post transcriptional modifications
Download as PPTX, PDF73 likes62,579 views
This document summarizes post-transcriptional modifications in eukaryotes. It discusses how eukaryotic mRNA undergoes processing, including capping, splicing to remove introns, and polyadenylation. Splicing requires snRNPs and the spliceosome to recognize splice sites. Alternative splicing allows one gene to code for multiple proteins. tRNA and rRNA also undergo processing as they mature, including modification of bases and removal of sequences. Final mature mRNA, tRNA, and rRNA are then ready for translation.
post transcriptional modifications
- 1. POST TRANSCRIPTIONAL MODIFICATIONS Prepared by: Narasimha Reddy.P.K (2014-11-104) college of horticulture kerala agricultural university Vellanikkara,thrissur 1
- 2. Eukaryotic vs. Prokaryotic Transcription • In eukaryotes, transcription and translation occur in separate compartments. • In bacteria, mRNA is polycistronic; in eukaryotes, mRNA is usually monocistronic. – Polycistronic: one mRNA codes for more than one polypeptide – monocistronic: one mRNA codes for only one polypeptide • “Processing” of mRNA is required in eukaryotes for the maturation • No processing in prokaryotes(mRNA matures on transcription)
- 3. Introduction… Coupled transcription and translation mRNA processed and transported out of nucleus for translation
- 4. 4 DNA Introduction Transcription Pri -RNA tRNA mRNA rRNA Processing Pri - transcript Matured RNAs
- 5. 1. mRNA Processing • Capping (addition of a 5’ 7-methyl guanosine cap) •Splicing to remove intervening sequences (introns) • Polyadenylation (addition of a poly-A tail at the 3’) 5
- 6. 5’Capping 6 Pri-mRNA Guanyltransferase O-methyl transferase
- 7. Cap Functions Cap provides: 1. Protection from some ribonucleases degradation 2. Stabilizes mRNA 3. Enhanced translation and splicing 4. Enhanced transport from nucleus to cytoplasm
- 8. mRNA splicing • mRNA is called hnRNA (heterogenous nuclear RNA) before splicing occurs •The hnRNP proteins to help keep the hnRNA in a single-stranded form and to assist in the various RNA processing reactions • Exon and intron lengths & numbers vary in various genes • Exon (Expressed sequences)is any segment of an interrupted gene that is represented in the mature RNA product. • Intron (intervening sequences )is a segment of DNA that is transcribed, but removed from within the transcript by splicing together the sequences (exons) on either side of it. 8
- 9. 9 Splice Junction Consensus Sequence • GU-AG rule describes the presence of these constant dinucleotides at the first two and last two positions of introns of nuclear genes. • Splice sites are the sequences immediately surrounding the exon-intron boundaries • Splicing junctions are recognized only in the correct pairwise combinations
- 10. The sequence of steps in the production of mature eukaryotic mRNA as shown for the chicken ovalbumin gene.
- 11. mRNA splicing • Splicing is mediated by a large RNPs(Ribonucleoproteins) complex spliceosome • Spliceosome contains a specific set of base Uracil-rich snRNPs (small nuclear RNPs) associated with proteins (snRNA complex with protein) Function of snRNPs: • Recognizing the 5’ splice site and the branch site. • Bringing those sites together. • Catalyzing (or helping to catalyze) the RNA cleavage. 11
- 12. Spliceosome Complex • Splicing snRNPs: • U1: 5'- site recognition • U2: branch site recognition • U4: forms base paired complex & acts with U6 • U5: 3'- junction binding of U4-U6 complex • U6: complex with U4 makes spliceosome transesterase 12 spliceosomes recognize introns starting with 5'-GU and ending in AG-3’
- 13. Mechanism of Spliceosome Exon 1 Exon 2 AG 5′ 3′ 5′ splice site Branch site 3′ splice site U1 GU A U1 binds to 5′ splice site. U2 binds to branch site. A 5′ 3′ U4/U6 and U5 trimer binds. Intron loops out and exons are brought closer together. U1 snRNP U2 snRNP A U4/U6 snRNP U5 snRNP U2 5′ 3′ Intron loops out and exons brought closer together 13
- 14. Mechanism of Spliceosome U1 U4 5′ splice site is cut. 5′ end of intron is connected to the A in the branch site to form a lariat. U1 and U4 are released. A U5 U2 5′ 3′ 3′ splice site is cut. Exon 1 is connected to exon 2. The intron (in the form of a lariat) is released along with U2, U5, and U6 (intron will be degraded). U6 A U2 U6 U5 Intron plus U2, U5, and U6 Intron will be degraded and the snRNPs used 5′ 3′ Two connected Exon 1 Exon 2 exons again 14
- 15. Alternative splicing pre-mRNA are spliced in several different ways, allowing a single gene to code for multiple proteins The generation of different mature mRNAs from a particular type of gene transcript can occur by varying the use of 5’- and 3’- splice sites 15 Sex determination in the Drosophila
- 16. Polyadenylation 16 Polyadenylation Complex Consensus sequence for 3’ process
- 17. Polyadenylation of mRNA at the 3’ end CPSF: cleavage and polyadenylation specificity factor binds upstream AAUAAA poly(A) Signal 5’ end. CStF: cleavage stimulatory factor F interacts with a downstream GU- sequence & bound with CPSF forming a loop in RNA CFI & CFII: cleavage factor I & II. PAP: poly(A) polymerase stimulates cleavage at poly A site Bound PAP adds ≈12 A residues at a slow rate to 3’- OH group PABPII: poly(A)-binding protein II. PABPII (short poly A tail) accelerates rate of addition of A by PAP After 200–250 A residues have been added, PABPII signals PAP to stop polymerization Poly (A) tail controls mRNA stability & influences translation
- 18. Matured mRNA • 5’ cap • 5’ untranslated region • Start codon • Coding sequence • Stop codon • 3‘ untranslated region • Poly A tail 18
- 19. 2. tRNA Transfer RNA/ Soluble RNA/ supernatant RNA/ Adaptor RNA • Smallest among RNAs (75-93 nucleotides) • Recognizes codon on mRNA • Shows high affinity to amino acids • Carry amino acids to the site of protein synthesis • tRNA is transcribed by RNA polymerase III • tRNA genes also occur in repeated copies throughout the genome, and may contain introns. 19
- 20. Processing of tRNA 1. Removal of leader sequence & trailer 2.Replacement of nucleotide 3.Modification of certain bases: • Replacement of U residues at the 3′ end of pre-tRNA with a CCA sequence • Addition of methyl and isopentenyl groups to the heterocyclic ring of purine bases • Methylation of the 2′-OH group in the ribose of any residue; and conversion of specific uridines to dihydrouridine(D),pseudouridine(y) 4.Excision of an intron 20
- 21. tRNA PROCESSING AND MATURATION Ribozyme RNA can act as an Enzyme and catalyse reactions including its own replication
- 22. 3. Ribosomal RNA (rRNA) • In cell >80% of rRNA • Serves to release mRNA from DNA • Act as ribozymes in protein synthesis • Relatively G:::C rich • Ribosome • Prokaryotes – 70S (50S & 30S) • Eukaryotes – 80S (60S & 40S) • Prokaryotes – In 50S subunits - 23S & 5S :31 proteins In 30S subunits - 16S :21 proteins • Eukaryotes – In 60S sub-units – 28S, 5.8S and 5S :50 proteins In 40S sub-units – 18S :33 proteins 22
- 23. 23 Processing of ribosomal RNA • Processing of 45s molecules occurs inside nucleolus • 45s molecules tightly associated protein forming (RNPs) • Frist cleavage: occurs at site I & remove 5’ terminal leader sequence, produces 41s intermediate & 18s • Second cleavage: occurs 41s intermediate at site 3’ generates 32s intermediate • Final cleavage: separation of 32s intermediate into 28s, 5.8s • Processed rRNA 28s, 5.8s & 18s
- 24. 2nd Cleavage 24 Processing of ribosomal RNA
- 25. Processing of ribosomal RNA 25
- 26. 26 Synthesis of 5S rRNA • rDNA cistron for 5S rRNA is present outside Nucleolar organizer • Transcription requires RNA pol III + TFIIIA, TFIIIB & TFIIIC