The research goal of this section is to understand how nutrient availability
coordinates global patterns of gene expression as well as pathway-specific
regulation in bacteria. Such complex networks integrate synthesis of macromolecules
and regulate expression of the genomic repertoire. We focus on the roles
of two regulatory nucleotides that are widespread in bacteria and that
have recently been found in plants. Collectively called (p)ppGpp, they
are analogs of GTP and GDP with pyrophosphate residues on the ribose 3'-hydroxyl
group. Nutrient limitation, whether starving for amino acids, phosphate,
nitrogen, or energy sources, causes fluctuations of (p)ppGpp levels. A
regulatory role is assigned to (p)ppGpp because artificial induction of
(p)ppGpp without nutrient limitation gives many of the same regulatory
effects as starvation itself. Fluctuation of (p)ppGpp can also be an important
element in adaptive responses to starvation, such as insuring survival
by induction of the stationary phase-specific sigma factor (RpoS) of RNA
polymerase. Regulatory responses to (p)ppGpp are thought to occur by effects
on transcription, translation, and metabolism. We wish to understand these
mechanisms as well as how nutrient limitation leads to changes in (p)ppGpp
levels..
(p)ppGpp Effects on rpoS mRNA Translational Efficiency
Brown, Szalewska-Palasz, Murphy, Cashel in collaboration with T. Elliotta
We find that induction of RpoS by (p)ppGpp occurs by changing the efficiency
of translation of rpoS mRNA instead of through the transcriptional
effects on mRNA abundance. We have come to believe the dksA gene
is involved; in the absence of (p)ppGpp, a dksA deletion blocks
(p)ppGpp induction of RpoS, and overexpression of DksA induces RpoS. We
have found that the effects of (p)ppGpp and DksA are exerted on a region
of RpoS mRNA far upstream of the rpoS mRNA leader region, where
the SD sequence is known to be sequestered by RNA folding and regulated
by other factors. At the same time, DksA effects are puzzling because
increased (p)ppGpp does not induce DksA nor increased DksA induce (p)ppGpp.
Though at the transcriptional level, another suggestion of a relation
between DksA and (p)ppGpp is based on the finding that the five amino
acids required in a dksA deletion are a subset of those nine amino acids
that are required in a complete (p)ppGpp deficiency [(p)ppGpp0]. Finally,
RNA polymerase mutants that suppress the multiple amino acid auxotrophy
of (p)ppGpp0 strains also suppress the
five amino acid requirements of dksA deleted strains.
Suppressors of a (p)ppGpp-Deficiency Occur Exclusively in RNA Polymerase
rpoB, rpoC and rpoD Subunit Genes
Szalewska-Palasz, Murphy, Cashel
While most (p)ppGpp regulation is thought to act directly on RNA polymerase
initiation or elongation, verification of these notions with pure transcription
components has proven elusive. Therefore, we have taken an alternative
genetic approach of isolating mutants that suppress cellular phenotypes
in response to a complete deficiency of (p)ppGpp. Accordingly, we have
identified over 50 distinct lesions in the two largest RNA polymerase
subunits. Interestingly, by using the crystal structure of the RNA polymerase
core enzyme recently resolved by the Darst laboratory, we find that the
sites of these amino acid changes are almost exclusively on enzyme surfaces
deduced to involve DNA contacts. We have now isolated strains with multiple
lesions and can ask whether there are spatially grouped subclasses of
lesions that are functionally synergistic or antagonistic.
A (p)ppGpp-Synthesizing and -Degrading Enzyme from Streptococcus
Displays Reciprocal Regulation of Bifunctionality as an Intrinsic Enzymatic
Property
Mechold, Brown, Murphy, Cashel in collaboration with T. Hoggb
Most Gram-positive bacteria possess a single enzyme capable of both (p)ppGpp
synthesis and degradation. A futile metabolic cycle would occur if both
were fully active. This means that net activity requires a switch within
the enzyme that activates one activity while simultaneously curtailing
the opposing activity. We have found that the function of the C-terminal
half of the 84 kDa protein is to regulate the opposing functions present
in the N-terminal half protein in a reciprocal manner. To explore this
switch further, we have isolated within the N-terminal half of the protein
single missense suppressor mutations that restore reciprocal activation
and repression of separate domains encoding (p)ppGpp degradation and synthesis.
The N-terminal half protein, in which reside opposing catalytic domains,
has been crystallized and its structure determined. Our intragenic allele-specific
suppressors appear to map on the boundaries separating the two domains,
but the mechanism of reciprocal activation and inhibition built into the
structure remains to be elucidated.
Mapping the Ribosomal Binding Sites of RelA, the Major (p)ppGpp Synthetase
from E. coli
Glaser, Murphy, Cashel in collaboration with laboratory of Al Dahlbergc
Amino acid starvation by codon-specified uncharged tRNA binding has long
been known to induce synthesis of (p)ppGpp on the ribosome by the RelA
protein, but few details are available on the RelA-ribosome interaction.
We have now mapped RelA differences in dimethylsulfate modification of
rRNA by RelA binding. With recently solved ribosome structures from the
Noller laboratory as a guide, we have localized perturbations of rRNA
structure on the interior surfaces of both the large and small ribosomal
subunits. The DMS modifications accompanying RelA binding are found to
occur in the region of the CCA end of bound tRNA as well as in the decoding
region of A site bound tRNA, where the codons pair with anticodons. The
conditions for binding examined are not physiological because neither
mRNA nor codon-specified uncharged tRNA are present. Our next goal is
to ask how rRNA conformational perturbations are altered by the presence
of more physiological conditions and how specifically the RelA protein
senses the difference between the binding of codon-specified uncharged
and charged tRNA.
|
PUBLICATIONS
Vinella
D, Cashel M, D'Ari R. Selected amplification of the cell division
genes ftsQ-ftsA-ftsZ in Escherichia coli. Genetics 2000;156:1483-1492.
*Left the laboratory 2001
a Dr. Tom Elliott, Department of Microbiology
and Immunology, West Virginia University Medical School, Morgantown, WV.
b Tanis Hoggs, Institute of Molecular
Biotechnology, Department of Structural Biology and Crystallography, Jena,
Germany.
c Dr. Albert E. Dahlberg, Division of
Biology and Medical Science, Brown University, Providence, RI.
|