Protein Synthesis

E. coli Ribosome -70S particle, MW ~2.5 x 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

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

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 Tyrosyl-tRNA Puromycin Inhibition of Protein Synthesis

Science (2000) 289:920-30

Chemistry of Peptidyl Transfer Science (2000) 289: B B B

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

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

mRNA template DNA ? Gene Structure Analysis by EM

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

Pre-mRNA Splicing -splicing is nuclear (HeLa nuclear extracts) -requires Mg 2+ -requires ATP -(is co-transcriptional)

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

Pre-mRNA Splicing

Chemistry of Splicing 1st step2nd step 5’ exon 3’ exon

Splicing Time Course IVS-E2 IVS E1-IVS-E2

Complex Formation in HeLa Extract H A B C

Discovery of the Spliceosome Cell (1985) 42, S 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

snRNP Composition

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

Cell (1999) 96, snRNP Core Proteins B,D 1,D 2,D 3,E,F,G

5’ splice site3’ splice site poly-pyrimidine tract branch region Splicing Directed by Conserved Intron Sequences

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

U2 Auxiliary Factor -heterodimer, 65 kD, 35 kD subunits -U2AF 65 required splicing factor -U2AF 35, 3’ splice site U2AF 65 Domain Structure

Bridged Commitment Complex E Complex

Selection of 5’ Splice Site and Branch

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

RNA Rearrangment at 5’ Splice Site

Unusual Classes of Introns “AT-AC” minor spliceosome