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Volume 271, Number 24,
Issue of June 14, 1996
pp. 14572-14583
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Amino Acid Substitutions in the Two Largest Subunits of
Escherichia coli RNA Polymerase That Suppress a
Defective Rho Termination Factor Affect Different Parts of the
Transcription Complex*
(Received for publication, December 18, 1995, and in revised form, March 27, 1996)
Laura M.
Heisler
§,
Guohua
Feng
¶ ,
Ding Jun
Jin
'',
Carol A.
Gross
 and
Robert
Landick
¶
From the Department of Bacteriology, University of
Wisconsin, Madison, Wisconsin 53706, the ¶ Department of Biology,
Washington University, St. Louis, Missouri 63130 and the '' Laboratory
of Molecular Biology, NCI, National Institutes of Health,
Bethesda, Maryland 20892
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES
ABSTRACT
Among the earliest rpoBC mutations
identified are three suppressors of the conditional lethal
rho allele, rho201. These three mutations are
of particular interest because, unlike rpoB8, they do not
increase termination at all -dependent and
-independent terminators. rpoB211 and
rpoB212 both change Asn-1072 to His in conserved region H
of rpoB ( N1072H), whereas rpoC214 changes
Arg-352 to Cys in conserved region C of rpoC ( R352C).
Both substitutions significantly reduce the overall rate of transcript
elongation in vitro relative to wild-type RNA polymerase;
however, they probably slow elongation for different reasons. The
nucleotide triphosphate concentrations required at the T7 A1 promoter
for both abortive trinucleotide synthesis and for promoter escape are
much greater for N1072H. In contrast,  R352C and two adjacent
substitutions ( G351S and  S350F), but not N1072H, formed open
complexes of greatly reduced stability. The sequence in this region of
 modestly resembles a region of Escherichia coli DNA
polymerase I that contacts the phosphate backbone of DNA in
co-crystals. Core determinants affecting open complex formation do not
reside exclusively in  , however, since the Rifr
mutation rpoB2 in also dramatically destabilized open
complexes. We suggest that the principal defects of the two
Rho-suppressing substitutions may differ, perhaps reflecting a greater
role of region H in nucleoside triphosphate-binding and nucleotide
addition and of  region C in contacts to the DNA strands that could
be important for translocation. Although both probably suppress
rho201 by slowing RNA chain elongation, these differences
may lead to terminator specificity that depends on the rate-limiting
step at different sites.
INTRODUCTION
The two largest subunits of Escherichia coli RNA
polymerase,  (Mr 155,063) and (Mr 150,538) are homologous to the largest and
second largest subunits, respectively, of all multisubunit RNA
polymerases (Allison et al., 1985 ; Briggs et al.,
1985; Falkenberg et al., 1987 ; Hudson et al.,
1988 ; Leffers et al., 1989 ; Patel and Pickup, 1989 ). Most
catalytic and regulatory functions of RNA polymerase appear to involve
these subunits. Thus and  are likely to form an enzymatic
platform for RNA synthesis common to all forms of life, making
elucidation of structure/function relationships in these subunits in a
well studied prokaryote an ideal approach to understanding the
mechanism of transcription. Sequence comparisons and recent
crystallographic studies of single subunit DNA and RNA polymerases
reveal broadly similar catalytic regions for both classes of enzyme
(Steitz et al., 1994 ; Pelletier et al., 1994 ;
Beese et al., 1993 ; Sousa et al., 1993 ;
Kohlstaedt et al., 1992 ). Although E. coli RNA
polymerase has proven refractory to study by x-ray crystallography,
owing principally to its large size and multisubunit complexity,
low-resolution electron crystallography of E. coli RNA
polymerase and yeast RNA polymerase II suggests that the multisubunit
RNA polymerases also share this basic architecture, most notably a
central nucleic acid binding channel large enough to encompass the DNA
double helix (Darst et al., 1989 , 1991 ; Polyakov et
al., 1995 ). Most progress in identifying functionally important
regions of the enzyme, however, has come through study of altered
function mutants and sites of cross-linking between the subunits and
nucleotide analogs.
Of the three core subunits, most is known about , largely because it
is the target of a widely studied antibiotic that inhibits
transcription, rifampicin (Rif).1
Rifr mutants in particular have been a focus of
study for decades since they exhibit a number of pleiotropic
phenotypes, most notably effects on pausing and termination (Fisher and
Yanofsky, 1983 ; Jin et al., 1988 ). Several more recent
genetic studies have identified sites throughout rpoB,
generally in evolutionarily conserved regions, that affect pausing and
termination (Landick et al., 1990 ; Tavormina et
al., 1996 ). In addition, substitutions in have been isolated
that suppress substitutions in Rho and NusA, suggesting that is a
site of functional, and possibly physical interaction with these
transcriptional regulators (Guarente and Beckwith, 1978 ; Guarente,
1979 ; Jin and Gross, 1989 ; Ito et al., 1991 ; Ito and
Nakamura, 1993 ). Others have used reverse genetics and high resolution
cross-linking to identify domains in that affect a number of
aspects of polymerase function (Grachev et al., 1987 , 1989 ;
Mustaev et al., 1991 , 1995 ; Kashlev et al., 1990 ;
Lee et al., 1991 ; Martin et al., 1992 ) and thus
suggest the functional importance of highly conserved regions.
Several lines of evidence suggest that the  subunit also is
intimately involved in RNA chain synthesis. Early physical studies
showed that purified  subunit is capable of binding DNA and heparin
(Zillig et al., 1970 ), and several cross-linking studies
have demonstrated that  is in the immediate vicinity of short and
intermediate length transcripts (Hanna and Meares, 1983 ; Dissinger and
Hanna, 1991 ; Borukhov et al., 1991a ), as well the DNA
template (Okada et al., 1978 ; Chenchik et al.,
1982 ). Substitutions in  affecting termination, suppression of
nusA and rho mutants, DNA replication, and phage
growth have been described, although, with a few exceptions, the
particular amino acids affected have not been identified (Nomura
et al., 1984 ; Rasmussen et al., 1983 ; Tanaka
et al., 1983 ; Petersen and Hansen, 1991 ; Robledo et
al., 1991 ). More recently we reported several clusters of
substitutions in conserved regions of  that alter pausing and
termination (Weilbaecher et al., 1994 ),
streptolydigin-resistance was found to result from amino acid
substitutions in one of these clusters that corresponds to the
amanitin-resistance region of the largest subunit of RNA polymerase II
(Severinov et al., 1995 ), and conserved aspartic acid
residues that appear to chelate active-site Mg2+ ions were
identified using the  homolog in yeast RNA polymerase III (Dieci
et al., 1995 ).
To understand the roles of and  in transcription better, we
have studied mutations isolated as allele-specific suppressors of
rho201 by Guarente (1979) and mapped to the rpoBC
locus. These mutations were localized to either rpoB or
rpoC in a previous study and renamed rpoB211,
rpoB212, and rpoC214 (Jin and Gross, 1989 ). Unlike the
rpoB8(Rifr) mutant that was also isolated
as a suppressor of rho201 (Guarente and Beckwith, 1978 ) as
well as independently by several other criteria (reviewed in Jin and
Gross (1988) ), these mutations do not cause general defects in
termination, but rather alter termination only subtly at a few
-dependent and -independent terminators (Jin and
Gross, 1989 ).
To continue this investigation, we identified the precise amino acid
substitutions in rpoB211, rpoB212, and rpoC214,
finding that both rpoB211 and rpoB212
specify N1072H and that rpoC214 specifies  R352C, and
purified both mutant RNA polymerases. A current model of
Rho-dependent termination (Jin et al., 1992 ;
Platt, 1994 ) postulates that the extent of termination is determined by
``kinetic'' coupling between extrusion of the nascent RNA by the
elongating RNA polymerase molecule and movement of Rho along the RNA
toward the complex. That this relationship is the basis of at least
some modes of rho-suppression was demonstrated for
rpoB8( Q513P): Q513P RNA polymerase elongates
at a significantly slower rate than wild type, and its hypertermination
defect is correlated to its elongation rate (Jin and Gross, 1991 ). We
wanted to determine whether a reduction in elongation rate was a
plausible basis of isolation of these RNA polymerase mutants, or if
they suppress rho201 by a different mechanism (such as
disruption of interaction with Rho). If these mutants are defective in
transcription itself, we wanted to ask whether they and Q513P
exhibit similar or distinct transcriptional defects.
Here we show that N1072H and  R352C elongate more slowly in
vitro and argue, based on their effects on abortive initiation and
open complex stability, that they probably do so by principally
affecting distinct interactions of RNA polymerase with different
components of the transcription complex.
MATERIALS AND METHODS
Chemicals and Enzymes
Adenosyl 5 -uridine (ApU) and
isopropyl- -D-thiogalactopyranoside were from
Sigma. -ANS-UTP was a kind gift of M. Thomas
Record. NTPs were purchased from Boehringer Mannheim or Pharmacia (high
performance liquid chromatography-purified). Radioactive
[ -32P]UTP (800 Ci/mmol) and [ -32P]ATP
(3000 Ci/mmol) were obtained from DuPont NEN,
[ -32P]CTP (410 Ci/mmol) was from Amersham.
Polynucleotide kinase and restriction enzymes were from New England
BioLabs. Sequenase Version 2.0 was purchased from U. S. Biochemical
Corp. AmpliTaq was obtained from Perkin-Elmer Instruments and Tfl
polymerase from Epicentre.
Single-stranded DNA-agarose and Q-Sepharose FF were purchased from
Pharmacia and S-300HR from Sigma. Magic Miniprep
columns were from Promega. Nitrocellulose filters (BA85, 24 mm, 0.45-m
pore size were purchased from Schleicher and Schuell.
Bacterial Strains, Plasmids, and Bacteriophage
The bacterial strains used in this study are E. coli
K12 derivatives and are listed in Table I. Plasmids and
bacteriophage used in this study are listed in Table II.
T7 D111 phage stock was from Studier and phage DNA was prepared as
described (Studier, 1975 ).
Bacterial Techniques
Cells were grown in L broth (Difco Laboratories). LB plates were
prepared as described (Miller, 1992 ). Antibiotics were added at the
following concentrations, unless otherwise indicated: ampicillin (Amp)
50 µg/ml; kanamycin (Kan) 30 µg/ml; tetracyline (Tet) 20 µg/ml.
Competent cells were prepared by CaCl2 shock (Mandel and
Higa, 1970 ), or by electroporation (Dower et al., 1988 ) and
were stored at 80 °C. Electroporation was performed with a Bio-Rad
GenePulser using 0.2-cm Bio-Rad GenePulser cuvettes.
Marker rescue of the cold-sensitive growth phenotype of
rpoC214 was as described (Jin and Gross, 1989 ) using
plasmids pRL611-pRL617 (Table II).
DNA Techniques
Restriction enzyme digests and agarose gel electrophoresis were
as described by Sambrook et al. (1989) . Plasmid DNA
purification was as described by Sambrook et al. (1989) or
by purification on Qiagen or Magic Miniprep columns.
DNA for Nitrocellulose Filter Binding
End-labeled DNA was
prepared by PCR essentially as described by Dombroski et al.
(1992) , using 30 cycles of 1 min at 95 °C, 1 min at 55 °C, and 1 min at 72 °C and purifying the DNA by elution from nondenaturing
10% polyacrylamide gels as described by Sambrook et al.
(1989) . Quantitation of labeled DNA was as described by Dombroski
et al. (1992) .
Nitrocellulose Filter Binding Assays
DNA binding was
determined by measuring retention of polymerase-DNA complexes on
nitrocellulose filters. All data represent the average of at least 3 duplicate experiments, unless stated otherwise. Binding buffer (BB: 50 mM KCl, 40 mM Tris-HCl, pH 7.9, 0.1 mM EDTA, 1 mM -mercaptoethanol) was made
according to the specifications of Pfeffer et al. (1977) for
the heparin competition experiments; for the excess DNA competition
experiments, the KCl concentration was increased to 100 mM
(see ``Results''). The composition of the wash buffer (WB) was
identical to that of BB, except that KCl was omitted. The experimental
procedure was essentially as described (Pfeffer et al.,
1977 ; Hinkle and Chamberlin, 1972 ). Briefly, 0.25-1 nmol of
end-labeled DNA ( 2 × 105 cpm/pmol) was preincubated
with a 4-fold molar excess of active RNA polymerase in 1 × BB at
37 °C for 10 min. Duplicate 25-µl aliquots were filtered and
washed with 250 µl of 1 × WB prior to the addition of competitor
(t = 0). The reaction mixture was split into two tubes:
competitor was added to one, the other was the no competitor control.
Duplicate aliquots were removed at the indicated times after competitor
(or at regular intervals in the no competitor controls) and filtered at
10-15 mm Hg. Filters were washed once with 250 µl of 1 × WB,
dried under an infrared heat source, and subjected to liquid
scintillation counting. Background retention was typically <3% of the
total counts and was subtracted from the experimental values.
Experimentally determined dissociation rates were corrected for
baseline dissociation when necessary (calculated from the control
reactions described above) by subtracting the dissociation rate of the
no-competitor control.
PCR Amplification and Sequencing of Chromosomal
Mutations
Marker-rescue of the cold-sensitive growth phenotype of
rpoC214 was done as described for rpoB211 and
rpoB212 (Jin and Gross, 1989 ). Mutations rpoB211,
-212, and rpoC214 were sequenced by PCR
amplification of the identified regions. Primers were made flanking the
regions, and PCR reactions were essentially as described above.
Double-stranded PCR product was gel purified from 2% agarose gels in
Tris acetate-EDTA buffer and eluted according to Sambrook et
al. (1989) . Purified fragment was sequenced according to the
Sequenase procedure for sequencing double-stranded plasmid DNA.
Biochemical Techniques
Purification of RNA Polymerase
Strains CAG 14179, 14180, and 14181 (Table I) were grown in LB + antibiotics in a 6-liter
fermenter (University of Wisconsin Pilot Plant) and harvested in
mid-log phase (OD600 1). Protein purification of
25 g of cells (wet weight) was essentially as described by Lowe
et al. (1979) and Burgess and Jendrisak (1975) , with several
modifications: cell suspensions were sonicated in several 30-s
intervals instead of being subjected to high speed sheer (described in
Thompson et al. (1992) ); the TGED + 1 M NaCl
polymin P eluate was precipitated with ammonium sulfate as described
(Burgess and Jendrisak, 1975 ) and resuspended in TGED to a conductivity
equivalent to 0.25 M NaCl (in TGED), then loaded on a 10-ml
single-stranded DNA-agarose column. Holoenzyme, eluted in TGED + 1 M NaCl, was precipitated, resuspended, and loaded on a
120-ml S-300 HR sizing column. The holoenzyme fractions were pooled and
eluted from an 8-ml Q-Sepharose FF column in TGED over a gradient from
0.25 to 0.75 M NaCl; the polymerase fraction typically
eluted at 0.5 M NaCl.2 Highly
pure fractions of holoenzyme (>95%) were pooled and dialyzed against
polymerase storage buffer (TGED, containing 50% glycerol + 0.1 M NaCl) and stored in aliquots at 80 °C.
RNA Polymerase Micropreparations
Crude preparations
of  S350F and  G351D RNA polymerases from strains RL676
were from strain RL676 transformed with appropriate plasmids
(Weilbaecher et al., 1994 ) using a variation of the RNA
polymerase microprep protocol of Gross et al. (1976)
described by Tavormina et al. (1996) . Similar preparations
of G1074D and H526Y (rpoB2) RNA polymerases were
generously provided by P. Tavormina.
Polymerase Activity Assays
Activity of polymerase
preparations was determined both by the Chamberlin T7 D111 assay and
by the fluorescence abortive initiation assay of Bertrand-Bergraff
et al. (1994), using -ANS-UTP (data not shown). The
results of both assays indicated that the wild-type preparation was
45-50% active and the N1072H and  R352C preparations were
between 20 and 25% active. Q513P RNA polymerase was purified from
strain CY15023 (Horn and Yanofsky, 1981 ) by the method of Hager
et al. (1990) and determined as described above to be
~25% active.
In Vitro Transcription Elongation Assays
T7 D111 assays
(Chamberlin et al., 1979 ) were carried out at 30 °C and
at 0.4 mM NTPs. The PR-tR1
assays, originally described by Chen and Richardson (1987) , were done
according to the modifications described by Heisler et al.
(1993) . Reactions were carried out at 37 °C; 1 × NTPs equals 0.2 mM ATP, CTP, GTP, and 0.02 mM UTP.
Abortive Initiation Apparent NTP Km Assays
We used
an abortive initiation assay similar to that originally developed by
McClure et al. (1978) , a more detailed description of which
will be published separately.3 0.25 pmol of
the 142-bp fragment of pCL185 (T7A1 promoter; Feng et al.
(1994) ; see Table II) was incubated with 1 pmol of active RNA
polymerase in 1 × T7A1 buffer (20 mM Tris acetate, pH 8.0, 20 mM NaCl, 14 mM MgCl2, 14 mM -mercaptoethanol; Levin et al. (1987) ) and
25 µg of acetylated bovine serum albumin/ml at 37 °C for 10 min.
Reactions were initiated by the addition of ApU and
[ -32P]CTP (final specific activity 6 Ci/mmol) at
the concentrations indicated. Aliquots were withdrawn at 2-min
intervals (from 2 to 10 min) into a 4-fold excess of transcription stop
buffer (final concentration, 1 × TBE, 3.5 M urea, 0.025%
xylene cyanol, 0.025% bromphenol blue). 4.5-µl samples were
electrophoresed on 15% denaturing PAGE, and wet gels were exposed to a
Molecular Dynamics PhosphorImager screen at 80 °C to prevent
diffusion of the bands. The results were quantitated on a Molecular
Dynamics PhosphorImager and normalized to 14C standards.
Apparent substrate constants for nucleotides were determined assuming a
random-order, rapid-equilibrium mechanism (Siegel, 1975 ). Since only
the pathway in which NTP-binding preceded ApU-binding was significant,
only the apparent equilibrium constants for binding NTP first
(KNTP) and for binding ApU second
(K ApU) are reported. The apparent equilibrium
constant for binding GTP (KGTP) was determined
by varying [GTP] at high [ApU].
Promoter Escape
Transcription assays and PAGE were
essentially as described for abortive initiation assays, except that
ApU was added at 0.5 mM in all reactions, and GTP, ATP,
[ -32P]CTP, and 3 -deoxy-UTP were added at the
concentrations indicated. Aliquots were withdrawn into stop solution at
the times indicated and electrophoresed on 15% urea-polyacrylamide
gel, as described above. Control experiments done in the absence of
3 -dUTP (data not shown) demonstrated that the results were unchanged
in the presence of the chain terminating NTP, except that there was no
readthrough of the 17 nucleotide product (see also Sagitov et
al. (1993) ). The identities of the products as indicated in Fig. 4
were confirmed by Feng et al. (1994) using 5 -AUC, 5 -AUCG,
and 5 -AUCGA generated in transcription reactions with ApU and
[ -32P]CTP only, ApU, CTP, and
[ -32P]GTP only, or ApU, CTP,
[ -32P]GTP, and 3 -deoxy-ATP only, and showing (i) that
they co-migrated with the abortive products as assigned in Fig. 6 and
(ii) that addition of a 5 PO4 increased the mobility of
these short transcripts to that of 5 -pAUC, 5 -pAUCG, and 5 -pAUCGA,
confirming that they were initiated with ApU. Note that the mobilities
of short RNAs (less than 5 nucleotides in length) lacking a 5
PO4 are reversed on a 15% urea-polyacrylamide gel (Feng
et al., 1994 ; Krummel, 1990 ; Levin et al., 1987 ;
Cai and Luse, 1987 ).
Fig. 4.
Escape from the T7 A1 promoter by WT,
N1072H, and  R352C RNA polymerases. Transcription reactions
were performed as described under ``Materials and Methods.''
Numbers above the lanes indicate the times at which aliquots
were removed into stop buffer. Samples were electrophoresed on 15%
denaturing PAGE, as described under ``Materials and Methods.''
A, reactions at 5 µM. Sizes of transcription
products are indicated based on control experiments, described
elsewhere (Feng et al., 1994 ). Note that small transcripts
without 5 PO4 migrate aberrantly in 15% PAGE, such that
the sizes of the transcripts appear to be reversed (see Levin et
al. (1987) and ``Materials and Methods''). B,
reactions at 50 µM. The specific activity of
[ -32P]CTP was the same as for 5 µM
reactions shown in panel A (see ``Materials and
Methods'').
Fig. 6.
NTP concentration dependence of productive
and abortive transcripts as a function of [NTP] for WT, N1072H,
and  R352C RNA polymerases. Transcription reactions were
performed and quantified as described in the legend to Fig. 5 and under
``Materials and Methods.'' The amounts of productive and abortive
RNAs present at 2 min were used to prepare these plots, which represent
the averages of two experiments, that typically differed by less than
30%. Ten fmol is equivalent to 1 transcript per input template.
A, total dU17 for WT ( ),  R352C ( ), and N1072H
( ) RNA polymerases. B, ratio of total abortive
transcripts to dU17 (productive) transcripts for WT ( ),  R352C
( ), and N1072H ( ) RNA polymerases.
RESULTS
rpoB211, -212, and rpoC214 Are Located in Conserved Regions
Important in Transcription
Jin and Gross (1989) describe the
localization of rpoB211 and rpoB212 to a 650-bp
region in the C-terminal portion of rpoB. We amplified this
region by PCR and determined the sequence changes of these chromosomal
mutations (see ``Materials and Methods''). These two mutations
contain identical single base pair substitutions and may have
been isolated as siblings in the original selection, or as two
independent isolates (Guarente, 1979 ). The amino acid change, N1072H,
occurs in conserved region H, which has been subjected to
extensive mutational analysis (Sagitov et al., 1993 ; Fig.
1). Mutations in this region affect many aspects of
transcription.
Fig. 1.
Conserved regions of and  subunits
and locations of significant features and substitutions examined.
Conserved regions A-I in and A-H in  are indicated by
shading and with letters. Regions less strongly
conserved are indicated by shading alone. N1072H (rpoB211,
212) in is located in region H and R352C (rpoC214)
in  is located in conserved region C. In addition to the regions of
sequence conservation, the following relevant sites are noted (N
terminus to C terminus). For , regions in which mutations conferring
resistance to rifampicin have been isolated (amino acids 142-146;
Severinov et al. (1993) and Lisitsyn et al.
(1984) ; amino acids 503-533, 560-564, 687; Jin and Gross (1988) and
Severinov et al. (1993) ); the streptolydigin-resistance
region (amino acids 543-546: Heisler et al. (1993) and
Severinov et al. (1993) ); a deletable hinge region (amino
acids 940-1040; Borukhov et al. (1991b) ); sites targeted by
the 5 end of a cross-linkable nucleotide analog (Lys-1065 and
His-1237; Grachev et al. (1989) and Mustaev et
al. (1991) ). For  , a region of weak sequence similarity to a
DNA-binding region in DNA polymerase I (342-387; Allison et
al. (1985) ); the streptolydigin-resistance region in  which
corresponds to the amanitin-resistance region of the largest subunit of
polymerase II (Severinov et al., 1995 ); a site to which the
3 end of the nascent RNA has been cross-linked (between amino acids
932 and 1020; Borukhov et al. (1991a) ).
Prior to this study, rpoC214 had not been mapped within
rpoC. Marker rescue of the cold-sensitive phenotype of this
mutant by plasmids containing internal fragments of rpoC
(see ``Materials and Methods'') allowed us to localize this mutation
to an interval of rpoC between amino acids 305 and 695 (region 2; Weilbaecher et al. (1994) ). PCR amplification and
sequencing identified an amino acid change from Arg to Cys at amino
acid 352. This amino acid lies in a region of weak sequence similarity
to a putative DNA-binding region in the Klenow fragment of DNA
polymerase I (Ollis et al., 1985 ) and is adjacent to several
rpoC mutations isolated for their effects on termination
(Weilbaecher et al. (1994) ; Fig. 1).
N1072H and  R352C Decreased Elongation Rate in
Vitro
We purified the mutant enzymes according to
variations in the procedures of Burgess and Jendrisak (1975) and Lowe
et al. (1979) (see ``Materials and Methods'') and examined
their rates of elongation on T7 D111, a deletion of T7 phage DNA that
contains only one E. coli E 70 promoter, T7 A1
(Studier et al., 1975). In this assay, wild type RNA
polymerase reached the T7 Te terminator 6.5 min after addition of
nucleotides and heparin or at 15 nucleotides/s, whereas N1072H and
 R352C elongated RNA at 6 and 8 nucleotides/s, respectively (Fig.
2). We also isolated RNA polymerase from two
rpoC mutants containing amino acid changes adjacent to
R352C: S350F and G351S (Weilbaecher et al., 1994 ). These
mutant enzymes elongated at rates comparable to that of  R352C
( S350F, 10.5 nucleotides/s and  G351S, 8 nucleotides/s; data not
shown).
Fig. 2.
Transcript elongation of WT, N1072H, and
 R352C RNA polymerases on a T7 D111 template. Transcription
on the T7 D111 template was assayed as described (Chamberlin et
al., 1979 ). Incorporation of [ -32P]UTP is plotted
as a function of time after initiation of a single round of
transcription. The intercept of the two slopes indicates the time
required for RNA polymerase to reach the T7te terminator located 6160 nucleotides downstream of the T7A1 promoter. WT ( ), N1072H ( ),
and  R352C ( ).
To confirm the elongation defect of mutants N1072H and  R352C, we
tested transcription on a Pr-tR1 template.
This template contains a number of well characterized pause sites in
addition to the tR1 terminator (Chen and Richardson,
1987 ). At 1× NTPs (0.2 mM), wild type reached the
terminator by 2 min, while N1072H required 4× NTPs and  R352C,
2× NTPs, to match the transcription rate of wild type at 1× NTPs
(Fig. 3). Neither mutant exhibited fundamental
differences in the pattern of pausing at any concentration of
nucleotides (the patterns are far more similar that different,
especially since apparently new minor pauses in the mutant lanes may be
difficult to detect in the 0.5-min wild-type sample; Fig. 3). Thus, the
reduced elongation rates of these mutants result from slower elongation
at pause sites that also are recognized by the wild-type enzyme, rather
than recognition of new pause sites. Furthermore, these defects in
elongation rate are magnified at lower NTP concentrations and do not
appear to be template-specific since the magnitudes of their elongation
defects were similar on the two templates studied. In these regards,
both mutants resemble Q513P(rpoB8) ( Q513P elongates at
~0.25 the rate of wild-type; Jin and Gross (1991) ).
Fig. 3.
Transcript elongation from PR
to tRI of WT, N1072H, and  R352C RNA
polymerases. Transcription reactions were performed as described
under ``Materials and Methods'' with 1, 2, or 4 × NTPs (1× = 0.2 mM ATP, CTP, GTP, and 0.02 mM
[ -32P]UTP). The reactions were sampled at the times
indicated above the lanes. The positions of the readthrough
(RT) transcript and the tR1 termination sites
I (at nucleotide 288 relative to the start site of transcription), II,
and III are indicated, as are the locations ( ) of previously
characterized pause sites (Chen and Richardson, 1987 ).
N1072H and  R352C Increased the Apparent Km for both
Priming and Substrate Nucleotides during Initiation
In principle,
the defects in elongation we found could either reflect a fundamental
catalytic defect, which should be evident at all nucleotide addition
events, or might be specific to the elongation phase of transcription,
such as would be the case if they affected movement of the DNA through
the enzyme. In an attempt to distinguish these possibilities, we
examined the mutant RNA polymerases using a steady-state abortive
initiation assay first described by McClure et al. (1978) .
This assay takes advantage of the cycling of RNA polymerase in an open
complex when the presence of a limited set of nucleotides forces the
enzyme to synthesize and release a single short transcript (usually a
dimer or trimer) repetitively. By restricting nucleotide addition to a
single new phosphodiester bond in the open complex, we hoped to learn
both whether the defects affected the catalytic center and if they also
were manifest during initiation. We measured the synthesis of the
trimers ApUpC or ApUpG on the T7A1 promoter (or a single bp variant, C + 3 G) by systematically varying the concentrations of the priming
dinucleotide ApU and the substrate NTP (either CTP or GTP; see
``Materials and Methods''). Double reciprocal plots of reaction
velocity versus substrate concentration were linear over the
entire range tested, indicating that there was no substrate activation
or inhibition (data not shown). However, the kinetic parameters
determined must be regarded as apparent substrate constants,
rather than true dissociation constants for binding of ApU and NTPs
since we cannot be certain the reactants bound in rapid equilibrium
(see McClure et al., 1978 ; Smagowicz and Scheit,
1978 ).3 On both templates, the mutant RNA polymerases
exhibited substantially high apparent substrate constants for both ApU
primer and NTP substrates (Table III).
KApU for wild-type was ~0.325 mM,
in agreement with values determined for KATP on
this promoter (Smagowicz and Scheit, 1978 ). Both mutants yielded
slightly increased KApU values (~1
mM; Table III), suggesting that these amino acid changes
may somewhat perturb the primer sub-site of the catalytic center (site
i, see Fig. 8; Erie et al. (1992) ), either
directly or indirectly.
Fig. 8.
Schematic model of the structure of a
transcription complex. The four regions indicated by the
letters A-D correspond to possible parts of the
transcription complex that might be affected by amino acid
substitutions in the or  subunits that alter chain elongation
(see ``Discussion''). The non-template strand is depicted as extruded
from the transcription complex and reannealing with the template strand
just after it emerges from the complex to reflect the fact that the
position that exoIII stops when digesting the template strand
corresponds to the position of the non-template strand that becomes
accessible to modification by the single-strand specific reagents
KMnO4 or diethylpyrocarbonate (see Lee and Landick, 1992 ;
Lee et al., 1994 ). A, contacts to single-stranded
RNA or DNA in exit channels. B, contacts to the RNA or
template DNA strands that align them in the active site. Whether or not
the RNA and DNA are extensively paired in this region (e.g.
8-12 bp) is controversial and not important to our analysis here.
However, several results suggest their relationship to polymerase
differs here from that in region A (see Lee and Landick, 1992 ; Borukhov
et al., 1993 ; Feng et al., 1994 ; Lee et
al., 1994 ; Nudler et al., 1994 ; Chan and Landick, 1994 ;
Wang et al., 1995 ; Chamberlin 1995 ; Zaychikov et
al., 1995 ). C, catalytic center consisting of two
subsites: i, for RNA terminus and i+1, for
NTP-binding site (see Erie et al., 1992 ). D,
duplex DNA-entry channel and helix-unwinding site.
In agreement with determinations on this and other promoters, the
wild-type KNTP values were approximately an
order of magnitude smaller than KApU
(KCTP = 47 µM,
KGTP = 80 µM, Table III).
Although both the and  substitutions increased these values,
interestingly, they were approximately 6-fold greater than wild-type
for N1072H and about 2-3-fold greater for  R352C, depending on
the nucleotide in question (Table III).
Thus, the abortive initiation assay uncovered at least one component of
the transcriptional defect of these mutant RNA polymerases, namely a
decrease in apparent affinities for both priming nucleotide and
substrate NTP that is consistent with their reduced elongation rate. We
suspected, however, that the mutants might affect other aspects of RNA
polymerase function because, although N1072H has a somewhat greater
defect in elongation rate and KNTP during
abortive initiation, the growth rate of  R352C is slower than that
of N1072H. If elongation were the only aspect of transcription
affected, N1072H would be the more deleterious allele. Furthermore,
 R352C demonstrated a more severe hypertermination phenotype
in vivo (Jin and Gross, 1989 ), suggesting that its reduced
elongation rate is not the sole determinant in its suppression of the
rho-201 phenotype.
The Mutants Differed in the NTP Concentration Required for
Efficient Promoter Escape
Promoter escape, or the transition from
abortive to productive synthesis, is another component of the
transcription cycle that is sometimes altered by amino acid changes in
RNA polymerase. Reductions in promoter escape appear to be correlated
with increased pausing and a reduced elongation rate (Kashlev et
al., 1990 ; Sagitov et al., 1993 ). Interestingly, during
initial assays of the N1072H and  R352C RNA polymerases, we found
that both were defective in forming halted elongation complexes at
position 16 of a T7 A1 promoter template at low nucleotide
concentrations (pCL185 G16 complexes; data not shown; see Feng et
al. (1994) ). To characterize the NTP concentration dependence of
promoter escape, we monitored the appearance of abortive and productive
transcripts as a function of time in the presence of ApU, 3 -dUTP, and
several different concentrations of ATP, CTP, and GTP (see ``Materials
and Methods''). These conditions limited productive transcription to
the formation of a dU17 complex. Representative time courses for the
wild-type and two mutant polymerases at low (5 µM) and
high (50 µM) NTP concentrations are shown in Fig.
4 and complete quantified data from these experiments in
Fig. 5. Productive initiation is reflected by appearance
of the dU17 transcript, whereas abortive initiation yielded three short
RNAs, ApUpC (the major product), ApUpCpG, and ApUpCpGpA (Fig. 4; see
``Materials and Methods'' and Feng et al. (1994) ). Other
bands between the abortive and full-length products are most likely
paused intermediates (Krummel and Chamberlin, 1989 ), since they chased
through the time course of the experiment; these have not been included
in the quantification. We first discuss the transition to productive
initiation exhibited by wild-type polymerase and then consider how the
mutants alter this pattern.
Fig. 5.
Amounts of productive and abortive
transcripts produced by WT, N1072H, and  R352C RNA
polymerases. Graphic representation of the accumulation of
dU17-mer and total abortive products in the experiment shown in Fig. 4
were determined by quantitiation on a Molecular Dynamics PhosphorImager
(see ``Materials and Methods''). Filled boxes, dU17-mer;
open boxes, total abortive products. A, wild-type
RNA polymerase at 5, 10, 20, and 50 µM NTPs (constant
specific activity of [ -32P]CTP). B,
N1072H RNA polymerase as in A. C,  R352C RNA
polymerase, as in A.
Quantification of the accumulation of productive and abortive products
by wild type reveals that the transition from abortive to productive
initiation is a time-dependent process (Fig.
5A). The lag observed in attaining the plateau level
reflects the time required for escape from abortive cycling; with each
round of initiation, a certain fraction of complexes clears the
promoter. This switch between abortive and productive mode is
demonstrated graphically by the biphasic accumulation of abortive
transcripts. At early times (often <10 s), accumulation of abortive
transcripts was rapid due to the relatively large number of complexes
engaged in synthesizing abortive products. The inflection points in the
accumulation of abortive products and full-length transcripts
correspond to the point at which the limit of productive initiation was
reached (less than 1 mol of transcript/mol of template, indicating that
transcription has been limited to a single round). The small amount of
abortive transcript that continued to be synthesized at a constant rate
beyond the inflection point probably arose from a subpopulation of
templates that somehow form another open complex at high ratio of
active RNA polymerase to DNA (4:1 in our experiments) or from a
subpopulation of polymerase molecules that are slow to clear the
promoter. Kubori and Shimamoto (1996) recently reported detection of
the latter class of complexes using heparin to block initiation by
multiple polymerases. However, rigorously establishing that
initiating complexes can become trapped in abortive mode will require
showing unambiguously that the template DNA does not harbor a second
polymerase molecule that inhibits promoter escape by the first.
Nucleotide concentration influences the transition to productive
synthesis in two ways, which are probably not independent. First, it
takes longer to make the transition to productive transcription at
lower [NTP] (Fig. 5, compare 5 and 50 µM). Second, many
more abortive transcripts are made per productive transcript at lower
[NTP]. These two points are demonstrated graphically in Fig.
6. The transition to productive mode is efficient only
at higher nucleotide concentrations, requiring the production of >3
abortive transcripts/productive transcript at the lowest nucleotide
concentration (5 µM; Fig. 6A). However,
essentially every polymerase-promoter complex eventually made a
productive transcript, even at the lowest nucleotide concentration (5 µM, Fig. 6B).
N1072H is altered in its nucleotide dependence for the abortive to
productive transition. This mutant dramatically over-synthesized
abortive products (Fig. 5B) relative to the wild type (Fig.
5A). At the lowest nucleotide concentrations, N1072H
synthesized approximately 60 abortive products for every productive
product, which is more than an order of magnitude greater than the
abortive/productive ratio of wild-type polymerase (Fig. 6A).
As a consequence, N1072H was significantly slower than wild type in
making the transition to productive initiation (compare Fig. 5,
panels A and B). Indeed, at 5 µM
NTP only one-fourth of the mutant complexes (as compared to wild type)
succeeded in making the productive transcript (Fig. 6B). We
note that the fraction of polymerases repetitively engaged in producing
abortive transcripts at low NTP concentrations is not a dead-end
population; addition of high nucleotide concentrations as much as 10 min after the reaction is started allows their transition to productive
initiation (data not shown). These aberrant responses of N1072H were
ameliorated by higher nucleotide concentrations; higher NTP
concentrations increased the fraction of polymerases that make
productive transcripts (Fig. 6A) and decreased the ratio of
abortive to productive transcripts (Fig. 6B) such that at
the highest NTP concentration, the fraction of N1072H complexes that
made productive transcripts approached that made by the wild-type
complexes.
 R352C is also defective in the abortive to productive transition
(Fig. 5C); however, in this case the effect of increasing
nucleotides is less straightforward. At low NTPs, a smaller fraction of
mutant than wild-type polymerases made the transition to productive
initiation (5 µM, Fig. 6A), and the
abortive/productive ratio for the mutant was slightly greater than for
wild type (Fig. 6B). Although these deficiencies were
slightly ameliorated by increased nucleotide concentrations, much of
the defect of  R352C for promoter escape did not appear to be
responsive to increased nucleotides (Fig. 6A). We return to
this point under ``Discussion.''
Mutations in rpoC and in the Rifr Region of rpoB Cause
Defects in Promoter Binding
The rpoC mutations we
investigated lie in a region of weak sequence similarity to a region in
E. coli DNA polymerase I that may be in the vicinity of the
DNA-binding channel. Because of the possible impact of DNA-binding
defects on termination as well as on the promoter escape defect noted
above, we examined the DNA-binding properties of these mutants. We used
nitrocellulose filter binding to measure stability of RNA polymerase
binding to an end-labeled DNA fragment containing the T7 A1 promoter.
Chamberlin and co-workers (Pfeffer et al., 1977 ) have
demonstrated that polymerase-promoter complexes at this promoter
exhibit very different kinetics of dissociation depending on the choice
of competitor; T7 A1 complexes are extremely sensitive to heparin, most
likely owing to invasion of the open complex by the polyanion, while
these same complexes are stable in the presence of passive competitors
such as excess DNA (Hinkle and Chamberlin, 1972 ). In light of these
observations, we examined the properties of wild-type and mutant
complexes in the presence of each type of competitor. To test for
possible correlations with an altered termination phenotype, we also
tested the stability of complexes formed by Rifr mutants
with known termination defects: Q513P(rpoB8), which
hyperterminates on all terminators examined, and
H526Y(rpoB2), which hypoterminates.
We present the results of the DNA-binding properties of wild type and
mutants using heparin as a competitor in Table IV,
second column. Results of a representative set of experiments are shown
in Fig. 7. As demonstrated by Pfeffer et al.
(1977) , the wild type complexes were disrupted with a
t1/2 of approximately 30 min in the presence of 10 µg/ml heparin (Table IV). The mutants examined fell into 3 categories
with respect to their stability in this assay. N1072H and G1074D,
another elongation defective mutant in region H (Tavormina et
al., 1996 ), exhibited t1/2 values that are only
slightly decreased relative to wild type (less than 2-fold faster),
consistent with their primary defect being on the process of nucleotide
addition. Two of the mutations in rpoC,  R352C,
 S350F, as well as H526Y, caused pronounced changes in
t1/2 of disruption, reducing the stability of the
binary complex by approximately a factor of 5. We will discuss the
implications of the magnitude of the defect for H526Y below.
Q513P and  G351S exhibited intermediate defects
(t1/2 approximately twice as fast as wild type).
 G351S, located between the two rpoC mutants that had a
large effect in this assay, exhibited a t1/2 that is
similar to that seen for Q513P.  G351S may be an amino acid
substitution that does not significantly affect RNAP:DNA binding. That
both Rifr mutants affect DNA binding, and that it is
the hypoterminating allele H526Y that is less stable, suggests that
there is no straightforward correlation between promoter binding and
termination. The mutants that have a large effect in this assay,
however, may profoundly affect the architecture of the open complex
such that it is more susceptible to invasion by heparin.
Fig. 7.
Dissociation of open complexes formed by WT,
N1072H, and  R352C RNA polymerases at the T7 A1 promoter.
RNA polymerase was incubated with the end-labeled 142-bp fragment of
pCL185 containing the T7A1 promoter (see ``Materials and Methods'')
at 37 °C for 10 min prior to the addition of heparin to a final
concentration of 10 µg/ml or poly(dA-dT) to 10 µg/ml. Aliquots were
removed (in duplicate) and filtered through nitrocellulose filters at
10 mm Hg vacuum pressure. Filters were washed once with 250 ml of 1 × wash buffer, dried, and counted. Background was subtracted from each
measurement. Averages of at least 3 duplicate experiments (except where
noted) are presented in Table III with standard deviations. , WT;
, N1072H; ,  R352C. A, dissociation of T7 A1
promoter open complexes in the presence of heparin. B,
dissociation of T7 A1 promoter open complexes in the presence of
poly(dA-dT).
Qualitatively similar results were seen when approximately 10 µg
poly(dA-dT)/ml was used as a cold competitor (Table IV, third column;
also Fig. 7). When these experiments were done under the same solution
conditions as the heparin challenge experiments, we observed much
slower dissociation rates for wild type than those measured when
heparin was used as competitor, consistent with the hypothesis that
heparin actively invades the open complex, while the DNA competitor
acts as a trap for enzyme molecules that dissociate (data not shown).
Because of the high degree of inaccuracy in these measurements, we
increased the salt concentration (from 50 to 100 mm), thus increasing
Kd (Hinkle et al., 1972 ). Again, the
mutants fell into essentially the same classes, with the same two
rpoC mutants exhibiting the most profound effects on
Kd, the region H mutant showing little deviation
from the wild-type value, and Q513P and  G351S falling between
these two extremes (Table IV, third column).
DISCUSSION
We identified the amino acid substitutions of three mutant alleles
of rpoB and rpoC that were originally isolated by
Guarente (1979) as suppressors of rho201. rpoB211 and
-212 encode a change from Asn to His at amino acid 1072 in
conserved region H of rpoB, and rpoC214 encodes a
change from Arg to Cys at amino acid 352 in conserved region C of
rpoC. Unlike another suppressor of rho201,
rpoB8( Q513P), neither N1072H nor  R352C greatly
increase termination at most -dependent and
-independent terminators, but both appear to be somewhat specific
for the -dependent terminator, trpt , used
for their isolation. Furthermore, all three of the
rho201-suppressing mutants, Q513P, N1072H, and
 R352C fail to suppress another conditional lethal rho
mutation, rho-15 (Das et al., 1978 ), whereas
another class of polymerase mutants, is specific for rho-15
(Jin and Gross, 1989 ). We have purified the N1072H and  R352C
mutant enzymes and investigated their properties in vitro.
Our most important findings are that (i) although their phenotypes
might have been consistent with specific interactions at
trpt , N1072H and  R352C exhibit general defects in
elongation and transcriptional activity that are consistent with their
compensating for a reduced activity of Rho by slower elongation, (ii)
the precise nature of the defects caused by these substitutions (and
thus the likely role in transcription of the corresponding conserved
regions of the and  subunits) differ; (iii) the specificity of
N1072H and  R352C for trpt and rho201 most
probably reflect differences in the rate-limiting steps for termination
in these cases that are affected differently by the particular
structural perturbations caused by these two substitutions. To discuss
these points, we refer to a simple diagram of the structure of a
transcription elongation complex (Fig. 8).
N1072H and Conserved Region H in May Be Located Near the
Catalytic Center
Our studies of N1072H are consistent with the
idea that conserved region H may be in the proximity of the catalytic
center (C, Fig. 8). Our failure to detect significant effects of this
mutation on the stability of the open complex (Table IV; Fig. 7) and of
the ternary complex (data not shown) suggests that DNA binding is not
perturbed in this mutant. Although this region could interact with the
nascent transcript, at least some of the mutant defect is manifest even
at the initial nucleotide binding step. The initiating N1072H mutant
encounters the same decision points as the wild-type enzyme but
releases abortive transcripts much more often, resulting in a higher
ratio of abortive to productive transcripts and inefficient conversion
to an elongation complex. The decreased affinity of the mutant for NTPs
clearly could contribute to favoring release over elongation since the
defect is greatly reduced at higher concentrations of NTPs. Finally,
N1072H increases apparent KApU ~3-fold and
KNTP ~6-fold during abortive initiation.
Perturbation of the catalytic center appears able to explain all of
these effects of this mutant. However, apparent, but only moderate,
perturbation of binding sites both for the priming nucleotide and the
substrate NTP argues against direct contacts between N1072 and
either reactant. Instead, a general deformation of the active site
caused by N1072H could be an indirect effect of the mutation. For
example, N1072H could alter an amino acid contact that positions one
of the secondary structure elements that actually form the active
site.
This interpretation is consistent with other studies of conserved
region H, one of nine colinear regions of sequence conservation found
in homologs from bacteria to humans (Sagitov et al.
(1993) and references therein). Cross-linking studies indicate that
conserved region H is likely to be positioned close to the 5 face of
the priming nucleotide; an evolutionarily invariant lysine residue
within this segment (Lys-1065 in and probably Lys-979 in
Saccharomyces cerevisae polII) is within 2 Å of the
-phosphate of the initiating NTP (Grachev et al., 1987 ,
1989 ; Mustaev et al., 1991 ; Riva et al., 1990 ).
The observation that the nascent chain can be elongated by only two
residues after cross-linking to this site is further evidence for its
proximity to the nucleotide binding site (Mustaev et al.,
1993 ).
Studies of site-directed mutants in this region by the Goldfarb group
also support the location of this region of near the catalytic
center. The K1065R mutation exhibits a severe lethal phenotype.
Although it forms open complexes, it catalyzes the formation of only
one or two phosphodiester bonds, an effect similar to inhibition by
Rif. However, replacement of a number of conserved amino acids between
1063 and 1073, including Lys-1065, with alanine caused a less severe
phenotype (Sagitov et al., 1993 ). Single alanine
substitutions all retained significant transcriptional activity on
natural templates ( 15% of the wild-type polymerase in multiround
assays), although they deviated from wild type in a number of steps in
transcription, including promoter escape and the extent and pattern of
pausing. The mild phenotypes of most Ala substitutions in region H are
consistent with the view that it does not form the nucleotide addition
site per se, but is probably in its immediate vicinity and
is likely to affect RNA polymerase by perturbing the catalytic center
indirectly. For instance, changes in charged residues (e.g.
Lys-1065 to Arg and Asn-1072 to His) could disrupt catalytic function
by forming new H-bonds or charge-charge interactions that reposition
side chains near the active center.
Satigov et al. (1993) have suggested that mutations in this
region distort the active center, possibly by affecting the coupling of
nucleotide addition and translocation. Although our experiments do not
define the function of the region, they are most consistent with this
idea. Jin and Turnbough have recently described a Rifr
polymerase (R529C in ) that exhibits an increased apparent
KNTP for pyrimidines in promoter escape (Jin and
Turnbough, 1994 ). However, the fact that the N1072H mutant is more
defective than  R352C in the process of promoter escape, but
exhibits a comparable increase in KNTP
(cf. 6-fold for N1072H and 2-3-fold for
 R352C), suggests that additional factors, such as
context-dependent conformational changes, may contribute to
the phenotype of N1072H at such decision points. A more thorough
investigation of the block to elongation at these points is necessary
to better articulate the nature of the N1072H defect.
 R352C and Conserved Region C in  May Contact DNA Like a
Weakly Similar Sequence in a DNA-binding Region of DNA Polymerase
I
Our selection of experiments on  R352C were motivated in
part by finding it located adjacent to two termination-altering
mutations at amino acids 350 and 351 (Weilbaecher et al.,
1994 ) in a region of weak sequence similarity to a putative DNA-binding
region in DNA polymerase I. Our results support the view that this weak
sequence similarity reflects functional similarity of these two
sequences: substitutions in this region profoundly destabilize
polymerase-promoter complexes in the presence of competitor
molecules.
In DNA polymerase I, this region comprises -sheets 7 and 8 of the
thumb domain of the DNA-binding cleft and is immediately upstream of
helices J and K. The region homologous to amino acids 350-352 in 
forms part of a loop between these two sheets. Polesky et
al. (1990) have generated an alanine substitution of the arginine
(R668A) corresponding to Arg-352 in rpoC. This mutation has
profound effects on kcat (400-fold decrease),
Kd for DNA (20-fold decrease), and slight effects on
Km for dNTPs, suggesting that this arginine side
chain forms part of the catalytic site of polymerase I and may also be
in close proximity to the phosphate backbone of the DNA (Polesky
et al., 1990 ).
Our results suggest that this region of  may, like the R688A
mutation in DNA polymerase I, be involved in more than one important
function of polymerase. The convergence of effects on
KNTP with those on DNA binding might be
predicted in a region involved in nucleotide addition; at some point in
the process, addition of NTPs must be tightly coupled to DNA contacts.
Similarly, the failure of  R352C to convert efficiently from the
abortive to productive mode (Figs. 4, 5, 6), while not due to an obvious
increase in the release of abortive products, could reflect aberrant
contacts between the polymerase and either the transcript or the
template. These defects could, however, also be the indirect result of
alterations in the conformational changes that accompany the switch to
productive initiation.
It is premature to conclude that this region of  is involved in
nucleotide addition or stabilization of contacts to the template during
this process: the mutants that are described in this study do not have
strong enough phenotypes to unequivocally determine what aspects of
transcription are primarily altered. However, further study of this
region of  is warranted, particularly with a view to uncovering the
relationship between defects in DNA binding and nucleotide addition.
The relatively weak phenotype of  R352C could stem from its having
been isolated as a haploid, altered-function mutation, necessitating
that it retain significant function. We are currently generating the
R352A mutation in rpoC as well as several other
site-directed changes to evaluate more stringently the function of this
region. Interestingly, no significant differences were detected between
 R352C and wild type in an elongation assay designed to measure
dissociation of the ternary complex during elongation (Arndt and
Chamberlin, 1988 ; data not shown). It is conceivable that in the
ternary complex additional contacts between the polymerase and the RNA
or DNA mask the putative destabilizing effect of the mutation at
 R352C but that the structural pertubation still affects transcript
elongation.
DNA-binding Defects of Substitutions in  and in the
Rifr Region of Suggest the Existence of
DNA-binding Domains in Each Subunit
We have presented results
implying that regions in both rpoB and rpoC are
important in DNA binding, although we do not yet have sufficient
evidence to suggest that these regions contact DNA directly. Prior to
this study, only a few workers had addressed the DNA binding properties
of the core subunits of RNA polymerase. Martin et al. (1992)
describe the DNA binding defect of a deletion mutant of rpoB
(in conserved region C, Fig. 1) that fails to form stable complexes at
the promoter. Several groups have described the isolation and partial
in vitro characterization of cold-sensitive mutations in and  that appear to be defective in open complex formation
(Larionov et al., 1979 ; Gragerov et al., 1980 ;
Panny et al., 1974 ). Interestingly, Nomura et al.
(1984) describe mutations in rpoC, as well as a double
mutation in which includes the H526Y change, discussed below, that
appear to be important in promoter selectivity. Unfortunately, none of
the rpoC mutations examined in these earlier studies have
been mapped or sequenced.
The three mutations in rpoC and two mutations in the
Rifr region of rpoB that we analyzed
significantly affected the stability of the open complex to challenge
by both heparin and poly(dA-dT) (Table IV). In both cases,  R352C
and  S350F, as well as H526Y, formed open complexes that were
significantly less stable than wild type in the presence of
competitors, consistent with, but not proving that these residues
stabilize contacts to the DNA.
The result obtained with H526Y is particularly interesting. The T7
A1 open complex containing H526Y is as susceptible to both types of
competition as the rpoC mutants. Recent experiments by
Goldfarb and co-workers (Mustaev et al., 1995 ) in which Rif,
substituted with a cross-linkable side chain, is bound to RNA
polymerase at the T7 A1 promoter indicate that the bound Rif molecule
cross-links to the template strand of the DNA between positions 2 and
3 relative to the start site of transcription. These observations,
taken with our findings for H526Y and Q513P, suggest that the
Rifr region of rpoB is in the immediate vicinity
of the DNA when polymerase is bound at the promoter and may be involved
in stabilizing contacts to the DNA in the open complex. In another
study,3 we found evidence suggesting that the
Rifr region, or at least the residue affected by the
Q513P mutation, is important in correctly positioning the 3 end of
the nascent transcript in the ternary complex (i.e. makes
contacts in the promoter distal segment of region B, Fig. 8). This same
region could be involved in contacting the DNA in the absence of
transcription (i.e. in the binary promoter complex). An
interaction between the Rifr region and DNA is also
consistent with observations by Chamberlin and co-workers (Hinkle
et al., 1972 ) that the affinity of RNA polymerase for Rif is
reduced by 2 orders of magnitude when polymerase is bound to DNA.
Whether or not these regions contact DNA directly, their effects on the
stability of binary complexes suggest that they must at least
communicate with regions that stabilize polymerase-DNA
interactions.
Suppression of rho Mutations May Occur by Distinct
Mechanisms
Jin et al. (1992) previously
described a model to explain the kinetic interplay between RNA
polymerase and Rho. In this view, the movements of polymerase and Rho
on DNA and RNA, respectively, are kinetically coupled such that
alterations in the translocation rate of either polymerase or Rho
affect termination efficiency. This model has been invoked to explain
the hypertermination phenotype of rpoB8 ( Q513P), which
elongates more slowly in vivo and in vitro, and
exhibits increased termination on all Rho-dependent (and
intrinsic) terminators tested. Indeed, rpoB8 was also
isolated (as rpoB203) as a suppressor of rho201
(Guarente and Beckwith, 1978 ).
Both Guarente and Beckwith (1978) and our previous study (Jin and
Gross, 1989 ) concluded that the rpoB211 and
rpoC214 mutations do not exhibit a general termination
defect such as that observed for rpoB8. Although
reconstruction experiments verified that both mutants increased
termination at the trptt terminators used in their
selection, only rpoC214 ( R352C) increased termination on
other terminators, and this defect was not particularly pronounced.
Furthermore, in contrast to Q513P, these mutants show only a
marginal increase in termination at intrinsic terminators in
vitro (data not shown). These differences in phenotype suggested
that a study of the defects of these two mutants might reveal
additional characteristics of the termination decision.
We were therefore not surprised to discover that while both N1072H
and  R352C elongate more slowly in vitro, neither
demonstrates an elongation defect as severe as that measured for
Q513P. Furthermore, although N1072H demonstrates the greater
elongation defect of the two,  R352C exhibits the more significant
termination-altering properties. We suggest that the specific effects
of the mutants on the trptt terminator can best be
understood in the context of the mutant defects elaborated above for
the transition between abortive synthesis and productive elongation.
Termination versus elongation is a decision similar to
abortive versus productive initiation. In both, release
competes with RNA chain elongation. The same properties of the mutants
invoked to explain their defects in making the transition to productive
elongation may account for their altered termination properties. Thus,
the defect of N1072H in nucleotide addition, and the altered
template binding properties of  R352C may affect their ability to
terminate. Moreover, such defects are likely to be manifest only at
those terminators whose particular rate-limiting steps are affected by
this defect, giving rise to the terminator specific properties of the
mutants. For similar reasons, region H mutants exhibit severe defects
in forming productive elongation complexes only at certain promoters
(Sagitov et al., 1993 ). Our findings complement other recent
studies examining the elongation properties of termination-altering
mutants that suggests that elongation rate is not the sole determinant
of the probability of termination (Landick et al., 1990 ;
Weilbaecher et al., 1994 ). Furthermore, it is becoming
increasingly clear that contacts to additional protein factors,
particularly NusG, play an important role in determining the efficiency
of Rho-dependent termination, at least at some terminators.
Further studies are underway, both in our groups as well as several
others, to unravel the intricacies of the elongation to termination
decision at both Rho-dependent and intrinsic termination
sites.
FOOTNOTES
*
This work was supported by National Institutes of Health
Grants GM 38660 (to R. L.) and AI 19635 (to C. A. G.). The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
``advertisement'' in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
Present address: Third Wave Technologies, Inc., 2800 S. Fish
Hatchery Rd., Madison, WI 53711.
Present address: Campbell Taggart, Inc.; 1101 Wyoming St., St.
Louis, MO 63110.
Present address: Dept. of Stomatology, University of
California, San Francisco, San Francisco, CA 94143.
Present address and to whom correspondence should be addressed:
Dept. of Bacteriology, 1550 Linden Dr., University of
Wisconsin-Madison, Madison, WI 53706-1567. E-mail:
landick{at}macc.wisc.edu.
1
The abbreviations used are: Rif,
rifampicin; ApU, adenosyl 5 -uridine; NTP, nucleotide triphosphate;
PCR, polymerase chain reaction; bp, base pair(s); PAGE, polyacrylamide
gel electrophoresis. -ANS-UTP, uridine 5 triphosphate containing
1-aminonaphthalene 5-sulfonate attached via a phosphoamidate
bond.
2
J. Jendrisak, personal
communication.
3
G. Feng, L. M. Heisler, and R. Landick,
manuscript in preparation.
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