Williams BCH 444 2014 Lectures 3-6

To explain these findings blobel and sabatini

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Unformatted text preview: that is about 20 amino acids larger than the authentic secreted light chain. To explain these findings, Blobel and Sabatini hypothesized in 1971 that secretory versus other proteins are distinguished NOT by differences in their respective mRNAs and NOT through inherent differences between membrane or free ribosomes, but rather by differences in their newly-synthesized N-terminal segments. They suggested that secretory pathway proteins possess an Nterminal signal sequence that directs the translating polyribosomes to the ER membrane where they attach and discharge the nascent polypeptide into the ER lumen. Known as the SIGNAL HYPOTHESIS. 6 Key experiments to support the Signal Hypothesis Methodology: In vitro translation systems Initiation system centrifugation 100,000 x g -  poly A+ RNA fraction from myeloma tumour (encodes mostly Ig light chain) -  purified large and small ribosome subunits -  pH 5 enzymes - contains charged tRNAs, initiation and increasing [sucrose] elongation factors -  14C amino acids -  buffer, ATP, GTP, ATP-regeneration system Used to study new translation on free ribosomes programmed with added mRNA rough microsomes Readout system -  rough microsomes or detached ribosomes (detergenttreated rough microsomes) from myeloma tumour -  mRNA is present in the ribosomal fractions (encodes Ig light chain) detergent -  pH 5 enzymes - contains charged tRNAs, initiation and -  sucrose gradient centrifugation elongation factors amino acids 14C -  buffer, ATP, GTP, ATP-regeneration system Used to complete ongoing translation of mRNA by ribosomes bound to, or detached from, the ER membrane. mRNA 5 3 In both systems, analyze translation products by SDS-PAGE detached ribosomes NH2 7 – -  Translation of Ig light chain mRNA on free ribosomes in an initiation system (lane 1) results in product larger than secreted Ig L chain (lane 2). Confirmed previous work. translation by rough microsomes translation by detached ribosomes std of secreted IgG L translation of Ig Light chain in initiation sys Key experiments to support the Signal Hypothesis -  Ongoing translation of myeloma mRNA in a readout system with rough microsomes results exclusively in the smaller product (lane 5) and it is protected from exogenous protease, i.e., membrane-enclosed (lane 6). (i) cleavage of putative signal is a microsome-associated event + – + protease (ii) translation on rough microsomes is tightly coupled with translocation into membrane-enclosed vesicles -  Polyribosomes detached from rough microsomes are engaged in the translation of both large and small forms of IgG light chains (lane 3); both forms are accessible to exogenous protease (lane 4) Speculate that ribosomes at right (3 ) end of mRNA are translating light chains that have already had their N-terminal signals removed whereas ribosomes at the 5 end of the mRNA are translating light chains with signals still intact 1 2 3 4 5 6 X protease 8 Key experiments to support the Signal Hypothesis Kinetics of readout translation using detached polyribosomes with new initiation inhibited by ATA ATA = aurintricarboxylic acid no membranes in this experiment suggests that:...
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This note was uploaded on 03/27/2014 for the course BCH 444 taught by Professor Mccallan during the Spring '14 term at University of Toronto- Toronto.

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