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Journal of Bacteriology, December 1998, p. 6719-6728, Vol. 180, No. 24
Department of Microbiology, University of
Virginia School of Medicine, Charlottesville, Virginia 22908
Received 20 August 1998/Accepted 2 October 1998
The level of the vitamin B12 transport protein BtuB in
the outer membrane of Escherichia coli is strongly reduced
by growth in the presence of cobalamins. Previous analyses of
regulatory mutants and of btuB-lacZ fusions indicated that
the primary site of btuB gene regulation was at the
translational level, and this required sequences throughout the
240-nucleotide (nt) leader region. Cobalamin-dependent regulation of
transcriptional fusions was of a lesser magnitude but required, in
addition to the leader, sequences within the first 100 nt of the coding
sequence, termed the translated regulatory region (TRR). To analyze the
process of transcription-level regulation of btuB in
E. coli, the levels and metabolism of btuB RNA
were analyzed by S1 nuclease protection assays, and mutations that
alter the coupling of translational and transcriptional control were
analyzed. Expression of transcriptional fusions was found to correlate
with changes in the level of intact btuB RNA and was
related to changes in the metabolic stability of the normally
long-lived RNA. Mutational analysis showed that the btuB
start codon and a hairpin structure that can sequester the
Shine-Dalgarno sequence are necessary for cobalamin-dependent regulation and that translation of the TRR is necessary for extended RNA stability and for expression of the transcriptional fusion. The
absence of regulation at the stage of transcription initiation was
confirmed by the findings that several truncated btuB RNA fragments were expressed in a constitutive manner and
that the normal regulatory response occurred even when the
btuB promoter and upstream sequences were replaced by the
heterologous bla and lac promoters.
Transcription driven by phage T7 RNA polymerase was not regulated by
cobalamins, although some regulation at the translational level was
retained. Cobalamin-dependent changes in RNA structure were suggested
from the RNase III-dependent production of a transcript fragment that
is made only in the presence of cobalamin and is independent of the
regulatory outcome. These results indicate that the primary control of
btuB expression by cobalamin occurs at the level of
translation initiation, which directly affects the level and stability
of btuB RNA in a process that requires the presence of the
intact translated regulatory region.
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Coupled Changes in Translation and Transcription during
Cobalamin-Dependent Regulation of btuB Expression in
Escherichia coli
*
Corresponding author. Mailing address: Department of
Microbiology HSC#441, School of Medicine, University of Virginia,
Charlottesville, VA 22908. Phone: (804) 924-2532. Fax: (804) 982-1071. E-mail: rjk{at}virginia.edu.
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