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Volume 272, Number 23,
Issue of June 6, 1997
pp. 14969-14974
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
Single-stranded RNA Recognition by the Bacteriophage T4
Translational Repressor, RegA
(Received for publication, February 26, 1997, and in revised form, April 3, 1997)
David
Brown
,
Joelle
Brown
§
,
ChulHee
Kang
¶
,
Larry
Gold
§
and
Patrick
Allen
From the Department of Molecular, Cellular, and
Developmental Biology, University of Colorado, Boulder, Colorado 80309, § NeXstar Pharmaceuticals, Inc., Boulder, Colorado 80301, and ¶ Department of Biochemistry and Biophysics, Washington State
University, Pullman, Washington 99164
The T4 protein, RegA, is a translational
repressor that blocks ribosome binding to multiple T4 messages by
interacting with the mRNAs near their respective AUG start codons.
Other than the AUG, there are no obvious similarities between the
affected mRNAs. High affinity RNA ligands to RegA were isolated
using SELEX (systematic evolution of
ligands by exponential enrichment). The
selected RNAs exhibited the consensus sequence 5 -AAAAUUGUUAUGUAA-3 .
The AUG was invariant, suggesting that it is the primary effector of
binding specificity. The UU immediately 5 to the AUG and the upstream
poly(A) tract were highly conserved among the selected RNAs. Boundary
and footprinting experiments are consistent with the consensus sequence
defining the RegA-binding site. Interestingly, chemical modification
and nuclease digestion data indicate that the RNA-binding site is
single-stranded, as if RegA discriminates between targets based on
their primary sequence, not their secondary structure. Minor variations
from the consensus at positions other than the universally conserved
AUG have little effect on RegA binding, but accumulation of mutations
has a profound effect on the interaction. Comparison of the in
vivo targets for RegA to the SELEX-generated consensus suggests a
repression pattern whereby the translation of individual messages is
sequentially halted until the least similarly affected message, the
regA gene itself, is repressed.

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Copyright © 1997 by the American Society for Biochemistry and Molecular Biology.
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