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Pre-mRNA processing
Biochem and medical genetics
| Question | Answer |
|---|---|
| Main mRNA modifications | Capping the 5' end Splicing of introns Cleavage and polyadenylation (3' end) |
| RNA pol II transcription cycle | Initiation - S5 phosphorylation Promotor escape - further S5 phosphorylation Promotor proximal pause - S2 and S5 phosphorylation Productive elongation - S2 phosphorylation Termination |
| What triggers capping | After initiations RNA polymerase associates with negative elongation factors and arrests at a promotor proximal position At the same time the polymerase interacts with capping enzymes that cap the emerging mRNA Triggered by S5 phosphorylation by TFIIH |
| How does capping occur | Phosphatase activity cleaves the final phosphate from RNA A GMP nucleotide is fused to the 5' end of mRNA in a 5'-5' linkage The G is subsequently methylated and it binds to a protein complex called the cap binding complex |
| Why is capping needed | Protects against premature degradation by 5' exoribonucleases Facilitates translation initiation Allows RNA to be grabbed by proteins and transported out nuclear pores |
| What triggers splicing | Phosphorylation of S2 and S5 by pTEFb at the pol II CTD and the negative elongation factors results in release of the paused polymerase and allows for recruitment of the spliceosome that will splice the mRNA |
| Structure of protein coding genes | Coding region is split into exons by non-coding intron which need to be removed Also contain promotors, 5' and 3' UTRs and a poly(A) site |
| Splicing reaction | Splicing is an RNA mediated trans-esterification reacting resulting in two breakages and re-joining of RNA backbone Mediated by small nuclear ribonucleic acid protein complex Two nucleophilic attacks to give fusion of exons and expulsion of intron lariat |
| How do spliceosomes know where to splice | Borders between exons and intron all look very similar at all splice sites 5'splice site, branch point, pyrimidine traps and 3' splice site are all identified by spliceosomes |
| Spliceosomes | Large complexes of protein and RNA RNA components are five small nuclear RNAs with around 200 proteins This directs excision or splicing of intron sequences and fusion of exons |
| How does splicing work | Spliceosomes bind to RNA at specific sites e.g. pyrimidine traps by complementary base pairing with their RNA components This results in an mRNA molecule with a continued open reading frame - introns removed and exons in a linear fashion |
| Why have introns | Separating exons allows for alternative splicing This allows for an increase in proteins within differentiated cells and allows genes to code for multiple proteins |
| What is alternative splicing | Allows permutation of exons, enabling cells to generate several proteins from a single gene All introns are removed, exons can also be emitted Plays an important role in specific gene expression |
| Regulation of alternative splicing | Positive and negative splice factors can influence splicing outcomes by enhancing or supressing splice site recognition by the spliceosome This can promote or inhibit exclusion of exons |
| Splicing enhancers | Association of enhancer proteins with pre-mRNA makes it more likely that the splicing factors recognise the 3' and 5' splice sites that flank an exon Makes it more likely that an exon is included |
| Splicing inhibitors | Negative factors binding to specific sequences can prevent recognition of both the 3' and 5' splice sites that flank an exon and splicing will skip the exon This results in exclusion of the exon in the final mRNA |
| Alternative splicing in Drosophila | Can generate more that 38000 Dscam isoforms (twice as many as encoded) This is likely to contribute to specificity of neuronal connectivity DSCAM is a homologue of human Down syndrome adhesion molecule - codes for a receptor |
| What triggers 3' modification | At the end of transcription the CTD is phosphorylated at S2 and engages with cleavage and polyadenylation factors that endonucleolytically cleave pre-mRNA and poly-adenylate it This induces transcription termination |
| What signals direct 3' end formation | CIS elements that direct 3' modification are bipartite. The major signal is AATAAA, which is found 30b upstream of the cleavage and polyadenylation site. A second signal is a downstream sequence of GT rich repeats after the cleavage site |
| Polyadenylation machinery | A large multiprotein complex make sup cleavage and polyadenylation machinery that assembles at poly-A sites at 3'ends AAUAAA and DSE sequences on pre-mRNA Cleavage occurs before polyadenylation |
| Process of polyadenylation | Poly(A) polymerase adds 200-250 adenosines in a reaction without a template. The poly(A) tail is covered by poly(A) binding proteins This creates a uniform 3' end to all protein encoding mRNAs needed for stability |
| Structure of mature mRNA | Both ends are modified to protect against premature degradation Contains a 5' and 3' UTR An open reading frame of exons |
| Mutations in Splicing and disease | Mutations affecting splice sites can lead to reduced or abolished splicing of introns - faulty mRNA contains introns This give an incorrect protein output Mutations affecting splicing factors have many consequences |
| Mutations in Poly(A) signals and disease | Can reduce or increase cleavage and polyadenylation Total mRNA output can be affected |
| Lynch Syndrome | C - T mutation in exon 6 of the MLH1 gene creates a new 5' splice site resulting in emission of four nucleotides at the end of exon 6 resulting in a final mRNA with a changed reading frame introducing a premature stop codon in exon 7 - nonsense mutation |
| Thalassemia | Mutations in the poly(A) sequences in beta and alpha globin genes lead to a reduction in 3' modification as it cannot be recognised by poly(A) machinery This causes a reduction in protein made, leading to an excess of the other protein which aggregates |
| How influenza virus targets poly(A) machinery | Cannot produce caps so 'cap snatches' - increases its own translation and prevents host gene translation Polyadenylation via repeated copying of U stretches created export and translation competent mRNA (DNA will not need a poly A tail added) |
| mRNA vaccines | Have to adhere to mRNA modifications mRNA must contain a cap, 5' UTP, open reading frame, 3' UTR and poly(A) tail Can modify DNA templates to produce mRNA already containing these modifications |
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