JBC, Vol. 270, Issue 19, 11181-11189, May, 1995
Control of the Escherichia coli rrnB P1 promoter strength by ppGpp
X Zhang and H Bremer
Molecular Program, University of Texas at Dallas, Richardson 75083- 0688, USA.
Fusions of the rrnB P1 and P2 promoters, and of the tandem P1-P2
combination, to a wild-type lacZ gene were constructed on plasmids and
recombined into the mal region of the bacterial chromosome, close to the
normal location and in the normal orientation of rrnB. The upstream
activator region (Fis-binding sites) was always present with the P1
promoter, and all constructs contained the box A antitermination site of
rRNA genes. Using these constructs, beta-galactosidase specific activities
were measured in Escherichia coli strains carrying either both ppGpp
synthetases, PSI and PSII (relA+ spoT+), or only PSII (delta relA spoT+),
or neither (delta relA delta spoT), using different media supporting growth
rates between 0.6 and 2.8 doublings/h at 37 degrees C. The
beta-galactosidase activities were used to estimate the relative strength
of the rrnB P1 promoter in comparison to the isolated rrnB P2 promoter.
Promoter strength (transcripts initiated per min per promoter per free RNA
polymerase concentration) was distinguished from promoter activity
(transcripts initiated per min per promoter). In ppGpp- synthesizing
(wild-type) bacteria, the relative strength of the rrnB P1 promoter
increased nearly 10-fold with increasing growth rate from 0.17 to 1.5, but
in the ppGpp-less double mutants it decreased by 20% from 1.7 to 1.5. Thus,
at low or zero levels of ppGpp, the P1 promoter was 1.5-1.7 times stronger
than the isolated P2 promoter. These results indicate that the normal
growth rate control of the rrnB P1 promoter strength requires ppGpp, and
that the strength is reduced at basal levels of ppGpp found during
exponential growth. No additional ppGpp- independent control of the rrnB P1
promoter strength was evident. From the beta-galactosidase data and
previously determined values of rRNA gene activities, the activities of the
isolated rrnB P1 and P2 promoters, and of the P2 promoter in the tandem
combination, were estimated. With increasing growth rate, the activity of
the isolated P2 promoter increased 6-fold from 6 to 33 initiations/min,
while the activity of the isolated P1 promoter increased 24-fold from 2 to
54 initiations/min. The increasing activity of the isolated P2 promoter is
assumed to reflect the increasing RNA polymerase concentration at constant
promoter strength, whereas the steeper increase in P1 promoter activity
reflects increases in both polymerase concentration and promoter strength.
When in tandem with P1, the P2 promoter activity is inferred to decrease as
the P1 promoter activity increases.
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