DYNAMICS AND TOPOGRAPHY OF YEAST RIBOSOMES

  • Lee, John C (PI)

Project: Research project

Project Details

Description

The objective is to elucidate the structure-function relationship of the
eukaryotic ribosome. The overall aim is to exploit recent advances
indicating that functional characteristics of the ribosome is closely
related to conformational changes in, and dynamic properties of its complex
structure. We will focus on the dynamics of the yeast ribosome and its
subunits. Specific aims are to: 1. continue our studies in defining the
contribution of individual proteins to specific ribosomal functions by
partial reconstitution in vitro, 2. selectively label ribosomal proteins
with fluorescent probes under non-denaturing conditions and reconstitute
biologically active ribosomal subunits with fluorescent-labeled protein(s),
focusing on those proteins whose functions have been demonstrated, 3.
characterize the properties of ribosomal subunits containing a specific
fluorescent-labeled protein by both steady-state and nanosecond
fluorescence, 4. study variations in fluorescence properties as the
fluorescent ribosome interacts with other components of the protein
synthetic machinery, 5. determine the kinetics of tritium exchange of yeast
ribosomal subunits that are in different functional states and attempt to
identify individual proteins whose dynamic properties are changed as the
ribosome is engaged in different biological activities using low pH HPLC,
and 6. identify protein neighborhood of selective ribosomal proteins,
focusing on the "acidic", phosphoproteins and ribosomal subunit interface
proteins. The dynamics of the ribosome will be studied using techniques
that have been applied successfully to monitor dynamics of proteins, viz.
fluorescence and tritium exchange to define global and local changes in
ribosomal proteins. These changes will be correlated with specific
functional states in which the ribosome is engaged during protein
synthesis. Protein topography will be determined by chemical crosslinking
using several reversible bifunctional reagents. Crosslinked products will
be identified by 2-D polyacrylamide gel electrophoresis, HPLC and
immunoblotting. A noval approach, using radiolabeled heterobifunctional
asymmetric and cleavable reagent, will be used to identify subunit
interfacial proteins. Information derived from the fluorescence and
tritium exchange studies will be correlated with functional as well as
static information of the ribosome.
StatusFinished
Effective start/end date7/1/8611/30/96

Funding

  • National Institutes of Health: $123,621.00
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health: $135,642.00
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health

ASJC

  • Medicine(all)
  • Biochemistry, Genetics and Molecular Biology(all)

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