pp 17 Flashcards
What are the differences between mRNA processing between bacteria and eukaryotes?
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
What are exons
Blocking of coding sequences
What are introns?
Blocks of non-coding sequences
How and when are introns removed?
Before the coding sequence in the exons is to be translated in the cytoplasm, removed by splicing in the nucleus
How have introns been evolved?
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.
What are the DNA names during the process of splicing?
Genomic DNA - promoter region, exons, introns
Pre-mRNA - after transcription of genomic DNA
Spliced mRNA - leaving of the introns, only exons
What is the evidence for splitting of genes into exons and introns?
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
What is splicing?
post-transcriptional process that occurs in the nucleus, resulting in the removal of introns
What chemicals are needed to remove introns from genes?
- 2’-OH of adenosine wihin intron attacks 5, phosphate of guanosine at 5’ splice site
- this results in free 3’-OH from the first exon attacks phosphate group 5’ of second exon
What does the first step of splicing result in?
Formation of an intermediate called a lariat (lasso)
What does the splicing sites require to get spliced?
specific sequences at 5’ and 3’ ends of the intron and also at the internal site in the intron
What type of consensus sequences does the processing machinery require?
- 5’ splice site (A/C, A, G)
- Internal branch point (C, U, A/G, A, C/U
- 3’-splice site (C, A, G)
What 40S macromolecular structure splices introns?
spliceosome
What does the spliceosome consists of?
Proteins and small nuclear RNA’s (snRNA’s)
How does the spliceosome work?
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
What is the mechanism of spliceosome?
- U1 binds to the 5’ splice site, U2 binds to the branch point
- U4-U6 displaces U1 at the 5’ splice site, U4 dissociates
- U6 and U2 catalyze attack of the branch point on the 5’ splice site
- 5’ splice site attacks the 3’ splice site, completing reaction
Which snRNPs are important for the spliceosome?
5’ splice site: U1, U5, U6
Branch point: U2
3’ splice site: U5 U2AF (associated factor of U2
What is required for recruitement of snRNP to form spliceosome?
RNA-binding proteins (splicing factors)
What do the splicing factors recognize?
boundary between an exon and intron and form a complex that bridges acorss either the exon or intron depending on organisms
When is exon definition seen?
seen in organisms with large introns and relatively small exons
When is intron definition seen?
seen in organisms with small introns
Why is C-terminal domain of RNA polymerase II used in splicing?
facilitates the assembly of splicing factors to pre-mRNA
What transports RNA out and into the nucleus?
Exportins and importins, mRNA leaves to get translated, some non-protein coding RNA’s are processed in cytoplasm and then need to get returned
What associates with exportins?
spliceosomes deposit an exon junction complex (EJC) on the newly spliced mRNA that will associate with exportins
What is alternate splicing?
Same primary transcript but different mature mRNA’s and different proteins. In humans, 95% of multi-exon genes can undergo alternative splicing.
What is the example of alternative processing of rat calcitonin?
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
What does the example of sex-determination of Drosophila?
Females are made with the help of Sx1 protein and Tra protein and Tra-2 proteins, males arent made with functional proteins.
What does self-splicing introns mean?
Some introns catalyze their own splicing without the need for spliceosome snRNP
What are the two groups of self-splicing introns?
- catalytic RNA that self splice via a mechanism dependent on a free guanosine nucelotide
- catalytic RNA that slice via a mechanism dependent upon an adenine nucelotide within the intron, forming a lariat
