From the Division of Biological Sciences and Center for Molecular Genetics, University of California, San Diego, La Jolla, California 92093-0634
Received for publication, November 10, 2002, and in revised form, December 18, 2002
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ABSTRACT |
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Bacteriophage T4 late promoters, which consist of
a simple 8-base pair TATA box, are recognized by the gene 55 protein
(gp55), a small, highly diverged member of the All multisubunit RNA polymerases require proteins that are
specialized for promoter identification and for initiation of
transcription. The eukaryotic and archaeal RNA polymerases employ
extrinsic proteins whose assemblies on DNA mark the promoter and
recruit RNA polymerase, whereas the bacterial RNA polymerases use their
tightly bound Much of the information about the function of The experiments that are presented here deal with the bacteriophage T4
gene 55 protein, gp55, a highly diverged, small (185 amino acids) The recognizable but weak homology of gp55 with the We have subjected the region of gp55 that is proposed to be homologous
to Proteins--
Gene 55 has been modified for this work by
insertion of an N-terminal kinase tag and a C-terminal His6
tag into the wild type gene in expression vector pET21b. The
corresponding protein is referred to throughout as "wild type"
gp55. Oligonucleotide-directed mutagenesis was used to generate
mutations at amino acids 68-83 that are specified in Fig. 1. (The
mutant protein with amino acid 68 changed from lysine to alanine is
referred to as gp55-K68A, for example.) The wild type and mutant genes
55 were overexpressed in E. coli BL21(DE3) grown at 37 °C
to an absorbance at 600 nm of 0.6-0.8 and induced with 1 mM isopropyl-
Untagged E. coli RNA polymerase core, RNA polymerase core
with a His6-tagged Co-immunoprecipitation of gp55 with RNA Polymerase
Core--
Wild type gp55 was phosphorylated in its N-terminal tag with
bovine heart protein kinase A (5 units) in buffer containing 20 mM Tris-HCl, pH 7.6, 100 mM NaCl, 10 mM MgCl2, and 450 µCi of
[ Single-round Transcription--
DNA for single-round
transcription was derived from pDH310, which contains a transcription
unit defined by the T4 gene 23 late promoter and the phage T7 early
transcription terminator and yields an ~420-nucleotide transcript.
Supercoiled or blunt end linear DNA was used for basal transcription.
For sliding clamp activated transcription, pDH310 was first linearized
with EcoO109 endonuclease and reacted with exoIII to generate
~60-100-nucleotide 5'-overhanging ends. The overhanging end upstream
of the T4 late promoter was removed with SmaI endonuclease,
and the resulting transcription template was purified as described
(32).
Single-round transcription at 25 °C was performed as described (23).
For basal transcription, 36 pmol of C-terminally
His6-tagged wild type or mutant gp55 was incubated on ice
with 6 pmol of RNA polymerase in 40 µl of transcription buffer (240 mM potassium acetate, 33 mM K-Hepes, pH 7.8, 10 mM magnesium acetate, 1 mM dithiothreitol, 150 µg/ml bovine serum albumin), transferred to 25 °C, and added to
0.75 pmol of linear pDH310 DNA in 40 µl of transcription buffer
(pre-equilibrated at 25 °C). At specified subsequent times,
transcription was initiated by transferring a 10-µl aliquot to 5 µl
of NTP mix (final concentrations, 1 mM GTP, 1 mM ATP, 0.1 mM CTP, 0.1 mM
[ KMnO4 Footprinting--
DNA (bp Aggregation of Mutant gp55--
Aggregation was assessed by gel
exclusion chromatography on Superose 12 essentially as described (23).
Briefly, gp55 was diluted (400-600-fold) out of its storage buffer
into column buffer (20 mM Na-Hepes, pH 7.8, 7 mM MgCl2, 500 mM NaCl, 10% (v/v)
glycerol, 0.006% (v/v) Tween 20, 4 µM Na3
EDTA, 10 mM Modeling--
gp55 amino acids 42-119 were aligned to
Tth An alanine scan mutagenesis of amino acids 68-83, the gp55
segment previously aligned with family proteins that replaces
70 during the final phase of the T4
multiplication cycle. A 16-amino acid segment of gp55 that is proposed
to be homologous to the
70 region 2.2 has been subjected
to alanine scanning and other mutagenesis. The corresponding proteins
have been examined in vitro for binding to
Escherichia coli RNA polymerase core enzyme and for the
ability to generate accurately initiating basal as well as sliding
clamp-activated T4 late transcription. Mutations in the amino acid
68-83 segment of gp55 generate a wide range of effects on these
functions. The changes are interpreted in terms of the multiple steps
of involvement of gp55, like other
proteins, in transcription.
Effects of mutations on RNA polymerase core binding are consistent with
the previously proposed homology of amino acids 68-82 of gp55 with
70 region 2.2 and the recently determined structures of
the Thermus thermophilus and Thermus aquaticus
70-RNA polymerase holoenzymes.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunits for a strictly comparable purpose. A single
major
protein,
70 in Escherichia coli, is
used to recognize most of a genome's promoters, but even the smallest
bacterial genomes encode additional
proteins recognizing distinct
promoter sequences, and some of the larger genomes encode dazzling
numbers of accessory
factors (1). All
proteins are multivalent
and multifunctional; they bind to RNA polymerase core enzymes and to
DNA, almost invariably recognizing two separate DNA sites. Many of the
proteins, possibly all of them, also are targets of accessory ligands
that regulate their activity and/or cellular compartmentation. The
segmented pattern of amino acid sequence conservation among
proteins (2, 3), defining homology segments 1.1, 1.2, 2.1-2.5, 3.1, 3.2, 4.1, and 4.2, is associated with common functions of these proteins.
proteins and much of
the recent key information about mechanism of action comes from the
analysis of E. coli
70.
70 is
a four-domain protein; each structural domain occupies a separate site
on the surface of the RNA polymerase core enzyme (4-9). Homology
segments 2 and 4, which are located in separate structural domains,
also bind specifically to separate DNA sites (the
10 and
35
promoter elements) centered ~1 and 3.2 turns upstream of the
transcriptional start site. The core of the polymerase holoenzyme
provides the scaffold that constrains the appropriate spacing of
homology segments 2 and 4 for promoter recognition. Polymerase core
binding also changes the internal structure of
70,
disrupting an interaction between homology segments 1.1 and 4 that
blocks DNA binding by homology segment 4 (10), separating segments 2 and 4 (4), and allowing site-specific binding to the melted
nontranscribed strand as well as double-stranded DNA of the
10
promoter element (11-13). The structures of E. coli
70 structure domain 2 (extending from homology segments
1.2 to 2.4) and, very recently, of three structure domains of
Thermus aquaticus (Taq)
A
(comprising homology segments 1.2-2.4, 3.0-3.1, and 4.1-4.2, respectively) have been determined (5, 14). The locations of E. coli
70 homology segments 1.1 (comprising the
fourth structure domain of
), 2, 3.1, 3.2, and 4 have been modeled
onto the structure of RNA polymerase core in the holoenzyme and in open
promoter complexes on the basis of an extensive survey of spatial
separations in solution, determined by fluorescence resonance energy
transfer (6). The culmination of all this effort has been the
determination of structure of
A holoenzymes from
Taq and Thermus thermophilus and of a
Taq holoenzyme complex with fork junction DNA representing
an open promoter complex (8, 9, 15).
family protein. gp55 confers the ability to recognize T4 late
promoters, which consist of an 8-base pair TATA box (TATAAATA in the
nontranscribed strand) centered one helical turn upstream of the
transcriptional start site. T4 late promoters entirely lack the
35
binding site that is characteristic of the other
family promoters,
and gp55 has no segment homologous to
region 4, which contains the
corresponding
35 site-binding domain (homology segment 4.2). T4 late
genes are activated by an apparently unique mechanism that connects
their transcription to concurrent DNA replication through the action of
the sliding clamp (gp45) of the phage DNA polymerase holoenzyme. From
the point of view of mechanism, the salient feature of gp45 is that it
activates T4 late transcription in a topologically but not physically
DNA-bound state. Consequently, gp45 does not play a direct role in
marking its conjugate T4 late promoters. Like its cellular homologs
(16, 17), the T-even phage family sliding clamp is ring-shaped (18, 19). Loading the sliding clamp activator onto DNA at primer-template junctions or single strand nicks and gaps in DNA is done by its clamp
loader, the T4 gene 44/62 protein complex (gp44/62). The gp45 sliding
clamp interacts with a C-terminal hydrophobic-acidic epitope of gp55;
gp33, the T4-encoded, RNA polymerase core-bound co-activator of late
transcription, and T4 DNA polymerase have similar C-terminal epitopes
(20, 21). The C termini of gp55 and gp33 are both required for sliding
clamp activation of T4 late transcription (21-24).
family proteins
is confined to segment 2 (amino acids 381-451 in E. coli
70). The corresponding amino acid 45-115 segment of
gp55 includes those parts of the protein that are most strongly
protected from peptide bond cleavage by hydroxyl radical when bound to
RNA polymerase core and that are supposed to be homologous to
70 segments 2.1 and 2.2 (25). This
70
region is known to be involved in binding to the RNA polymerase core
(12, 13, 26-29).
70 segment 2.2 (amino acids 68-83) to alanine
scanning and other mutagenesis. The corresponding mutant proteins have
been examined in vitro for ability to bind to E. coli RNA polymerase core and also for function in basal as well as
sliding clamp-activated T4 late transcription. The very wide range of
phenotypes that is generated by mutations in this short segment is
interpreted in terms of the multiple roles that
plays in promoter
recognition, initiation of transcription, and transcriptional activation.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-thiogalactopyranoside for
4 h. The cells were harvested and lysed, and the insoluble gp55
proteins were recovered from inclusion bodies that were washed twice in
buffer containing 50 mM Na-Hepes, pH 7.8, 5 mM
-mercaptoethanol, 1 M NaCl, and 0.2% (v/v) Triton X-100
before solubilization in buffer containing 40 mM Tris-HCl,
pH 8, 6 M guanidine HCl, 10 mM
-mercaptoethanol, and 10% (v/v) glycerol. The extracted proteins were applied to nickel-nitrilotriacetic acid-agarose, and washed on the
column with solubilization buffer. gp55 was eluted in solubilization buffer with 200 mM imidazole, stored at
20 °C at
concentrations of 150-700 µM, and diluted from this
storage buffer directly into transcription buffer (specified below)
before each transcription or RNA polymerase binding experiment
(introducing 3-18 mM guanidine into the reaction medium,
as specified below).
' subunit, T4 gp45 (sliding clamp),
gp44/62 complex (clamp loader), gp32 (single-stranded DNA-binding
protein), and gp33 (late transcription co-activator) were purified as
specified or referenced elsewhere (23, 30) and stored at
20 °C.
Restriction endonuclease, exoIII, protein A-coupled Sepharose beads,
and protein kinase A were purchased.
-32P]ATP (carrier-free). Unincorporated radioactivity
was removed by gel filtration on Biogel P-6 in buffer containing 40 mM Na-Hepes, pH 7.6, 100 mM NaCl, 10 mM MgCl2, 10 mM
-mercaptoethanol, 10% (v/v) glycerol, and 0.1% (v/v) Tween 20. Monoclonal antibody R4A2 to the
subunit of E. coli RNA
polymerase core, a generous gift from R. R. Burgess, was
conjugated to protein A-Sepharose beads following a standard method
(31). 32P-Labeled wild type gp55 (1.5 pmol) and 0.5 pmol of
E. coli RNA polymerase core were mixed in 10 µl of IP
buffer (100 mM NaCl, 40 mM Tris-HCl, pH 7.8, 10 mM MgCl2, 10% (v/v) glycerol, 0.1% (v/v)
Tween 20, and 266 µg/ml bovine serum albumin) for 30 min in the
presence or absence of 3 or 6 pmol of unlabeled mutant competitor gp55
in siliconized Eppendorf tubes that had been preblocked at 4 °C
overnight with IP buffer. 20 µl of a 25% slurry of anti-
antibody-conjugated protein A-Sepharose beads suspended in buffer IP
was added to each tube and allowed to equilibrate at 4 °C for 1 h, with rocking. Supernatant fluid was removed from pelleted beads, and
the latter were washed three times with 600-µl aliquots of IP buffer.
The pelleted beads were aliquoted for scintillation counting of bound
32P-gp55. Background was determined with samples lacking
core RNA polymerase. Each experiment was also accompanied by control
samples to determine competition by 3, 6, or 12 pmol of wild type
unlabeled gp55 and to examine the effect of 6 pmol of each unlabeled
mutant gp55 on competition by 6 pmol of unlabeled wild type gp55.
-32P]UTP (4000 cpm/pmol), 25 µg/ml rifampicin in
transcription buffer). Transcription was allowed to proceed for 8 min
and halted by adding 150 µl of stop buffer (20 mM
Na3EDTA, 40 mM Tris-HCl, pH 8, 250 mM NaCl, 0.4% (v/v) SDS, 250 µg/ml yeast RNA) containing
a small quantity of labeled DNA fragment as a sample recovery marker. The transcripts were purified, resolved, and quantified as described (25). For sliding clamp-activated transcription, the DNA mix contained,
in addition to 0.6 pmol of DNA, 6.1 µg of gp32 and 3 mM
dATP in 40 µl of transcription buffer, and the protein mixture contained, in addition to RNA polymerase core and gp55, 30 pmol of
gp33, 40 pmol of gp44/62 complex, and 22 pmol gp45 (trimer). The gp55
storage buffer introduced 3-14 mM guanidine HCl into the
reaction medium for formation of open promoter complexes.
150 to +150
relative to the transcriptional start site in pDH310), 32P
end-labeled in the nontranscribed strand, was generated by PCR and
purified by nondenaturing gel electrophoresis, essentially as described
(33). The samples, in 10 µl of transcription buffer containing 10 fmol of probe DNA, 50 ng of poly(dG-dC):poly(dG-dC), 200 fmol of
E. coli RNA polymerase core, and 1.2 pmol of wild type or
mutant gp55, were incubated for 20 min at 25 °C. KMnO4 (final concentration, 12 mM) was added to each reaction
mixture followed, 1 min later, by 125 µl of stop buffer (20 mM Na3EDTA, 40 mM Tris-HCl, pH 8, 250 mM NaCl, 0.5% (v/v) SDS, 250 µg/ml yeast RNA, and
200 mM
-mercaptoethanol). The reaction mixtures were extracted with phenol-chloroform, DNA was precipitated with ethanol, cleaved with piperidine, and analyzed on 6% polyacrylamide gel containing 7 M urea, as described (33). DNA cleavage at
T-4 of the nontranscribed strand was quantified by phosphorimage
scanning and normalized to cleavage generated by wild type gp55
holoenzyme after subtraction of background DNA cleavage in a sample
lacking RNA polymerase core.
-mercaptoethanol, 5 µM
phenylmethylsulfonyl fluoride, and 0.5 µg/ml each of leupeptin and
pepstatin) also containing cytochrome c (12.5 kDa),
ovalbumin (45 kDa), bovine serum albumin (68 kDa), and ferritin (450 kDa) as size markers. A 200-µl volume (containing 100 pmol of
protein) was loaded onto a Superose 12 column equilibrated and
developed in column buffer. Column fractions were analyzed by SDS-PAGE
with silver staining. Monomeric gp55 elutes between cytochrome
c and ovalbumin.
A amino acids 184-260 with a single gap
opposite gp55 residue Trp67. gp55 residues 42-66
and 68-119 were threaded into the Tth holoenzyme structure
(1IW7) by sequential mutagenesis and rotamer optimization, followed by
energy minimization using the GROMOS96 forcefield module of Swiss-Pdb
Viewer (34). gp55 Trp67, which was not included in the
modeling, would lie in the loop between the region 2.1 and region 2.2
helices. This threaded structure was used for examining the effects
of individual gp55 mutations following the same process as above. The
Noncovalent Bond Finder Module of Protein Explorer and visual
inspection was used to assess the effects of gp55 mutation.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
70 segment 2.2, was
carried out; alanine at amino acid 78 was replaced with Gly, the
putatively corresponding residue at
70 amino acid 411. Charge-reversing mutations at amino acids 77 (Glu
Arg) and 81 (Lys
Asp) were also introduced (Fig.
1). The corresponding C-terminally
His6-tagged proteins with an additional N-terminal kinase
tag (RRASV inserted between the first and second amino acids
of gp55) were overproduced in E. coli and purified from
inclusion bodies following the method previously used to purify the
corresponding wild type protein ("Experimental Procedures").
View larger version (7K):
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Fig. 1.
Alignment of amino acids 68-83 of gp55
with 70 homology segments 2.2 of
E. coli
70, Tth
and Taq
A, based on
the alignment of the
70 protein
family (2, 3, 50). Only those amino acids of Tth and
Taq
A that differ from E. coli
70 are indicated. Amino acids 68-72, 74-76, and 78-83
of gp55 were changed to alanine, Ala77 was changed to the
aligned
70 amino acid Gly, and additional
charge-reversing or radical mutations were introduced at amino acids
72, 77, and 81, as shown. Amino acids in
70 segment 2.2 that are implicated in RNA polymerase core binding (28) are indicated
by asterisks, and gp55 mutations found to strongly affect
core binding are identified by triangles.
Binding to RNA Polymerase Core--
Each of these proteins was
assessed for its ability to bind to E. coli RNA polymerase
core, using a co-immunoprecipitation/competition assay devised by Sharp
and co-workers (28) to screen 70 mutations for the same
function. Wild type gp55 was 32P-labeled in its N-terminal
kinase tag and used at a concentration sufficient to nearly saturate
the RNA polymerase core. The ability of a 2- and 4-fold excess of
unlabeled (unphosphorylated) wild type and mutant gp55 to compete with
this binding was compared by incubating labeled and unlabeled gp55 with
core and then separating core-bound gp55 on protein A-Sepharose beads
coated with monoclonal antibody directed against the RNA polymerase
subunit (the generous gift of R. R. Burgess). The quantity of
32P-labeled gp55 co-immunoprecipitating with core decreased
by ~70% in the presence of a 2-fold excess of unlabeled gp55 (Fig.
2). Mutant proteins considered to be
defective in core binding were those that diminished binding of
32P-gp55 by less than 50% at 2-fold excess. As a control,
mutant proteins were also tested for interference with competition by unlabeled wild type gp55 at high excess (a 4-fold excess each of the
wild type and mutant proteins, compared with an 8-fold excess of wild
type protein).
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The results of the analysis are presented in Fig. 2 and in Table I, where degrees of defective binding are indicated by a + to +++ scale, compared with ++++ for the wild type gp55. The mutant proteins found to be most defective in core binding were gp55-E70A, gp55-D74A, gp55-G82A, and gp55-L83A, but gp55-G82A and gp55-L83A also showed evidence of interference with competition by unlabeled wild type gp55 at high concentration. Such interference might be due to protein aggregation, perhaps resulting from misfolding under these conditions (see "Experimental Procedures"). Accordingly, these most core binding-defective proteins (with the exception of the transcriptionally highly active gp55-L83A), as well as gp55-M71A and gp55-I72K, were also tested for aggregation by sizing on Superose 12. Only gp55-G82A showed substantial aggregation, with ~50% of protein eluting as multimeric complexes (data not shown). Despite an apparent tendency to aggregate at the relatively high concentrations used for column chromatography, the severe defect in core binding of gp55-G82A is unlikely to be entirely due to aggregation.
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Basal Transcription--
Each of these proteins was examined for
basal transcription of linear duplex DNA containing the T4 late
transcription unit in the previously constructed and extensively
analyzed plasmid pDH310 (35). The T4 late promoter in pDH310 (from T4
gene 23, which encodes the major phage head protein) and the
transcriptional terminator from phage T7 gene 1 define a transcription
unit that yields an ~420-nucleotide RNA product. Promoter complexes
were formed at 25 °C for the times indicated in Fig.
3 (A and B) and then allowed to execute a single round of transcription as specified under "Experimental Procedures." Most of these mutant proteins were
quantitatively deficient in basal transcription, relative to wild type
gp55, but some, including gp55-Q69A and gp55-I76A, were comparably
active with the corresponding wild type protein. All of these proteins
are compared quantitatively (for activity after 20 min of promoter
opening) in Fig. 4 and Table I.
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It was especially interesting to see that gp55-E77A and gp55-L83A were
respectively 3- and 2-fold more active transcriptionally than wild type
gp55 (Table I and Fig. 4), because they form transcriptionally competent promoter complexes more rapidly (Fig. 3B and data
not shown). The existence of this surprising but consistently observed advantage was also confirmed in experiments with gp55-E77A to examine
the pseudo-first order rate constant of formation of DNA competitor-resistant nitrocellulose membrane-retained
polymerase-promoter complexes. In the example that is shown in Fig.
5, the gp55-E77A holoenzyme formed these
complexes 3.7 times more rapidly at 37 °C than did the wild type
gp55 holoenzyme.
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Basal transcription of negatively supercoiled pDH310 DNA was also surveyed in a standard assay, in which a single round of transcription at 25 °C in standard reaction buffer was quantified after allowing 10 min for formation of open promoter complexes (Fig. 4, open columns, Table I, and "Experimental Procedures"). DNA supercoiling was found to diminish the transcriptional defects of gp55 variant proteins. Restoration of activity was only partial for the more deficient proteins (gp55-K68A, gp55-E70A, gp55-M71A, gp55-I72A, gp55-I72K, gp55-D74A, gp55-A78G, and gp55-K81A). The rest were active at 60% or greater of wild type level. Equally striking was the fact that the hyperactivity of gp55-E77A was diminished (~5-fold) from ~200 to ~40% and that the transcriptional activity of gp55-L83A (possibly reduced in the same proportion) was no longer significantly above that of wild type gp55.
Activated Transcription-- Mutant proteins were also analyzed for ability to respond to activation by the gp45 sliding clamp. The DNA template for these experiments was pDH310 DNA linearized at its EcoO109 site (~2.3 kbp upstream and 1.0 kbp downstream of the T4 late promoter) and treated with exoIII to create, on average, ~60-100-nucleotide 3' overhanging single-stranded ends. The double strand-single strand junctions of this DNA serve as loading sites for gp45 by its clamp loader, the gp44/62 complex, in an ATP hydrolysis-requiring process (36-41). The loading site located downstream of the late transcription unit provides the gp45 orientation on DNA that is required for transcriptional activation (32). Accordingly, the upstream loading site was removed by endonuclease cleavage at the SmaI site. Sliding clamp loading by the gp44/62 clamp loader is facilitated by the homologous T4 single-stranded DNA-binding protein, gp32 (42), which was also present. Coating single-stranded DNA with gp32 also diminishes nonspecific and nonproductive sequestration of RNA polymerase.
The T4 late promoter opens extremely rapidly under the influence of the
sliding clamp activator and gp33 co-activator (21) (Fig.
3D). Measuring the capacity for a single round of
transcription after allowing only 1 min for sliding clamp loading,
promoter complex formation, and opening thus is a way of monitoring the ability of variant gp55 to respond to the transcriptional activator. An
experiment comparing rates of formation of transcriptionally competent
promoter complexes with wild type gp55, gp55-E70A, and gp55-E77A is
shown in Fig. 3C. The results of the gp55 mutant screen are
compiled in Fig. 4 and Table I. Most of the mutant T4 late holoenzymes,
including those assembled with gp55-K68A, gp55-I72A, gp55-I72K,
gp55-A78G, gp55-S79A, gp55-I80A, gp55-K81A, and gp55-G82A, are
indistinguishable from the wild type late holoenzyme in activated
transcription, despite significant defects of the mutant gp55 in basal
transcription of linear DNA. Even the highly defective gp55-M71A and
gp55-D74A yield comparable activity (~70% of wild type), and
gp55-E77R is also relatively active in sliding clamp-activated
transcription. gp55-E77A and gp55-L83A, which are hyperactive for basal
transcription of linear DNA, lose this advantage for gp45-activated
transcription. gp55-K81D is less active in gp45-activated than in basal
transcription of linear DNA (relative to the wild type). This is a
potentially interesting phenotype because it suggests a defect that is
specific to the activation mechanism. Only gp55-E70A is largely
defective in gp45-activated transcription, although a very slowly
accumulating transcription capacity can be detected even for this
protein (Fig. 6).
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Promoter Opening and Promoter Clearance--
Open T4 late promoter
complexes undergo multiple rounds of abortive synthesis of short
oligonucleotides before they produce each transcript (43). At certain
promoters, the 70 holoenzyme forms an appreciable
fraction of open complexes that remain in the abortive mode and never
yield complete transcripts (44, 45). Thus, a quantitative comparison of
the ability to form open promoter complexes and complete transcripts
provides a way of determining whether a gp55 mutation primarily impairs promoter opening or promoter clearance.
Promoter opening was screened by KMnO4 footprinting of the
nontranscribed strand in basal promoter complexes of gp55 holoenzyme formed under the conditions of the basal transcription screen, that is,
during 20 min at 25 °C, and quantified as described under "Experimental Procedures." The principal outcome of the analysis is
that promoter opening and production of transcripts are closely correlated (Fig. 7 and Table I). This
indicates that defects in promoter clearance are not determining for
inactivity generated by these homology segment 2.2 mutations of
gp55.
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DISCUSSION |
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70 binds to the RNA polymerase core through
separate sites that are located in its homology segments 1, 2.2, 3, and
4 (7, 12, 46, 47). Formation of the
70 holoenzyme is a
multi-step process that is accompanied by a reorganization of
interactions within
70 (4, 12) leading to the
segregation of
70 structure domains to well separated
sites on the surface of the core polymerase. Homology segments 1.2-2.4
constitute one of these structure domains (5).
The weak homology of gp55 with 70 is essentially
confined to homology segment 2. The part of gp55 that is homologous
with
70 segments 2.1 and 2.2 is most strongly protected
by RNA polymerase from proteolytic cleavage by hydroxyl radical (25),
implying tight binding. Our analysis of mutations in gp55 focuses on
amino acids 68-83, comprising a segment that is homologous with
70 segment 2.2 (Fig. 1). This segment is completely
conserved in the newly sequenced phage RB69. Indeed, the entire amino
acid sequence of gp55 is highly conserved between phages T4 and RB69, with only 11.3% divergence, and an additional 4-amino acid insertion near the N terminus of RB69 gp55
(phage.bioc.tulane.edu).
Mutations at multiple sites in 70 homology segment 2.2 (L402F, D403N, D403A, Q406A, E407K, N409D, and M413T) disrupt the
70-core interaction (28). gp55 mutations at amino acids
71 and 72 (M71A and I72K), putatively corresponding with amino acids 404 and 405 of
70, were previously shown to generate
defects in binding to RNA polymerase core (25). Alanine substitution
mutants at all possible sites in the amino acid 68-83 segment have
been examined. Ala78 has been replaced with Gly, the amino
acid at the putatively corresponding location in
70 and
charge-reversing mutations at amino acids 77 (Glu
Lys) and 81 (Lys
Asp) have also been introduced.
As anticipated, many of these mutations affect gp55 binding to the RNA polymerase core (Fig. 2 and Table I). In addition to the previously analyzed gp55-M71A and gp55-I72K, mutant proteins with significant core binding defects include gp55-E70A, gp55-D74A, gp55-E77A, gp55-A78G, gp55-G82A, and gp55-L83A. Although aggregation could contribute to the core-binding defects of gp55-G82A and gp55-L83A, it is unlikely that it is solely responsible for very weak binding. Indeed, gp55-L83A is transcriptionally highly active under all of the tested conditions (Figs. 3A and 4). The scale that has been used to designate binding defects in Table I can be calibrated by reference to gp55-M71A and gp55-I72K, which have also been examined by affinity chromatography and found not to bind core enzyme in presence of 400 mM NaCl. (Core binding by gp55-I72K is also partly defective in presence of 250 mM NaCl (25).) E70A and D74A generate even more defective core binding than M71A and I72K.
These effects of mutations on core binding can be interpreted in terms
of the recently determined structures of T. aquaticus (Taq) and T. thermophilus (Tth)
A-RNA polymerase holoenzymes (8, 9, 15), which retain
the four-helix structure previously determined for E. coli
70 and Taq
A domain 2 (
2), comprising homology segments 1.2-2.4 (5, 14). The
-helical homology segments 2.2 in these holoenzyme structures interact directly with an antiparallel coiled-coil segment of
'
(E. coli amino acids 265-310; Tth amino acids
540-585).
The proposed alignment of gp55 amino acids 68-82 with homology
segment 2.2 (Fig. 1) has been tested by comparing the outcome of the
gp55 mutagenesis (Table I) with structure predictions based on
A segment 2.2-
' coiled-coil interactions within the
Tth and Taq holoenzymes. Tth and
Taq
A homology segments 2.2 are identical
(Fig. 1), and the
' coiled-coils differ only by two conservative
substitutions (Tth Leu581 is Val581
in Taq
'; Tth Leu582 is
Ile582 in Taq
'). The comments that follow
refer directly to the higher resolution Tth holoenzyme
structure but can be inferred from either structure.
There is a periodicity of effect of alanine substitutions in gp55 on
E. coli RNA polymerase core binding, with the E70A, D74A, and E77A mutations generating the greatest defects (Table I). In the
gp55-A alignment, gp55 Glu70,
Asp74, and Glu77 correspond with Tth
A Asp211, Glu215, and
Gln218, whose side chains face the
' coiled-coil.
Tth A Asp211 interacts with
Tth
' Arg550 and Arg553
corresponding to Arg275 and Arg278 in E. coli
', respectively. A region 2.2. helix-
' coiled-coil energy-minimized model with gp55 amino acids 68-83 threaded into the
structure in place of the corresponding segment of
A has
gp55 Glu70 also interacting with the same Arg side chains
in the
' coiled-coil. In a mutational analysis of the E. coli
' coiled-coil region, Arthur and co-workers (47) found Arg
Gln at amino acid 275 greatly diminishing the ability to assemble
70 holoenzyme in vivo and essentially
eliminating the ability of the
' amino acids 1-319 segment (which
includes the entire coiled-coil) to bind
70 in far
Western blotting.
Tth A Glu215 interacts with
'
Arg553; in the energy-minimized gp55 model, the
corresponding Asp74 is similarly juxtaposed with
'
Arg553. Tth
A Gln218
interacts with
' Lys556 (Arg281 in E. coli
'); in the model, the corresponding Glu77 of
gp55 is also located in proximity to
' Lys556. Thus, in
terms of the Tth holoenzyme structure, it is plausible that
eliminating any of these three favorable charge interactions between
gp55 region 2.2 and
' and substituting a shorter side chain in gp55
would diminish core binding.
The three-amino acid phasing of coiled-coil interaction is
interrupted beyond this point. gp55 Ile80 corresponds to
Tth
A Ile221, which is in
vicinity of
' Gln560, corresponding to
Leu285 in E. coli
'. Interactions between
these side chains should not contribute significantly to affinity.
Gln560 is in the loop connecting the antiparallel
helices of the
' coiled-coil, and the lack of effect of the
gp55-I80A mutation on core binding is consistent with the
Tth holoenzyme structure. Gly82 and
Leu83 of gp55 correspond with Tth
A Ala223 and Val224, which are
not in the immediate vicinity of
'. In the gp55 model, the
Gly82 and Leu83 side chains face in the
direction of the crossing region 2.1 and 2.3 helices. Thus, examination
of the model suggests that weak core binding of the gp55-G82A and
gp55-L82A mutants may be due to effects of these mutations on the
folding of gp55 region 2.
What is striking about the properties of these mutants is their
diversity of phenotype for basal and activated transcription and the
diversity of relationship between RNA polymerase core binding and
transcriptional activity. The existence of such a range of defect is
conceptually congruent with recent insights into the function of
70 homology segment 2.2 (13).
70 binds
the nontranscribed strand and duplex DNA of the
10 promoter region
(11) through its homology segment 2.4. The switch from double-stranded
DNA binding to single-stranded DNA binding requires a structural
transition that is generated by interaction of
70 with
the
' subunit of the core enzyme (48). The
' coiled-coil interacts with
homology segment 2.2, as already specified; segments 2.2 and 2.4 are apposed (14). Evidently, polymerase core binding by
homology segment 2.2 is coupled to the promoter opening capacity of
70 homology segment 2.4 (13), but how that coupling is
achieved is not yet clear.
Most of the transcriptional defects that are detected with linear DNA templates are strongly mitigated by the sliding clamp activator. Several factors may operate to produce this effect. First, the additional interactions of the sliding clamp-activated promoter complex, between gp55 and DNA-confined gp45 and between gp45 and core-bound gp33, should increase the effective affinity of gp55 for RNA polymerase core. Second, transcription is assayed with a 6-fold excess of gp55 over core. For sliding clamp-activated transcription, this is an at least 4-fold excess over the core-saturating concentration of wild type gp55. The excess can compensate for considerably weaker binding by mutant gp55. Third, it is conceivable that gp45 interaction exerts a direct function-restoring effect on the structure of some of these mutant proteins. The extremely weakly binding gp55-D74A could be a candidate for such an effect.
Overall, these gp55 homology segment 2.2 mutations generate an ~300-fold range of activity in basal transcription of linear DNA. The close correlation between promoter opening, as determined by permanganate footprinting, and transcriptional activity specifies that these gp55 mutations neither create nor eliminate barriers to the transition from abortive to productive transcript elongation.
Among the other phenotypes, three are particularly interesting. First, gp55-E70A is almost completely inactive for basal transcription of linear DNA and for sliding clamp-activated transcription but significantly active for transcription of supercoiled DNA. The gp55-E70A RNA polymerase opens the promoter slowly in supercoiled DNA but ultimately yields a nearly wild type level of single round transcription (data not shown). This result suggests that the E70A mutation also affects the ability to respond to the transcriptional activator. The primary interaction site of gp55 with the gp45 sliding clamp is its C-terminal hydrophobic/acidic epitope (21, 23). The transcriptional defect generated by the E70A mutation is therefore likely to reside in a step of the reaction pathway that follows gp55-gp45 interaction, most probably promoter opening. The fact that the defect of gp55-E70A manifests itself most severely in basal as well as activated transcription of linear DNA suggests that the sliding clamp activator and DNA underwinding relieve distinguishable rate limitations on the opening of T4 late promoters by the gp55-RNA polymerase holoenzyme.
Second, gp55-E77A and gp55-L83A are significantly defective in core
binding but hyperactive in forming open promoter complexes for basal
transcription of linear DNA (Figs. 2-4 and Table I). The
"advantage" of these mutant proteins is almost completely lost in
the context of basal transcription of supercoiled DNA (and cannot be
tested properly for gp45-activated transcription under our assay
conditions because the late promoter opens so rapidly with wild type
gp55 (24)). An inverse correlation between core binding and promoter
opening in basal transcription implies that the interaction of wild
type gp55 segment 2.2 with polymerase core, which is essential to its
function, nevertheless limits the rate of promoter opening. This is
reminiscent of the just-published observation that disulfide
bridge-locking 70 segment 2.1 or 2.3 with segment 2.2 yields holoenzymes that are functional but quantitatively deficient in
formation and stability of open promoter complexes. Evidently,
conformational flexibility within structure domain 2 of
70 facilitates DNA strand opening in the promoter
complex and initiation of transcription (49). Comparable structure
adaptations probably figure in gp55-dependent T4 late transcription.
Third, conversely, certain gp55 mutations significantly diminish
activity for basal transcription without significantly diminishing core
binding. gp55-I80A and gp55-K81A fall into this category, with the K81A
mutant protein showing the most pronounced phenotype. The K68A mutation
only barely affects core binding but makes unenhanced transcription
grossly defective. Activation by gp45 restores transcription to the
wild type level. Under reaction conditions that differ only slightly,
the sliding clamp activator increases the second order rate constant
for T4 late promoter opening by gp55-RNA polymerase ~340-fold (24).
For gp55-K68A, the activation ratio must be an additional order of
magnitude greater.
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ACKNOWLEDGEMENTS |
---|
We thank R. R. Burgess, N. Thompson, C. A. Gross, and M. M. Sharp for generous gifts of antibodies, M. M. Sharp for guidance, J. D. Karam, and H. Krisch for sharing information in advance of publication, and S. Kolesky and M. Ouhammouch for advice and helpful discussions.
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FOOTNOTES |
---|
* This work was supported by a grant from the NIGMS, National Institutes of Health.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.
Supported by a training grant in Cell Biology, Molecular Biology
and Genetics from the NIGMS, National Institutes of Health. To whom
correspondence may be addressed. Present address: Diversa Corporation,
4955 Directors Place, San Diego, CA 92121. E-mail: kwwong@diversa.com.
§ To whom correspondence may be addressed. E-mail: gak@ucsd.edu.
¶ To whom correspondence may be addressed. Present address: Faculté de Pharmacie, CNRS UMR 9921, 15 Avenue Charles Flahault, 34060 Montpellier Cedex 2, France. E-mail: jp_leo@yahoo.com.
To whom correspondence may be addressed. E-mail:
epg@biomail.ucsd.edu.
Published, JBC Papers in Press, December 20, 2002, DOI 10.1074/jbc.M211447200
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