JOURNAL OF BACTERIOLOGY, Nov. 2010, p. 5580–5587
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 192, No. 21
A Mutation within the ? Subunit of Escherichia coli RNA
Polymerase Impairs Transcription from Bacteriophage
T4 Middle Promoters?
Tamara D. James,1Michael Cashel,2and Deborah M. Hinton1*
Gene Expression and Regulation Section, Laboratory of Molecular and Cellular Biology, National Institute of Diabetes and
Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0830,1and Section on
Microbial Regulation, Eunice Kennedy Shriver National Institute of Child Health and
Human Development, National Institutes of Health, Bethesda, Maryland 20892-27852
Received 25 March 2010/Accepted 12 August 2010
During infection of Escherichia coli, bacteriophage T4 usurps the host transcriptional machinery, redirecting
it to the expression of early, middle, and late phage genes. Middle genes, whose expression begins about 1 min
postinfection, are transcribed both from the extension of early RNA into middle genes and by the activation of
T4 middle promoters. Middle-promoter activation requires the T4 transcriptional activator MotA and coac-
tivator AsiA, which are known to interact with ?70, the specificity subunit of RNA polymerase. T4 motA amber
[motA(Am)] or asiA(Am) phage grows poorly in wild-type E. coli. However, previous work has found that T4
motA(Am)does not grow in the E. coli mutant strain TabG. We show here that the RNA polymerase in TabG
contains two mutations within its ?-subunit gene: rpoB(E835K) and rpoB(G1249D). We find that the G1249D
mutation is responsible for restricting the growth of either T4 motA(Am)or asiA(Am) and for impairing
transcription from MotA/AsiA-activated middle promoters in vivo. With one exception, transcription from
tested T4 early promoters is either unaffected or, in some cases, even increases, and there is no significant
growth phenotype for the rpoB(E835K G1249D) strain in the absence of T4 infection. In reported structures of
thermophilic RNA polymerase, the G1249 residue is located immediately adjacent to a hydrophobic pocket,
called the switch 3 loop. This loop is thought to aid in the separation of the RNA from the DNA-RNA hybrid
as RNA enters the RNA exit channel. Our results suggest that the presence of MotA and AsiA may impair the
function of this loop or that this portion of the ? subunit may influence interactions among MotA, AsiA, and
Bacterial RNA polymerase (RNAP) is a highly conserved
enzyme that shares sequence and structural homology with
multisubunit polymerases from single-celled archaea to multi-
cellular eukaryotes (19, 55). In bacteria, an RNAP core con-
sisting of five subunits (?1, ?2, ?, ??, and ?) combines with a
specificity factor, ?, to form holoenzyme (13). A primary ?
factor, such as ?70of Escherichia coli, is used for promoter
recognition during exponential growth; alternate ? factors are
needed under specific growth conditions or times of stress (10,
34). In addition, proteins that bind to RNAP and/or the DNA
can influence the activity of polymerase (2, 4, 16, 18, 42).
During infection of E. coli, bacteriophage T4 relies on the
host transcriptional machinery for transcription from phage
early, middle, and late promoters (reviewed in references 16,
26, and 54). Immediately after infection, synthesis of early
RNA is driven by T4 early promoters, which contain strong
matches to ?10 and ?35 DNA elements recognized by ?70.
Expression of T4 middle genes is delayed, commencing after 1
to 2 min. Middle RNA is generated both from early promoters,
whose transcripts extend into middle genes, and from specific
middle promoters. While middle promoters contain the ?70-
dependent ?10 element, they have a ?30 element (MotA box)
rather than the ?35 element recognized by ?70.
Two T4-encoded early proteins, MotA, a transcription acti-
vator, and AsiA, a coactivator, are required for the activation
of the middle promoters through a process called ? appropri-
ation (reviewed in references 16 and 18). In this process, AsiA
binds tightly to the C-terminal portion of ?70, region 4 (31, 33,
44, 47), structurally remodeling this region (22) and preventing
its interaction with the ?35 element (3, 9, 27). This remodeling
also allows MotA to interact with the C-terminal end of ?70(6,
36), a portion of ?70that is normally inaccessibly contained
within RNAP (8, 21, 52). In addition, MotA interacts with a
DNA sequence in the ?30 region of middle promoter DNA
(15, 23, 41). Thus, AsiA/MotA serves as a molecular switch:
AsiA-bound RNAP is inactive at most host promoters (1, 7, 35,
43, 45, 46) while AsiA-bound RNAP/MotA activates middle
promoters (17, 32). Understanding how AsiA and MotA inter-
act with all portions of holoenzyme can provide insight into the
intrasubdomain cross talk of RNAP.
Because middle promoters generate much of the prerepli-
cative RNA needed for the synthesis of T4 DNA polymerase
and its associated proteins, T4 motA and asiA mutant phage
exhibit a strong phenotype. Amber (Am) and temperature
sensitive (Ts) mutants exhibit a delay in DNA synthesis and
form small plaques (25, 33) while motA and asiA deletions are
lethal (5, 37). Analyses of RNA isolated from motA(Am) mu-
tant infections reveal that these mutants are leaky; although
* Corresponding author. Mailing address: National Institute of Di-
abetes and Digestive and Kidney Diseases, Building 8, Room 2A-13,
National Institutes of Health, Bethesda, MD 20892-0830. Phone: (301)
496-9885. Fax: (301) 402-0053. E-mail: firstname.lastname@example.org.
?Published ahead of print on 20 August 2010.
transcription from many middle promoters is nearly elimi-
nated, for particular promoters, a low level of RNA can still be
detected (11, 24, 49, 51). The mutant E. coli strain TabG was
originally identified by its inability to support T4 motA mutant
growth (39). Although TabG has no significant growth defect
on its own, a T4 motA(Am) infection of TabG yields no de-
tectable plaques. This defect can be complemented by expres-
sion of a plasmid-borne motA gene (15). Pulitzer and cowork-
ers reported that the TabG mutation maps to a site within or
near the rpoB gene, the gene that encodes the ? subunit of core
In this paper we show that the rpoB gene of TabG contains
two mutations, E835K and G1249D, and that it is the G1249D
mutation that is responsible for restricting the growth of either
a T4 motA(Am) or asiA(Am) mutant. Furthermore, we show
that middle-promoter transcription is significantly impaired in
a T4 wild-type (wt) infection of the mutant rpoB strain and that
the G1249D substitution is responsible for this impairment. In
contrast, transcription from early promoters is not compro-
mised by the rpoB mutation. Structures of RNAP of thermo-
philic bacteria (28, 52) predict that the G1249 residue of the ?
subunit is located at the entry to the RNA exit channel, im-
mediately adjacent to a hydrophobic pocket (switch 3 loop),
which is thought to be responsible for the separation of the
RNA from the DNA-RNA hybrid (53). Our results suggest
that the presence of MotA and AsiA could impair the function
of this loop or that this portion of ? may influence interactions
among MotA, AsiA, and holoenzyme.
MATERIALS AND METHODS
Phage and strains. Wild-type T4D?, T4 amG1 (motA) (25), or T4 amS22
(asiA) (33) were used for infections. The E. coli B strains TabG (39), which is
restrictive for T4 motA(Am) and motA(Ts) growth, and NapIV (30) have been
The rpoB mutations present within the TabG strain were transduced into the
target strain BL21(DE3) (50) using phage P1vir to minimize gene transfer inef-
ficiency due to the E. coli B-K restriction-modification. Two markers, 50% linked
by transduction (29), were first transduced into an E. coli B606 strain from the
donor K-12 strain CF2024, giving a B strain CF15179 bearing btuB::Tn10 (Tcr;
tetracycline resistant; 20 ?g/ml) and rpoB(T563P) (Rifr, rifampin resistant; 100
?g/ml). Phage P1vir grown on the CF15179 B strain was used to transduce the
TabG B strain, selecting Tcrand screening for Rifsrecombinants to obtain strain
CF15192. This increases the likelihood that the rpoBC region beyond RpoB T563
is of TabG origin. Donor P1vir from CF15192 was then used to transduce the
target strain BL21(DE3), and Tcrrecombinants were screened by infecting with
T4 amG1 (motA) to isolate those that could not support T4 motA(Am) growth
(as occurs for the TabG strain). The entire rpoBC genes were sequenced from
one such transductant, strain B11, which revealed only two codon changes from
the wild type. Both occurred within RpoB (E835K and G1249D). Thus, B11 and
BL21(DE3) are isogenic strains except for the rpoB mutations and the
DNA. Plasmids containing either a wild-type rpoB from BL21(DE3) strain or
rpoB(E835K G1249D) from TabG were constructed using standard cloning pro-
cedures. DNA was isolated from the BL21(DE3) and the TabG strain using a
MasterPure DNA Purification Kit (Epicentre). Oligonucleotides directly up-
stream and downstream of the rpoB gene [4,029 bp in length; E. coli BL21(DE3)
chromosome positions 4249149 to 4253177; accession number NC_012947] con-
taining NdeI and BamHI sites, respectively, were used to amplify the rpoB gene.
The PCR product was digested with NdeI and BamHI and ligated with
pet28a(?) (Novagen) previously digested with those restriction enzymes.
The wild-type and rpoB(E835K G1249D) plasmids described above were used
to separate the rpoB(E835K G1249D) mutations into single mutant plasmids.
PCR products were obtained from both plasmids starting at rpoB bp position
2756 and ending at bp position 4029 followed by a BamHI site. These PCR
products were digested with BsgRI and BamHI, generating inserts from rpoB bp
3689 to 4029. The wt and mutant rpoB plasmids were then digested with the same
enzymes. The insert containing wt rpoB DNA from bp 3689 to 4029 was ligated
into the mutant rpoB plasmid, generating pE835K/wt1249. The mutant rpoB
DNA from bp 3689 to 4029 was ligated into the wt rpoB plasmid, generating
pwt835/G1249D. Thus, each plasmid expressed an rpoB gene with only one of the
rpoB genes and, when indicated, the adjacent rpoC gene were sequence veri-
fied in their entirety by the Facility for Biotechnology Resources of the FDA
using PCR products that were generated with primers upstream, downstream,
and within rpoB. Primer sequences are available upon request.
Complementation. The ability of various strains to complement T4 motA-
(Am)or asiA(Am) growth was determined as described previously (6), except
cells containing plasmids were grown in the presence of 20 ?g/ml kanamycin and
1.7 mM isopropyl-?-D-thiogalactopyranoside (IPTG). After growth to mid-log
phase, cells were plated with T4 on LB plates (containing antibiotic, when
plasmid was present).
Isolation of in vivo and in vitro RNA and primer extension analyses. RNA was
isolated from T4-infected cells using method II of Hinton (14). Cultures were
infected for 5 min; chloramphenicol (to 100 ?g/ml) was added immediately
before harvesting. When cells with the plasmid-borne mutant rpoB were used,
cells were grown in the presence of 1.7 mM IPTG.
Primer extension analyses were performed as described previously (11, 15)
using avian myeloblastosis virus (AMV) reverse transcriptase (Life Sciences,
Inc.), 5?32P-labeled oligonucleotides that annealed approximately 100 nucleo-
tides (nt) downstream of the 5? end of predicted mRNAs, and the same amount
of RNA (from 1 to 10 ?g) as measured by absorbance at 260 nm for each analysis
(primer sequences are available upon request). RNA was generated in vitro using
the plasmid pGEX-5X-3 DNA (Pharmacia Biotech), which contains the Ptac
promoter. Transcription reactions were performed as described previously (6)
except that the concentration of each ribonucleoside triphosphate was 200 ?M,
and no labeled ribonucleoside triphosphate was added. In the primer extension
analyses shown in Fig. 2B, this PtacRNA and a 5?32P-labeled primer, which
annealed 66 nt from the start of the PtacRNA, was included in the primer
extension analyses as a control. Labeled primer extension products were sepa-
rated on denaturing, polyacrylamide gels. After autoradiography, films were
scanned using a Powerlook 100XL densitometer, and various species were quan-
tified using Quantity One software from Bio-Rad, Inc.
For quality control purposes, we performed eight independent primer exten-
sion analyses for the gene 46 middle promoter (Pm46) using two independently
isolated RNA preparations of the wt T4 and T4 motA(Am) infections of
BL21(DE3) and B11; for Pe35.3, the gene 46 early promoter, we performed four
independent primer extension analyses using the two different RNA prepara-
tions. B11 and BL21(DE3) are the appropriate strains for this analysis since they
represent the P1 transductant/parent set. The values and errors for Pm46and
Pe35.3shown in Fig. 3 are derived from all of these analyses. Furthermore, Pm46
was also analyzed using different primers. With primers that annealed 222 and
419 nt downstream from Pm46, the amount of Pm46RNA from wt T4 infection
of B11 versus BL21(DE3) was 0.53 ? 0.09 and 0.56 ? 0.05, respectively, which
agrees well with the value of 0.42 ? 0.14 obtained with the Pm46primer used in
the experiments shown in Fig. 2 and 3 that annealed 98 nt downstream. In
addition, the average of values from six primer extension analyses using five
independently isolated RNA preparations of wt T4 infections of TabG versus a
wt rpoB strain [NapIV or BL21(DE3)] was 0.28 ? 0.11, again using the Pm46
primer that annealed 98 nt downstream. Finally, all of the analyses shown in Fig.
3 were performed using the same RNA preparations that were analyzed for Pm46
and Pe35.3RNA levels. Thus, the fact that the levels of the early RNAs (except
Pe148.6) did not decrease in the B11 infections relative to the BL21(DE3) infec-
tions provides an additional control for the differences seen with the middle
E. coli TabG contains two mutations within rpoB, the gene
encoding the ? subunit of RNA polymerase. TabG is an E. coli
B strain, which restricts the growth of the T4 motA(Ts) mutant
tsG1 at the nonpermissive temperature and the growth of the
T4 motA(Am) mutant amG1; the mutation in TabG needed
for this restriction was previously reported to be in or near
rpoB (39). We sequenced the rpoBC operon genes from chro-
mosomal DNA of the TabG and wild-type strain BL21(DE3).
Only two codon changes were found in the TabG strain, both
VOL. 192, 2010 rpoB MUTATION REDUCES T4 MotA-DEPENDENT TRANSCRIPTION5581
within rpoB: E835K and G1249D (Fig. 1). These substitutions
resulted from rpoB nucleotide changes of a G to A at position
2503 and a G to A at position 3746, respectively.
Previous workers reported that the TabG strain had no
growth defects (39). To examine this finding in more detail, we
compared the growth properties of the wt rpoB strain,
BL21(DE3), with those of B11, a P1 transductant of
BL21(DE3) which contains both of the TabG rpoB mutations.
We found no significant difference in the abilities of these
strains to grow on minimal or LB plates incubated at room
temperature, 30°C, 37°C, or 42°C (data not shown).
The G1249D substitution within the ? subunit restricts the
growth of both T4 motA(Am) and T4 asiA(Am) phage. We
compared the ability of T4 wt, T4 motA(Am), and a T4
asiA(Am), amS22, to plate on the wt rpoB strain BL21(DE3)
and on the rpoB mutant strains TabG and B11 (Table 1). wt T4
plaque size and morphology were normal when the phage were
plated on each of these strains, and, as expected, both T4
motA(Am) and T4 asiA(Am) produced small plaques on the wt
strain. However, the growth of either T4 motA(Am) or
asiA(Am) was severely restricted on the rpoB mutant strains,
TabG and B11. T4 motA(Am) produced barely perceptible
plaques on B11 and no detectable plaques on TabG, while T4
asiA(Am) produced no detectable plaques on either strain. We
conclude that the inability of TabG to support T4 motA(Am)
or asiA(Am) growth is due primarily to one or both of the rpoB
mutations. However, additional differences between TabG and
the B11 backgrounds appear to accentuate this effect, allowing
for the very tiny plaques seen in the T4 motA(Am) infections
The presence of wt ?, produced from the plasmid pwtrpoB,
restored the ability of T4 motA(Am) to form small plaques on
either TabG or B11 (Table 1). Conversely, in the wt strain,
which produces wt ? from its chromosome, synthesis of the
plasmid-encoded mutant ? from pE835K/G1249D further de-
creased the plaque size of T4 motA(Am) although it did not
eliminate plaque formation. These results are consistent with
the previous conclusion (39) that the TabG allele is recessive to
wt in its ability to support T4 motA(Am) growth.
To determine whether both ? subunit mutations are neces-
sary for TabG restriction of T4 motA(Am) and T4 asiA(Am)
phage, we constructed rpoB plasmids containing only one of
the substitutions: pE835K/wt1249 or pwt835/G1249D. In each
case, expression of the plasmid-borne rpoB was under the con-
trol of an IPTG-inducible promoter, and SDS-PAGE demon-
strated that each of the mutant ? proteins was produced at a
FIG. 1. Location of TabG rpoB mutations. Schematic representation of the E. coli rpoB gene showing conserved regions (A, B, C, etc.) in dark
blue and positions of the TabG substitutions E835K and G1249D within the ? flap and adjacent to the switch 3 loop, respectively. Sequences of
the residues surrounding the ? flap and the switch 3 loop are given for the wt E. coli, for the E. coli TabG and B11, and for the T. thermophilus
(T. th.) ? proteins.
TABLE 1. T4 plaque phenotypes on E. coli strains
StrainChromosomal rpoB Plasmid
T4 motA(Am)T4 asiA(Am)
bNT, not tested.
cSmaller plaques than those made by T4 motA(Am) in BL21(DE3) without plasmid.
5582JAMES ET AL.J. BACTERIOL.
high level in the presence of IPTG (data not shown). With the
addition of IPTG in the B11 background, the presence of
pE835K/wt1249 complemented either T4 motA(Am) or T4
asiA(Am)growth while the presence of pwt835/G1249D did
not (Table 1). We conclude that it is the G1249D substitution
within rpoB that significantly restricts growth of either T4
motA(Am) or T4 asiA(Am).
rpoB(E835K G1249D) specifically reduces transcription
from T4 middle promoters. Our finding that the TabG muta-
tion is a substitution within the gene for the ? subunit of
RNAP suggested that this mutation might exert its effect at the
level of T4 transcription. To investigate the effect of the TabG
rpoB allele on the production of T4 prereplicative transcripts,
we isolated RNA from wt or T4 motA(Am) infections of two wt
rpoB strains [NapIV and BL21(DE3)] and two rpoB(E835K
G1249D) mutant strains (TabG and B11). RNA was isolated 5
min after infection at 37°C, a time point when middle tran-
scripts are the predominant species, but there are residual
early transcripts, and late transcripts have started to be syn-
thesized (reviewed in references 12, 40, and 48). The amount
of transcription from a number of T4 early and middle pro-
moters was then determined using primer extension analyses.
Previous work has shown that the T4 middle gene 46, which
encodes a nuclease important for T4 recombination, is ex-
pressed from both the early promoter (Pe35.3), located 678 bp
upstream, and the MotA/AsiA-dependent middle promoter
(Pm46), located 30 bp upstream of the gene; in addition, tran-
scription from promoters farther upstream, including Pm47,
also extend into the gene (11, 20, 24, 26) (Fig. 2A). As ex-
pected, primer extension analyses performed with RNA from
FIG. 2. The level of T4 gene 46 RNA generated from the MotA-dependent middle promoter Pm46is reduced by the TabG rpoB allele.
(A) Schematic of gene 46 region from T4 map units 36576 (middle promoter Pm47) to 34916 (position of primer) (map units are from reference
26). The relative positions of genes and identified promoters in this region are shown. Transcription from Pm47, Pe35.3, and Pm46yields primer
extension products of 1,660, 746, and 98 nt, respectively. (B) Gels showing the primer extension products of gene 46 RNA. RNA was isolated from
wt T4 (?) or T4 motA amG1 (am) infections of the indicated wt rpoB (?) strains, BL21(DE3), NapIV, or rpoB(E835K G1249D) mutant (m)
strains, B11 or TabG. PtacRNA and a32P-labeled primer that anneals 66 nt from the start of the PtacRNA were added to the primer extension
analyses of the T4 RNA (lanes 1 to 4 and 6 to 9) as a control. The analysis in lane 5 contained the PtacRNA and Ptacprimer alone.
VOL. 192, 2010 rpoB MUTATION REDUCES T4 MotA-DEPENDENT TRANSCRIPTION 5583
wt T4 infection of wt rpoB (wt T4/wt rpoB) revealed products
corresponding to transcription from both Pe35.3and Pm46(Fig.
2B, lanes 1 and 6), and transcription from Pm46was dependent
on MotA (Fig. 2B, lanes 3 and 8).
The primer extension analyses of gene 46 RNA isolated
from wt T4 infections of either of the mutant ? strains (T4/B11
or T4/TabG) revealed patterns that were qualitatively similar
to those seen with the wt T4/wt rpoB infections (Fig. 2B, lanes
2 and 7 versus lanes 1 and 6). Transcription was observed from
promoters far upstream, from the early promoter Pe35.3, and
from the MotA/AsiA-dependent middle promoter Pm46. How-
ever, while the amount of transcription from Pe35.3was similar
in these lanes, the amount of Pm46was significantly reduced
relative to that seen in infections of the wt strains (Fig. 2B,
compare lane 1 versus 2 and lane 6 versus 7).
To ensure that the differences in RNA levels were not due to
experimental error, we checked our analyses in a number of
ways. First, for the primer extension analyses shown in Fig. 2B,
we included RNA made in vitro from a plasmid that contains
the Ptacpromoter and an additional32P-labeled primer that
anneals 66 nt downstream of the predicted transcription start.
Primer extension using the PtacRNA and the primer alone
revealed a major product of 66 nt as well as slightly larger
minor products (Fig. 2B, lane 5). Addition of the PtacRNA and
primer to the in vivo RNA yielded similar levels of the Ptac
primer extension products in all the lanes (Fig. 2B, lanes 1 to
4 and 6 to 9). Second, we performed multiple primer extension
analyses using independently isolated RNA preparations of wt
T4 and T4 motA(Am) infections (see Materials and Methods
for details). The values and errors for Pm46and Pe35.3that are
shown in Fig. 3 are derived from all of these analyses. Thus, we
conclude that in a wt T4 infection of the rpoB mutant strain
B11, there is a specific decrease in the level of Pm46RNA
relative to that observed in an infection of the wt rpoB parent
strain BL21(DE3) while the level of Pe35.3RNA does not
To investigate whether the findings with gene 46 RNA were
general for T4 prereplicative transcription, we performed
primer extension analyses of RNA isolated from wt T4 infec-
tions of B11 or its parent strain BL21(DE3) using primers that
FIG. 3. Transcription from MotA-dependent T4 middle promoters is specifically reduced in the rpoB(E835K G1249D) mutant strain B11 versus
the wt rpoB strain BL21(DE3). Histograms show the levels of RNA from various middle promoters (top two panels) or early promoters (bottom
panel) made in the indicated infections: solid black, wt T4/BL21(DE3); solid red, wt T4/B11; open black, T4 motA(Am)/BL21(DE3); open red,
T4 motA(Am)/B11. A portion of the gel displaying the indicated primer extension products for each promoter is also shown. [Values for T4
motA(Am) infections were obtained using longer exposures.] For cases in which the primer extension analyses were performed three or more times,
an error bar is shown. In the other cases, values from two analyses were averaged. PmuvsXRNA has multiple ends due to the iterative addition
of As at the start of the transcript (15).
5584 JAMES ET AL. J. BACTERIOL.
annealed downstream of various T4 middle and early promot-
ers (Fig. 3). As indicated in the legend of Fig. 3, values were
obtained from two or more independent primer extension ex-
periments; those analyses performed three or more times are
shown with error bars. We observed the same general trend as
was seen for gene 46 transcription (Fig. 3, first and second
lanes of each panel). Transcription from all of the tested mid-
dle promoters, except Pm32, was reduced 2- to 8-fold while
transcription from early promoters, except Pe148.6, was the
same or enhanced. The fact that the levels of the early tran-
scripts (except Pe148.6) did not decrease in the B11 infections
relative to the BL21(DE3) infections indicates that the overall
level of T4 RNA is not generally suppressed in the B11 infec-
tions. Furthermore, the levels of RNA from the early promot-
ers for MotA (P161.1) and AsiA (P158.7) did not decrease,
indicating that the mutant ? protein in B11 did not simply
lower the level of motA or asiA RNA. Taken together, our
results indicate that the rpoB allele in TabG specifically impairs
transcription from T4 middle promoters.
As with Pm46(Fig. 2B, lane 3), we observed a small amount
of RNA from many middle promoters in the T4 motA(Am)
infections of the wt rpoB strain (Fig. 3, third lane of each
middle promoter panel). This low level of middle promot-
er RNA has been observed by other investigators in T4
motA(Am) infections of wt E. coli (11, 24, 49, 51). Previous
work has demonstrated that deletion of motA is lethal (5),
suggesting that the motA(Am) mutation is leaky. Thus, this
mutant is expected to generate a small amount of full-length
MotA protein, allowing a low level of middle-promoter tran-
scription. We find that a T4 motA(Am) infection of B11 (rpoB
mutant) further reduces this residual middle promoter tran-
scription (Fig. 3, compare the third and fourth lanes of each
middle promoter panel). These results suggest that it is this
further impairment of middle-promoter activation by the
TabG mutant ? protein that is responsible for preventing the
growth of T4 motA(Am) and indicates that middle promoter
activity is essential for T4 development.
The G1249D mutation within rpoB is responsible for the
defect in T4 middle-promoter transcription. We repeated the
primer extension analyses, isolating RNA from wt T4 infec-
tions of B11 grown in the presence of IPTG and a plasmid with
only one of the rpoB substitutions: either pE835K/wt1249 or
pwt835/G1249D. Each of these plasmids produced similar lev-
els of the singly mutant ? protein, as judged by SDS-PAGE
(data not shown). Using the same RNA preparations, we an-
alyzed RNA from both T4 early and middle promoters (Fig. 4).
In the absence of a plasmid, middle-promoter transcripts were
again reduced in B11 relative to BL21(DE3) (Fig. 4, lanes 1
and 2). The rpoB plasmid with the wt glycine at 1249 (lane 3)
restored the level of middle RNA to that observed in a wt T4
infection of the wt rpoB strain (lane 1), whereas the plasmid
with the mutant G1249D did not (lane 4). Thus, we conclude
that it is the G1249D mutation that impairs T4 MotA/AsiA-
As seen before (Fig. 3), the levels of transcription from the
early promoters Pe35.3, P158.7, and P161.1, which are immedi-
ately upstream of the early genes 46.2, asiA, and motA, respec-
tively, were similar or higher in the T4/B11 than in the T4/wt
rpoB infections (Fig. 4, lanes 1 versus 2). Furthermore, the
presence of either singly mutant plasmid affected these early
RNAs similarly, raising the levels even further over those seen
in the T4/wt rpoB infection (lanes 3 and 4). These results
indicate that simply increasing motA and asiA transcription
cannot compensate for the impairment of middle promoter
transcription imparted by the mutant ? protein.
Bacteriophage T4 gene expression is controlled primarily at
the level of transcription through the activity of phage early,
middle, and late promoters. Middle transcription, which gen-
erates the prereplicative RNAs needed for the synthesis of
phage replication proteins, begins approximately 1 to 2 min
after infection at 37°C (reviewed in references 26 and 54). Two
T4 early proteins, the transcriptional activator MotA and
the coactivator AsiA, are required for transcription from
nearly 60 known T4 middle promoters (reviewed in refer-
ences 16 and 18).
The E. coli mutant TabG was originally isolated as a strain
that significantly restricts the growth of the T4 motA amber
mutant, amG1, and the motA temperature sensitive mutant,
tsG1 (39). TabG also restricts T4 asiA(Am), amS22, as we have
demonstrated here. Pulitzer and coworkers mapped the TabG
mutation to a site in or near rpoB. However, the nature and
exact location of the lesions as well as the reason why TabG
restricts T4 motA(Am) were unclear. We have shown that
TabG rpoB contains two substitutions, E835K and G1249D.
Though these mutant residues are separated by over 1,200 bp
in the rpoB DNA sequence (Fig. 1), they are relatively close in
the structures of RNAP from the thermophilic bacteria, Ther-
mus thermophilus and Thermus aquaticus (28, 52) (Fig. 5). The
residue corresponding to E835 in E. coli (R707 of T. ther-
mophilus) is located near the DNA-RNA hybrid at the base of
a loop within the ? subunit called the ? flap, which forms one
FIG. 4. The rpoB mutation G1249D is responsible for the defect in
MotA/AsiA-activated transcription in a wt T4 infection. Portions of
gels showing primer extension products were obtained after wt T4
infections of the following: wt rpoB strain BL21(DE3) (lane 1), the
double mutant rpoB(E835K G1249D) strain B11 (lane 2), B11 contain-
ing pE835K/wt1249 (lane 3), and B11 containing pwt835/G1249D.
VOL. 192, 2010 rpoB MUTATION REDUCES T4 MotA-DEPENDENT TRANSCRIPTION 5585
wall of the RNA exit channel. Our results suggest that the
E835K mutation has no significant effect on the growth of T4
or the host, at least under our conditions. G1249 (G1011 of T.
thermophilus) is a highly conserved residue, located on the
opposite side of the RNA exit channel. Although this mutation
does not appear to be deleterious for the host or for transcrip-
tion from T4 early promoters, our analyses reveal that G1249D
confers a defect in MotA/AsiA-dependent transcription. Based
on the structure of a T. thermophilus RNAP elongating com-
plex (53), G1249 is immediately adjacent to a structural feature
called the switch 3 loop (T. thermophilus ? residues 1012 to
1022, corresponding to E. coli residues 1250 to 1260) (Fig. 1
and 5). This portion of ? is located at the upstream end of the
RNA-DNA hybrid. It has been suggested that switch 3 loop
interacts with the RNA, guiding it away from its interaction
with the DNA and leading it into the RNA exit channel (53).
Thus, this loop is thought to perform an extremely important
function for RNAP. However, since the G1249D change is not
yet associated with a phenotype other than its effect on MotA/
AsiA transcription, this change appears to be an alteration that
is relatively benign for the host.
Activation of T4 middle promoters by MotA and coactivator
AsiA occurs through a process called ? appropriation (re-
viewed in references 16 and 18). In this process, AsiA binds
tightly to free ?70, structurally remodeling region 4, located in
the C-terminal portion of the ?70protein. When the AsiA/?70
complex associates with core, this remodeling prevents ?70
from interacting with the ?35 region of promoter DNA as well
as the ? flap. Consequently, MotA can interact with its binding
site, the MotA box, centered at ?30 of middle promoter DNA,
as well as with residues at the very end of the C terminus of ?70,
which would normally be inaccessible due to their interaction
with the ?-flap tip. Recent work has indicated that AsiA also
binds the ? flap, replacing the normal interaction between ?70
region 4 and this portion of the ? protein (57). A direct inter-
action between AsiA and MotA has also been suggested (56)
although other work indicates that they do not directly interact
(6, 38). Thus, the RNAP/MotA/AsiA complex consists of mul-
tiple protein-protein interactions as well as a major conforma-
tional change surrounding ?70region 4. A thorough under-
standing of the roles of the ? flap and ?70region 4 and how T4
employs these regions to its advantage may provide insight into
how the activity of RNAP is regulated.
We can think of three simple models to explain how the
G1249D substitution, which will introduce both a negative
charge and a decrease in flexibility, might impair MotA/AsiA
activation. First, since this substitution lies on a face of ? that
is across from the ? flap (Fig. 5), it seems possible that it might
impair the association of the AsiA/?70complex with the core,
thus affecting the first step in ? appropriation, or impair an
association of AsiA with the ? flap. Alternately, the binding of
AsiA and MotA to RNAP could impose subtle conformational
alterations that extend to the switch 3 loop. In particular, the
presence of the additional negative charge from the G1249D
substitution might now affect the exit of the negatively charged
RNA if MotA-induced or AsiA-induced changes in RNAP
extend to the RNA exit channel. Finally, the G1249D substi-
tution may identify a region that directly interacts with either
AsiA or MotA. In vitro experiments with purified mutant
RNAP can test each of these hypotheses. Understanding how
the TabG mutation affects MotA/AsiA activation will help
elucidate the global arrangement of protein-protein interac-
tions needed for T4 middle gene transcription.
We thank C. Jones, K. Decker, L. Knipling, M. Hsieh, R. Bonocora,
and T. Cardozo for helpful discussions.
This research was supported in part by the Intramural Research
Program of the NIH, National Institute of Diabetes and Digestive and
Kidney Diseases (T.D.J. and D.M.H.) and by the NIH, Eunice
Kennedy Schriver National Institute of Child Health and Human De-
velopment (M.C.). T.D.J. was also supported by a Ford Foundation
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