post transcriptional modifications

simple post transcriptional modification ppt

1. POST TRANSCRIPTIONAL MODIFICATIONS Prepared by: Narasimha Reddy.P.K (2014-11-104 ) college of horticulture kerala agricultural university Vellanikkara,thrissur 1 WELCOME

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 N o processing in prokaryotes(mRNA matures on transcription)

3. Coupled transcription and translation mRNA processed and transported out of nucleus for translation Introduction…

4. 4 DNA Transcription Pri -RNA tRNA mRNA rRNA Processing Introduction Pri - transcript Matured RNAs

5. 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’) 1. mRNA Processing 5

6. 5’Capping 6 Guanyltransferase O-methyl transferase Pri-mRNA

7. Cap Functions Cap provides: Protection from some ribonucleases degradation Stabilizes mRNA Enhanced translation and splicing Enhanced transport from nucleus to cytoplasm

8. 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 . mRNA splicing 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. 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 . mRNA splicing 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. U1 3′ 5′ 5′ splice site 3′ splice site Branch site A GU Exon 1 Exon 2 U1 binds to 5′ splice site. U2 binds to branch site. AG 3′ 5′ A U4/U6 and U5 trimer binds. Intron loops out and exons are brought closer together. U1 snRNP U2 snRNP 3′ 5′ A U5 snRNP U4/U6 snRNP U2 Intron loops out and exons brought closer together Mechanism of Spliceosome 13

14. U1 U4 3′ 5′ 3′ 5′ 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. 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). A A U5 U6 U5 U6 U2 Intron plus U2, U5, and U6 Two connected exons Exon 1 Exon 2 U2 Intron will be degraded and the snRNPs used again Mechanism of Spliceosome 14

15. 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 Alternative splicing 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. 5’ cap 5’ untranslated region Start codon Coding sequence Stop codon 3‘ untranslated region Poly A tail Matured mRNA 18

19. 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. 2. tRNA 19 Transfer RNA/ Soluble RNA/ supernatant RNA/ Adaptor RNA

20. 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 Processing of tRNA 20

21. Ribozyme RNA can act as an Enzyme and catalyse r eactions including its own replication tRNA PROCESSING AND MATURATION

22. 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 3. Ribosomal RNA (rRNA) 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. 24 2 nd Cleavage 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

27. ThanQ ……