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RNA Metabolism
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RNA Metabolism

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  1. RNA Metabolism • DNA-dependent synthesis of RNA • RNA processing • RNA-dependent synthesis of RNA & DNA

  2. RNA (Ribonucleic Acid) Transcription: an enzyme system converts the genetic information in dsDNA into an RNA strand with a base sequence complementary to one of the DNA strand. • messenger RNA (mRNA) • transfer RNA (tRNA) • ribosomal RNA (rRNA)

  3. RNA Is Synthesized by RNA Polymerase Transcription in E. coli • encompasses ~35 bp of DNA (revealed by footprinting, p.985) • requires DNA template, NTP & Mg2+ • adds nucleotide units to the strand’s 3’-OH end in 5’ 3’ direction

  4. ~17 base pairs of DNA template are unwound

  5. Supercoiling of DNA brought about by transcription Positive supercoils form ahead of the transcription bubble, and negative supercoils form behind.

  6. The coding strand for a particular gene may be located in either strand of a given chromosome. e.g., adenovirus genome (36,000 bp) Many of the mRNA are initialy synthesized as a long transcript (25,000 nt), which is then extensively processed to produce the separate mRNA.

  7. Structure of E. coli RNA polymerase Lacks 3’ 5’ exonuclease activity error: 10-4 to 10-5 “holoenzyme”

  8. RNA Synthesis Is Initiated at Promoters Consensus sequence of typical E. coli promoters recognized by RNA polymerase holoenzyme containing s70

  9. RNA Polymerase Leaves Its Footprint on a Promoter “Footprinting”: a method that provides information about the interaction between RNA polymerase and promoters.

  10. Specific Sequences Signal Termination of RNA Synthesis • Not yet well understood in eukaryotes • At least two signals in E. coli: r(rho)-dependent and r-independent

  11. r-independent termination of transcription

  12. Eukaryotic Cells Have Three Kinds of Nuclear RNA Polymerase • RNA polymerase I: rRNA • RNA polymerase II: mRNA etc. • RNA polymerase III: tRNA, 5S rRNA etc.

  13. Common Sequences in Promoters Recognized by Eukaryotic RNA Polymerase II “Initiator sequence”

  14. RNA Polymerase II Requires Many Other Protein Factors for Its Activity

  15. Transcription at RNA Polymerase II Promoters • assembly • initiation • elongation • termination

  16. The Structure of TBP (gray) Bound to DNA (blue and white)

  17. RNA Polymerase Can Be Selectively Inhibited • actinomycin D: prok/euk. • rifampicin: prok. • a-amanitin: euk. pol II etc. Inserted into DNA between G/C

  18. A Complex of Actinomycin D and DNA G C

  19. RNA Metabolism • DNA-dependent synthesis of RNA • RNA processing • RNA-dependent synthesis of RNA & DNA

  20. Maturation of mRNA In a Eukaryotic Cell

  21. Phillip Sharp & Richard Roberts, 1977 The genes for polypeptides in eukaryotes are often interrupted by noncoding sequences (introns). i.e., “split gene” e.g., chicken ovalbumin gene

  22. Chicken ovalbumin gene Introns are removed by splicing intron: A-G exon: 1-7

  23. Introns • Group I: guanosine 3’OH as nucleophile • Group II: adenosine 2’OH in intron as nucleophile • Group III: dependent on snRNPs, pronounced “snurps” (small nuclear ribonucleoproteins), not self-splicing • Group IV: need ATP and endonuclease

  24. RNA Catalyzes Splicing Thomas Cech et al., 1982 (p.994) protozoan Tetrahymena thermophila the splicing mechanism of group I rRNA intron Sidney Altman et al., 1983 (p.1004) E. coli M1 RNA (377 nt) of RNase P cut tRNA

  25. Transesterification reaction: the first step in the splicing of group I introns

  26. Splicing mechanism of group I introns

  27. Splicing mechanism of group II introns “lariat”

  28. Splicing mechanism of group III introns in eukaryotic mRNA primary transcripts snRNAs (small nuclear RNAs)

  29. Assembly of spliceosomes snRNPs (“snurps”) = snRNA-protein complexes

  30. Splicing mechanism of group IV introns in yeast tRNA

  31. Eukaryotic mRNA Undergo Additional Processing • adding 5‘ cap • adding poly(A) tail

  32. cap 7-methylguanosine is added to the 5’ end of almost all eukaryotic mRNAs in 5’,5’-triphosphate linkage. Methyl groups (red) are sometimes found at the 2’ position of the first and second nt. (not in yeast) first second

  33. Generation of the 5’ cap adoMet = S-adenosylmethionine adoHcy = S-adenosylhomocysteine

  34. Addition of the poly(A) tail to the primary RNA transcript of eukaryotes

  35. Overview of the processing of a eukaryotic mRNA

  36. Multiple Products Are Derived from One Gene by Differential RNA Processing Alternative cleavage & polyadenylation Alternative splicing

  37. E.g., Alternative processing of the calcitonin gene transcript in rats calcium-regulating hormone (calcitonin-gene-related peptide)

  38. rRNAs and tRNAs Also Undergo Processing Processing of pre-rRNA in bacteria

  39. Processing of pre-rRNAs in vertebrates

  40. Processing of tRNAs in bacteria & eukaryotes

  41. Some modified bases of tRNAs, produced in post-trancriptional reactions

  42. Some Events in RNA Metabolism Are Catalyzed by RNA Enzymes Hammerhead ribozyme (only 41 nucleotides) requires Mg2+