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Journal of Bacteriology, April 1999, p. 2663-2665, Vol. 181, No. 8
Waksman Institute and Department of Genetics,
Rutgers, The State University, Piscataway, New Jersey
08854,1 and Institute of Molecular Genetics,
Russian Academy of Sciences, Moscow, Russia2
Received 27 January 1999/Accepted 12 February 1999
We localized five rpoC ( Cellular RNA polymerases (RNAP) are
multifunctional, multisubunit enzymes. Isolated subunits do not possess
RNAP partial functions (e.g., ability to melt DNA or bind nucleotide
triphosphates [26]). Thus, functional sites are formed
by allosteric changes and/or at subunit interfaces upon assembly of
RNAP. Understanding of intra- and intersubunit interactions during
enzyme assembly will be necessary to elucidate the mechanism of RNAP
action. In Escherichia coli, the dimeric Conditional mutations had been used to define sites in The tripartite interaction of Genomic DNA extracted from mutant cells was used to amplify by
PCR the whole rpoC gene. PCR products were cloned in the
pCYB2 expression vector (New England Biolabs). A control plasmid,
pCYB2
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Localization of Escherichia coli rpoC Mutations That
Affect RNA Polymerase Assembly and Activity at High
Temperature
ABSTRACT
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Abstract
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') mutations affecting
Escherichia coli RNA polymerase assembly. The Ts4, XH56,
and R120 mutations changed ' residues conserved throughout
eubacteria; the JE10092 mutation occurred in the hypervariable
region; rpoC1 (TsX) changed a universally conserved residue
and corresponds to yeast rpb1-1. Thus, distinct,
predominantly conserved ' residues participate in interactions
holding RNA polymerase together.
TEXT
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Abstract
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References
subunit
orchestrates RNAP assembly by interacting with the subunit
(8). The 2 subassembly then binds the '
subunit to form the catalytic core, 2'.
that
interact with other RNAP subunits. The rpoA112 (R45A)
mutation weakens the interaction with ; rpoA101 (C191R)
interferes with ' entry into the complex (7). Biochemical
and structural work indicates that Arg45 and
Cys191 directly contact and ', respectively (6,
25). Mutations in and in the yeast counterpart, RPB3, that
affect 2 formation in their respective systems occur
in homologous positions (2), suggesting that the RNAP
assembly pathway has been evolutionarily conserved.
' with 2 does not
easily lend itself to analysis. Consequently, we lack information
about regions of ' involved in RNAP assembly. Eighteen
temperature-sensitive strains with mutations in E. coli
rpoC, which codes for ', were isolated by Miller and
coworkers in 1976 (12). RNAP purified from some of the
mutant strains was temperature sensitive and/or failed to reassemble
after denaturation and renaturation (5, 6). Localization of
these rpoC mutations could pinpoint ' regions important
for assembly. Unfortunately, many original mutations were lost
(14a). We obtained six rpoC assembly
mutants
rpoC110, Ts4, XH56, R120, JE10092, and RpoC1from colleagues
and localized the mutations.
'WT, expressing wild-type rpoC was
also constructed. Five strains, R120, Ts4, JE10092, XH56, and RpoC1,
grew at a restrictive temperature (42°C) when transformed with
pCYB2'WT, confirming that rpoC mutations
caused temperature sensitivity of mutant strains. In contrast, RpoC110
strains obtained from two different sources failed to grow at 42°C in
the presence of pCYB2'WT. Plasmids expressing
rpoCR120, rpoCTs4,
rpoCXH56, rpoCJE10092, and rpoC1 did not support growth of the 397c
(rpoC397) temperature-sensitive strain (3) at
42°C; in contrast, 397c transformed with pCYB2'WT or
pCYB2'110 grew at 42°C. Therefore, we (i) discontinued
further analyses of rpoC110 and (ii) mapped the remaining
mutations by swapping fragments between mutant and wild-type plasmids
and assaying the ability of hybrid plasmids to support 397c growth at
42°C (Fig. 1). In this way, the
determinants of temperature sensitivity were shown to reside in
the 1,010-bp SalI-BspEI rpoC fragment
(R120, XH56, and JE10092), between the engineered NdeI site
at position 1 and the SnaBI site at codon 292 (Ts4), and
between the BspEI site at codon 1213 and the
HindIII site in the vector (RpoC1). These fragments and
the corresponding pCYB'WT fragments were sequenced.
Sequence comparisons revealed single-nucleotide differences between
the wild type and each of the mutants: Ts4 has a GGG (Gly)-to-GAG
(Glu) change at codon 181, XH56 has a CGT (Arg)-to-CAT (His) change at
codon 883, R120 has a GGT (Gly)-to-GAT (Asp) change at codon 900, JE10092 has a GGT (Gly)-to-GAT (Asp) change at codon 1033, and
RpoC1 has a GGT (Gly)-to-GAT (Asp) change at codon 1360. We
conclude that G181E, R883H, G900D, G1033D, and G1360D cause the
temperature-sensitive phenotypes of Ts4, R120, XH56, JE10092, and
RpoC1, respectively. Genetic contexts of these mutations are presented
in Fig. 2.
View larger version (36K):
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FIG. 1.
Localization of the R120 mutation to the
SalI-BspEI fragment of rpoC. (Top) The
E. coli rpoC gene and restriction sites used to construct
hybrid plasmids are shown (numbers indicate codon positions). (Bottom)
Plasmids pCYB2 'WT (labeled rpoC+)
and pCYB'R120 (labeled R120) and the indicated hybrid
plasmids were transformed into strain 397c. The transformants were
streaked on Luria-Bertani-ampicillin plates, and the plates were
incubated at 30 and 42°C overnight. The absence of growth of cells
labeled R120 SalI-BspEI at 42°C indicates that
the R120 mutation is within the 1,010-bp
SalI-BspEI rpoC fragment.
View larger version (28K):
[in a new window]
FIG. 2.
Genetic context of rpoC assembly mutants. The
thick bar represents the 1,407-amino-acid ' subunit of E. coli RNAP. Stippled boxes labeled A to H represent segments of
' highly conserved in evolution (1, 24). The E. coli hypervariable region (24), absent in homologues
from most bacteria, archaea, and eukaryotes, is shown by an open box.
The amino acid sequences of E. coli ' (E.c.)
harboring assembly mutations are expanded underneath. Homologous amino
acid sequences from Haemophilus influenzae
(H.i.), Pseudomonas putida (P.p.),
Helicobacter pylori (H.p.), Mycobacterium
leprae (M.l.), Thermotoga maritima
(T.m.), chloroplasts from tobacco (T.),
Sulfolobus acidocaldarium (S.a.), and yeast RNAP
1, 2, and 3 (Yp1, Yp2, and Yp3, respectively) are also shown. The dots
symbolize identity to the E. coli sequence. Amino acids
changed by assembly mutations, as well as three conserved cysteines
close to segment G, are highlighted by bold typeface. Note that
rpoC1 in E. coli and rpb1-1 in yeast
cause the same substitution.
The Ts4 mutation occurred between ' conserved segments A and B. Gly181 is the most conserved residue in the six-amino-acid
motif, MGAEA, that is present only in eubacterial '
subunits. RNAP purified from Ts4 cells is not temperature
sensitive, but biochemical analysis suggested that 'Ts4
is defective in association with 2 (21).
The XH56 and R120 mutations occurred close to conserved segment G,
involved in transcript elongation and cleavage (14). RNAPR120 is not temperature sensitive but fails to assemble
into active RNAP after denaturation (4).
'XH56 can be assembled into active enzyme in vitro, but
RNAPXH56 activity is temperature sensitive (5).
Further biochemical analysis will be necessary to determine why the
neighboring XH56 and R120 mutations result in different defects.
Residues changed by the XH56 and R120 mutations are strictly conserved
in eubacteria but are absent from archaeal and eukaryotic '
homologues. Three additional neighboring ' residues are conserved in
eubacteria: Cys888, Cys895, and
Cys898. Groups of cysteines can coordinate structural or
catalytic zinc ions in proteins (22). It is tempting to
speculate that Cys888, Cys895, and
Cys898 coordinate RNAP zinc and that the
temperature-sensitive mutations affect RNAP assembly by altering zinc binding.
The JE10092 mutation is a known mutation: E. coli rpoC907
also causes G1033D replacement and affects plasmid replication
(17). Compared to the wild type, a fourfold increase in
pBR322 copy number was observed in JE10092 grown at 39°C (data not
shown). A similar value was reported for RpoC907 (18),
confirming that the JE10092 and rpoC907 mutations are
allelic. RNAPJE10092 is assembly defective in vivo
(15) and is temperature sensitive (23). The
JE10092 mutation occurred in the evolutionarily hypervariable region of
', but in organisms where this region is present Gly1033
is conserved. Our recent analysis shows that the hypervariable region
has only limited ability to tolerate mutations and may be indirectly
involved in RNAP assembly (24).
The rpoC1 mutation was the first conditional mutation in
RNAP to be isolated (11). The mutation changes the
universally conserved Gly in segment H. Another temperature-sensitive
mutation affecting RNAP assembly, rpoC397, occurred upstream
of rpoC1 and resulted in substitution of 52 ' amino acids
distal to position 1355 with 23 unnatural residues (3).
RNAPRpoC1 is extremely temperature sensitive, but normal
activity of RNAPRpoC1 (and RNAP397C) can be
restored by simple addition of purified 'WT under
nondenaturing conditions (3, 16). Thus, mutant subunits bind
2 less tightly, suggesting that segment H forms the
2 binding site.
rpoC1 corresponds exactly to the yeast rpb1-1
mutation (19). Similar to rpoC1,
rpb1-1 makes yeast temperature sensitive, and all
transcription ceases upon temperature upshift. Suppressors of
rpb1-1 map in the conserved segment I of the homologue,
RPB2 (14). In E. coli, opr mutations
suppressing the temperature-sensitive phenotype of RpoC1 were isolated
(10). Mapping of these suppressors is currently underway.
In summary, our data demonstrate that point mutations in '
dramatically interfere with RNAP assembly. Four of five mutations changed evolutionarily conserved residues of ' and may define sites
of intersubunit contacts that hold the RNAP molecule together. Further
in-depth analysis of mutant enzymes and site-specific mutagenesis of
rpoC to generate new assembly-deficient RNAP should clarify
the role of these ' regions in RNAP assembly.
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ACKNOWLEDGMENTS |
|---|
This work was supported by NIH grant GM59295-01 and a Burroughs Wellcome Career Award to K.S.
We are grateful to R. Hayward (University of Edinburgh) for the R120, XH56, and Ts4 strains; to D. J. Jin (NIH) for JE10092; to E. Kalayeva (Moscow Institute of Molecular Genetics) for the T16 (rpoC1) strain; and to Y. Ohnishi (University of Tokushima) and the Yale Genetic Center for RpoC110 strains.
D.M. and E.C.N. contributed equally to this work.
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FOOTNOTES |
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* Corresponding author. Mailing address: Waksman Institute, 190 Frelinghuysen Rd., Piscataway, NJ 08854. Phone: (732) 445-6095. Fax: (732) 445-5735. E-mail: severik{at}waksman.rutgers.edu.
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