1 / 30

Protein Synthesis

Protein Synthesis. E. coli Ribosome. -70S particle, MW ~2.5 x 10 6 -dissociable into small (30S) and large (50S) subunits -30S contains 16S RNA, 21 polypeptides -50S contains 5S, 23S RNA + 31 polypeptides. “Although the ribosome has been crystallized…it is such

selah
Download Presentation

Protein Synthesis

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Protein Synthesis

  2. E. coli Ribosome -70S particle, MW ~2.5 x 106 -dissociable into small (30S) and large (50S) subunits -30S contains 16S RNA, 21 polypeptides -50S contains 5S, 23S RNA + 31 polypeptides “Although the ribosome has been crystallized…it is such a complex entity that it will be many years before its structure is known in molecular detail” - Voet and Voet, Biochemistry 1995

  3. Ribosome X-ray Structure Science (2000) 289:920-30

  4. Puromycin Inhibition of Protein Synthesis Tyrosyl-tRNA Puromycin “…is known to interfere with protein formation by interfering with the function of RNA in the cells involved. In goldfish studied by Bernard W. Agranoff at the University of Michigan long term memory was obliterated when the fish were given minute injections of puromycin. Since short-term memory is not much affected, it is concluded that the antibiotic interferes with the process by which memory becomes fixed in the brain” - Merck Index 1968

  5. Science (2000) 289:920-30

  6. Science (2000) 289:920-30

  7. Chemistry of Peptidyl Transfer B B B Science (2000) 289:947-50

  8. Split Genes and RNA Splicing Nobel Prize - Medicine or Physiology - 1993 studies on genetic structure of adenovirus 2 P.A. Sharp (Biology, MIT) Proc. Natl. Acad. Sci. U.S.A. (1977) 74, 3171-5

  9. Split Gene Structure -eukaryotes from yeast to humans -90% of human genes -intron length highly variable -exon length ~200 nt -Dystrophin

  10. Gene Structure Analysis by EM mRNA ? template DNA

  11. Discovery of Split Genes (1977) chicken ovalbumin 7700 bp mRNA 1872 nucleotides I III DNA IV VI 3 < 20% coding 5 4 1 6 VII 2 5’ II 7 3’ V Voet and Voet Biochemistry

  12. Pre-mRNA Splicing -splicing is nuclear (HeLa nuclear extracts) -requires Mg2+ -requires ATP -(is co-transcriptional)

  13. Analysis of in vitro Splicing of 32P-Labeled pre-mRNA IVS-E2 IVS E1-IVS-E2 E1-E2 E1

  14. Pre-mRNA Splicing

  15. Chemistry of Splicing 1st step 2nd step 5’ exon 5’ exon 3’ exon 5’ exon 5’ exon 3’ exon

  16. Splicing Time Course IVS-E2 IVS E1-IVS-E2

  17. Complex Formation in HeLa Extract C B A H

  18. Discovery of the Spliceosome -60S particle required for pre-mRNA splicing -spliceosome contains ribonucleoprotein particles (snRNPs - small nuclear) -U1, U2, U4/U6 U5 -each snRNP contains respective snRNA (U1, U2, U4/U6 U5) + associated proteins Cell (1985) 42, 345-53

  19. snRNP Composition

  20. Protein Components of the Spliceosome -~10-220 kD -structural roles, functional roles -conserved (core), unique -non-snRNP “Comprehensive proteomic analysis of the human spliceosome.” Nature 419, 182-185 (2002).

  21. snRNP Core Proteins B,D1,D2,D3,E,F,G Cell (1999) 96, 375-87

  22. Splicing Directed by Conserved Intron Sequences 5’ splice site 3’ splice site branch region poly-pyrimidine tract

  23. Early Steps in Spliceosome Assembly Recognition of the Pyrimidine Tract U2AF required for A complex

  24. U2Auxiliary Factor -heterodimer, 65 kD, 35 kD subunits -U2AF65 required splicing factor -U2AF35, 3’ splice site U2AF65 Domain Structure

  25. Bridged Commitment Complex E Complex

  26. Selection of 5’ Splice Site and Branch

  27. RNA Rearrangements in the Spliceosome -extensive U4/U6 interaction is replaced with a U2/U6 structure -U1 displaced at 5’ splice site by U6

  28. RNA Rearrangment at 5’ Splice Site

  29. Unusual Classes of Introns “AT-AC” minor spliceosome

More Related