MECHANISM OF TRP GENE REGULATION BY TRAP-RNA RECOGNITION

Project: Research project

Project Details

Description

Numerous studies have provided a detailed understanding of how regulatory
proteins interact with DNA, however relatively little is known about the
principles that dictate how sequence-specific RNA-binding proteins
recognize their RNA targets. Since fragile X syndrome is caused by defects
in an RNA-binding protein, and RNA-binding proteins are involved in
regulating HIV and adenovirus gene expression, results from the proposed
studies could have a Positive impact on human health. TRAP of Bacillus
subtilis is responsible for regulating expression of the trpEDCFBA operon
and trpG by binding to a series of closely spaced G/UAG repeats present in
each transcript. TRAP binds to a segment of the trp leader RNA that
includes the antiterminator, thereby promoting transcription termination.
TRAP also binds to a segment of the trpG transcript that includes the trpG
ribosome binding site (RBS). The mechanisms responsible for these
regulatory events will be characterized using a combination of genetic and
biochemical approaches. The first aim of the proposed research is to
determine the nucleotides in the G/UAG repeat sequences that are required
for TRAP binding, to define the nucleotide spacing requirements between
adjacent repeats, and to determine the number of repeats that are
necessary to form a stable TRAP-RNA complex. This will be accomplished by
testing the ability of various artificial RNA targets to serve as TRAP
binding sites in a filter binding assay. Similar experiments will be
performed to determine if TRAP can bind to double-stranded RNA in addition
to single-stranded RNA. The next aim is to demonstrate that TRAP binding
to the trpG RBS prevents translation. RNA footprint analyses will be
performed to identify the nucleotides of the trpG transcript that TRAP and
ribosomes interact with, and to demonstrate that bound TRAP prevents
ribosome binding. In vitro translation studies will be carried out to
determine if TRAP binding inhibits TrpG synthesis. The next goal is to
determine the time frame in which TRAP binds to the nascent trp leader
transcript. If TRAP binding occurs after the antiterminator is formed it
is likely that RNA polymerase pauses following its formation to prevent
readthrough past the termination signal. In the absence of pausing, TRAP
binding must be sufficiently rapid to prevent antiterminator formation.
Single-round transcription experiments will be carried out in vitro to
determine if RNA polymerase pauses following antiterminator formation.
NusA protein will be added to the transcription system to see if it plays
a role in polymerase pausing. Evidence for pausing will also be examined
in vivo. Lastly, a structure-function analysis will be performed to
identify amino acid residues of TRAP that are responsible for RNA binding,
L-tryptophan binding, and subunit oligomerization, as well as to identify
nucleotides that interact with specific amino acid residues of TRAP. TRAP
deficient mutants will be obtained using several strategies, including
genetic selections and site-directed methods. The defects associated with
various mutant TRAP proteins will be determined using available
techniques. A collaboration with William Royer will continue to determine
the crystal structure of the TRAP-RNA complex.
StatusFinished
Effective start/end date8/1/957/31/96

Funding

  • National Institute of General Medical Sciences

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