
3. U6 and U2 catalyze attack of the branch point on the 5’ splice site
4. 5’ splice site attacks the 3’ splice site, completing reaction

Question 17

Which snRNPs are important for the spliceosome?

Answer 17

5’ splice site: U1, U5, U6
Branch point: U2
3’ splice site: U5 U2AF (associated factor of U2

Question 18

What is required for recruitement of snRNP to form spliceosome?

Answer 18

RNA-binding proteins (splicing factors)

Question 19

What do the splicing factors recognize?

Answer 19

boundary between an exon and intron and form a complex that bridges acorss either the exon or intron depending on organisms

Question 20

When is exon definition seen?

Answer 20

seen in organisms with large introns and relatively small exons

Question 21

When is intron definition seen?

Answer 21

seen in organisms with small introns

Question 22

Why is C-terminal domain of RNA polymerase II used in splicing?

Answer 22

facilitates the assembly of splicing factors to pre-mRNA

Question 23

What transports RNA out and into the nucleus?

Answer 23

Exportins and importins, mRNA leaves to get translated, some non-protein coding RNA’s are processed in cytoplasm and then need to get returned

Question 24

What associates with exportins?

Answer 24

spliceosomes deposit an exon junction complex (EJC) on the newly spliced mRNA that will associate with exportins

Question 25

What is alternate splicing?

Answer 25

Same primary transcript but different mature mRNA’s and different proteins. In humans, 95% of multi-exon genes can undergo alternative splicing.

Question 26

What is the example of alternative processing of rat calcitonin?

Answer 26

Splicing of introns in thyroid, leaving exons 1,2,3,4 giving calcitonin
Splicing of introns in the brain leaving exons 1,2,3,5,6, giving CGRP

Question 27

What does the example of sex-determination of Drosophila?

Answer 27

Females are made with the help of Sx1 protein and Tra protein and Tra-2 proteins, males arent made with functional proteins.

Question 28

What does self-splicing introns mean?

Answer 28

Some introns catalyze their own splicing without the need for spliceosome snRNP

Question 29

What are the two groups of self-splicing introns?

Answer 29

1. catalytic RNA that self splice via a mechanism dependent on a free guanosine nucelotide
2. catalytic RNA that slice via a mechanism dependent upon an adenine nucelotide within the intron, forming a lariat