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(Received for publication, September 18, 1995; and in revised form, November 2, 1995) From the
A transcription termination factor (Rho) was purified from the
Gram-positive bacterium Micrococcus luteus, and the complete
gene sequence was determined. The M. luteus Rho polypeptide
has 690 residues, which is 271 residues more than its homolog from Escherichia coli. Most of the additional residues compose a
highly charged, hydrophilic segment that is inserted in a nonconserved
region between two conserved regions of the RNA-binding domain of the
known Rho homolog proteins. This segment extends from residues 49 to
311 and includes a stretch of 238 residues that contain no hydrophobic
side chains. Biochemical studies indicate that the M. luteus protein is very similar to E. coli Rho in terms of its
RNA-dependent NTPase activity and its sensitivity to the Rho-specific
inhibitor bicyclomycin. However, the M. luteus protein has a
less stringent RNA cofactor specificity. It also acts to terminate RNA
transcription with E. coli RNA polymerase on the
The orderly expression of the genetic information in DNA
segments into RNA molecules depends on the function of transcription
terminators. In Escherichia coli, one mechanism of
transcription termination is mediated in part by an essential protein
factor called Rho(1) . Rho factor from E. coli has
been studied since its discovery nearly 25 years ago(2) . The
Rho monomer is a 47-kDa protein. However, Rho factor functions as a
homohexamer (3) that can bind to a nascent transcript and
mediate its release by actions on the transcription complex that are
coupled to the hydrolysis of NTPs(1) . A recent phylogenetic
study by Opperman and Richardson (4) comparing rho genes isolated from organisms from several of the major branches
of bacteria suggests that Rho is ubiquitous throughout the bacterial
domain. An unexpected result was discovered during the analysis of the rho gene from Micrococcus luteus, a Gram-positive
soil bacterium that has an unusually high G + C DNA content
(74%)(5) . The M. luteus rho homolog was found to have
an open reading frame encoding a protein that was homologous to E.
coli Rho through a very long portion. However, the homology did
not extend all the way through the RNA-binding domain toward the amino
terminus of the protein. Because the region of homology starts in a
segment that has an in-frame GTG codon preceded by a sequence that is a
good match to a Shine-Dalgarno sequence, Opperman and Richardson
proposed that translation began at that GTG codon to yield a 41,733-Da
protein of 382 amino acids that is 52% identical (71% similar) to E. coli Rho. If this proposal were correct, the M. luteus Rho protein would be unusual in comparison with the other Rho
homologs as it would lack a conserved part of its RNA-binding domain.
Additionally, the protein would be The
data of Opperman and Richardson (4) were also consistent with
an alternative hypothesis, namely that the M. luteus Rho
polypeptide is much larger than the homologs from other organisms and
includes a large region with a very unusual amino acid sequence. The
DNA sequence determined in that work indicated that the open reading
frame extended upstream for at least 160 amino acid residues. However,
because the G + C content of that upstream region was To resolve this issue, we purified Rho protein from M.
luteus. Our studies show conclusively that the latter hypothesis
is correct and demonstrate directly that an organism that is
phylogenetically distinct from E. coli also has a factor that
can cause the termination of RNA transcription.
To identify a 203-kbp ( It was
determined that a BamHI/PstI double digest of M.
luteus genomic DNA produced a
Analysis of the final fraction
by electrophoresis on a 10% SDS-polyacrylamide gel (8) revealed
that it consisted of a single polypeptide in >95% purity with an
apparent M
Figure 1:
Gel electrophoretic analysis of the
RNA-dependent ATPase from M. luteus. The protein samples were
separated by electrophoresis on a 10% polyacrylamide gel with the
Laemmli buffer system(8) . Lane M, marker proteins (in
kilodaltons); lane Ec, 2 µg of E. coli Rho
protein; lane Ml, 4 µg of the RNA-dependent ATPase
purified from M. luteus.
A sample of this highly purified M.
luteus ATPase, analyzed by the Microsequencing Facility at Harvard
University, yielded an N-terminal amino acid sequence of TESTE, which
is different from the sequence of MAGIL at the N terminus of the
proposed M. luteus rho gene product(4) . This sequence
also did not match any other pentapeptide sequence in the segment of
the M. luteus rho gene that had been sequenced prior to this
work. However, a 42-kDa fragment generated from the 95-kDa protein by
partial digestion with trypsin had the N-terminal sequence GRPGPEVDE,
which did match a sequence located upstream of the previously proposed rho translational start site(12) . Together, these
results concerning the apparent size and N-terminal sequence of the M. luteus RNA-dependent ATPase suggested that the M.
luteus rho gene sequence reported by Opperman and Richardson (4) was not complete.
Figure 2:
Nucleotide and predicted amino acid
sequences of the M. luteus rho gene. The sequences were
determined by sequencing both DNA strands. The Shine-Dalgarno sequence
is indicated (underlined).
To
test whether the lower activity M. luteus Rho had with CTP was
related to the RNA cofactor used, the rate of CTP hydrolysis was
measured with poly(U) and was found to be
Figure 3:
Bicyclomycin inhibits M. luteus Rho ATPase activity. ATP hydrolysis at 37 °C was measured in
standard Rho ATPase reaction mixtures containing 58 nM Rho (M. luteus (
Figure 4:
M. luteus Rho terminates
transcription. Ternary transcription complexes stalled on a
To
show that the smaller transcripts were the result of M. luteus Rho action as a transcription termination factor rather than as a
ribonuclease, transcripts synthesized in the absence of Rho factor were
subsequently incubated with M. luteus Rho (Fig. 4, lane 11). Although a small amount of a 145-nucleotide RNA
appeared, the fact that no other RNA molecules appeared that had the
same sizes as the products made when M. luteus Rho was present
cotranscriptionally rules out the possibility that they were generated
by a ribonuclease activity. Because very few of the transcripts were
extended to the size of the readthrough RNA when M. luteus Rho
was present during transcription, the overall efficiency of termination
within the transcribed fragment was nearly 100% Two lines of
evidence suggest that the 145-nucleotide RNA arose as a result of a
contamination of M. luteus Rho with a ribonuclease. First, the
extent of appearance of the 145-nucleotide RNA was higher with other,
less pure preparations of the M. luteus factor (data not
shown). Second, it also appeared when the function of M. luteus Rho was inhibited by bicyclomycin (Fig. 4, lanes 12 and 13). A comparison of the distribution of
transcripts in reaction mixtures lacking Rho that had been quenched at
2, 5, and 8 s after initiation (Fig. 4, lanes
1-3) with the distribution of the terminated transcripts (lanes 9 and 10) indicated that, as with E. coli Rho, the preferred positions for termination stop points were at
the positions where RNA polymerase naturally pauses. However, with M. luteus Rho, the termination occurred at pause sites that
were farther upstream than the pause sites that were used as the
termination points by E. coli Rho. We have isolated a transcription termination factor from M. luteus that is phylogenetically related to transcription
termination factor Rho from E. coli. Although rho homologs have been identified from several different phylogenetic
branches of
bacteria(4, 25, 26, 27, 28) ,
this is the first demonstration that an organism that is distantly
related to E. coli actually expresses its rho homolog
gene. Although M. luteus Rho is similar to E. coli Rho in having a broad NTP substrate specificity, in its turnover
number with poly(C) as a cofactor, and in its sensitivity to inhibition
with bicyclomycin, it differs in having a less stringent RNA cofactor
specificity and in its specificity of termination during transcription
of a coliphage gene with E. coli RNA polymerase. We have also
found that M. luteus Rho differs from E. coli Rho in
containing an extended insertion of very unusual sequence and likely
structure within its RNA-binding domain. M. luteus belongs
to the phylogenetic branch called the high G + C Gram-positive
group. The G + C content of its DNA is An exceptionally unusual feature of M.
luteus Rho is the amino acid composition of the insert in the
RNA-binding domain between the two phylogenetically conserved sequence
segments that are found in the RNA-binding domain of all the known Rho
sequences. In M. luteus Rho, this insert is between Ile
Figure 5:
Schematic representation of the M.
luteus and E. coli Rho polypeptides. The M. luteus and E. coli Rho polypeptides have been drawn to scale.
The relative positions of amino acid insertions are compared with E. coli Rho and are indicated by diverging lines. The black area in M. luteus Rho represents the 263-amino
acid segment between Ile
The sequences of two other rho genes from this same group
of organisms have recently become available: the genes from Streptomyces lividans( M. luteus Rho also contains another smaller insertion sequence that runs
from Lys
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank(TM)/EMBL Data Bank with accession number(s)
L27277[GenBank].
Volume 271,
Number 2,
Issue of January 12, 1996 pp. 742-747
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
C Gram-positive
Bacterium Micrococcus luteus(*)
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
cro DNA template, but at much earlier termination stop points than
those recognized by E. coli Rho. Thus, the M. luteus protein functions as a true Rho factor, but with a different
specificity than that of E. coli Rho. We propose that this
altered specificity is consistent with its need to function on
transcripts that have a high content of G + C residues.
30 amino acids smaller than any
of the other predicted Rho factors that have been sequenced.
78%,
which is a value that is typical of intergenic spacer regions in M.
luteus DNA(5) , and because these upstream codons had a
very unusual bias favoring Arg, Asp, Gln, and Gly residues and lacking
hydrophobic residues, Opperman and Richardson argued that it was
unlikely to be part of the coding region for the M. luteus Rho
protein.
Materials
Restriction enzymes, T4 DNA ligase,
Vent DNA polymerase, and DNA polymerase I Klenow fragment were
purchased from New England Biolabs Inc. Sequenase version 2.0 was
purchased from U. S. Biochemical Corp. E. coli RNA polymerase
was purchased from Epicentre Technologies Corp. E. coli Rho
protein was provided by Lislott Richardson (Indiana University). NusG
was a gift from Barbara Stitt (Temple University). Bicyclomycin was
obtained from Fujisawa Pharmaceutical Co. Ltd. (Osaka, Japan). L-1-Tosylamido-2-phenylethyl chloromethyl ketone-treated
trypsin was from Worthington. Radioactive nucleotides were purchased
from ICN Radiochemicals. Ribonucleotides, deoxynucleotides, and
dideoxynucleotides were purchased from Pharmacia Biotech Inc.
7-Deaza-2`-dGTP was purchased from Boehringer Mannheim. Polynucleotides
were purchased from Miles Inc. Standard primers used for sequencing,
custom DNA oligonucleotides, and random deoxynucleotides
(d(N)
) were synthesized at the Institute for Molecular and
Cellular Biology at Indiana University by Lawrence Washington.ATPase Assay
ATPase activity was assayed
colorimetrically as described by Lanzetta et al.(6) .
Typically, 50 ng of protein were mixed with 100 µl of assay
solution (40 mM Tris-HCl, pH 7.7, 50 mM KCl, 10
mM MgCl
, 1 mM ATP, and 10 µg/ml
poly(C)). After 10 min at 37 °C, the release of P
from
ATP was detected by the addition of 800 µl of a mixture of 4.2%
ammonium molybdate, 0.045% malachite green, concentrated flame
photometer diluent (Bacharach, Inc.) (12.5:37.5:1) that had been
premixed and filtered through Whatman No. 1 filter paper. Color
development was quenched after 1 min by the addition of 100 µl of
34% citric acid. After 30 min at 20 °C, the absorbance at 660 nm
was measured. One unit is defined as the amount that hydrolyzes 1
µmol of ATP/min. This assay was found to be quantitative from 1 to
15 nmol of P.Protein Sequencing
10 µg of the intact protein
were bound to a strip of polyvinylidene difluoride membrane by direct
adsorption (7) . The N terminus was sequenced by William Lane
at the Harvard University MicroChem Laboratory. To obtain N-terminal
sequence information of a stable tryptic fragment, 16.8 µg of Rho
protein was digested in the presence of 105 nM trypsin for 3
min at 37 °C in a total volume of 30 µl. Proteolysis was
stopped by the addition of 10 µl of 4 
sample loading buffer
(252 mM Tris-HCl, pH 6.8, 8% SDS, 40% glycerol, 20%
2-mercaptoethanol, and 0.004% bromphenol blue) followed by immersion of
the mixture in a boiling water bath for 2 min. The products were
separated by electrophoresis on an 8% SDS-polyacrylamide
gel(8) , and the protein bands were transferred to
polyvinylidene difluoride membrane with a Pharmacia Biotech 2117
Multiphor II blotting apparatus. The transfer was carried out in Towbin
buffer (25 mM Tris-HCl, pH 8.2, 192 mM glycine, 15%
methanol) (9) for 1 h at 30 mA. The protein fragments were
visualized by Coomassie Blue staining. The band with an M
of 42,000 was removed, washed with double
distilled HO, air-dried, and stored at -20 °C.
N-terminal sequencing was performed by Brian VanWuyckhuyse at the
Protein Sequencing Facility at the University of Rochester Medical
Center (Rochester, NY).Isolation of the Upstream Portion of the M. luteus rho
Gene
Genomic DNA was prepared from M. luteus as
described by Wilson(10) . The method employs the detergent
hexadecyltrimethylammonium bromide to remove proteins and
polysaccharides. A typical yield from a saturated 100-ml culture was
0.7 µg of DNA.
)fragment
containing the sequence encoding the N-terminal region of the M.
luteus rho gene, 10 µg of genomic DNA were digested with
various combinations of restriction endonucleases, and the products
were separated by agarose gel electrophoresis. Fragments containing the
desired rho gene segment were identified by Southern
hybridization (11) with a 0.28-kbp HindIII/SmaI fragment from the plasmid
pMLRHOSK(12) , which had been radiolabeled to
a high specific activity with
P(13) .
2-kbp fragment that contained
the desired portion of the M. luteus rho gene. To clone this
DNA fragment, 50 µg of M. luteus DNA were digested with BamHI and PstI, and the fragments were size-selected
on a 1% agarose gel and ligated into pBluescript II SK
(Stratagene). Colonies of E. coli DH5
F` transformed
with these ligated plasmids were screened by nucleic acid hybridization (14) with the same HindIII/SmaI fragment used
for Southern analysis.DNA Sequencing
Both double-stranded and
single-stranded DNA templates were utilized for sequencing
reactions(15) . Double-stranded templates were prepared as
described in the Sequenase protocol manual (U. S. Biochemical Corp.).
Single-stranded DNA was generated from pBluescript derivatives by
infection with M13K07 helper phage(16) . Reactions were carried
out according to the manufacturer's instructions with the
following modifications. 7-Deaza-dGTP was used in place of dGTP to
reduce band compressions(17) , and a dideoxy-dGTP/7-deaza-dGTP
ratio of 1:15 and a dideoxy-CTP/dCTP ratio of 1:100 were used in the
termination mixtures to enhance dG and dC signals.RNA Transcription
A DNA fragment encoding the
cro gene was prepared by amplification of the segment of
the gene from residues -188 to +372 from pCBC1 DNA (
)with Vent DNA polymerase. This plasmid contains a mutation
of C to G at position 6 of the cro gene. Transcription complex
formation was carried out according to Burns and Richardson (18) with slight modifications. In a reaction volume of 60
µl, 3 pmol of E. coli RNA polymerase were incubated with 3
pmol of DNA in transcription buffer (150 mM potassium
glutamate, pH 7.8, 40 mM Tris acetate, pH 7.8, 4 mM Mg(OAc), 1 mM dithiothreitol, 0.02% Nonidet
P-40, 0.002% acetylated bovine serum albumin, 1% glycerol) for 3 min at
37 °C to form open complexes. The addition of 40 µl of NTP
mixture (4 µM GTP, 4 µM ATP, 4 µM [-P]UTP (350 nCi/pmol)) and incubation
at 16 °C for 20 min resulted in the formation of stalled ternary
complexes 24 residues downstream from the start point of transcription
of
P
(A complex). After the addition of 5
µl of rifampicin (1 mg/ml), the unincorporated NTPs were removed by
ultrafiltration (20 min, 500
g) with a Microcon-100
(Amicon, Inc.). The retentate (10 µl) was diluted to 200 µl
with transcription buffer containing 36 units of rRNasin (Promega). 10
µl of this diluted A complex solution were used per
reaction. After the addition of Rho factor, all four NTPs (including
CTP) were added to 200 µM, and the 20-µl reaction
mixture was incubated at 37 °C for 3 min. After the addition of 20
µl of 2
stop mixture (20 mM EDTA, 0.1% SDS, 0.5
mg/ml tRNA, 0.3 mg/ml proteinase K) and incubation at 37 °C for 20
min, the RNA was collected by ethanol precipitation and resuspended in
6 µl of loading dye (10 mM EDTA, 0.001% bromphenol blue,
0.001% xylene cyanol in 98% formamide). The entire sample was loaded on
a 6% polyacrylamide gel (20 40 cm) containing 50%
urea(14) , and the RNA transcripts were separated by
electrophoresis for 2 h at 30 watts.
Purification of an RNA-dependent ATPase from M.
luteus
From preliminary studies, we found that the ATPase
activity in partially fractionated extracts of M. luteus cells
was stimulated by the addition of RNA homopolymers and that poly(C) was
especially effective. Since E. coli Rho is an RNA-dependent
ATPase that is strongly activated by poly(C) (19) , we made use
of an assay for ATP hydrolysis with poly(C) present for purification of
a putative Rho factor from M. luteus. The purification
procedure, which is described in detail elsewhere (20) ,
involved chromatography of the crude extract on a Bio-Rex 70
cation-exchange column (Bio-Rad), concentration of the pooled fractions
containing poly(C)-dependent ATPase with a Centriprep-100
ultrafiltrator (Amicon, Inc.), and chromatography on heparin-Sepharose
CL-6B resin (Pharmacia Biotech Inc.). of 95,000 (Fig. 1), which is
approximately twice as large as the E. coli Rho polypeptide
and also significantly larger than the M. luteus rho gene
product (M = 41,733) proposed by Opperman
and Richardson(4) .
Location of the TESTE Sequence
The M. luteus
rho gene identified in the previous study (12) was cloned
as a 2.1-kbp SphI/SacI DNA fragment in the plasmid
pMLRHOSK. The remainder of the M. luteus rho gene was cloned as a 2-kbp BamHI/PstI DNA
fragment into pBluescript II SK
to create the plasmid
pBN10. We intentionally chose a fragment that would overlap partially
with the rho insert in pMLRHOSK
. The DNA
sequence of this fragment confirmed that the target DNA had been
successfully isolated and contained
1200 base pairs of additional
upstream M. luteus sequence. 111 base pairs (37 residues)
upstream from the SphI site, we found a segment encoding the
sought-after N-terminal pentapeptide sequence TESTE, which had been
identified from microsequencing of the purified RNA-dependent ATPase.
This result demonstrated that the RNA-dependent ATPase is indeed
encoded by the gene identified by Opperman and Richardson(4) .
Although the open reading frame continues upstream for 108 base pairs
from the codon of the first Thr residue, the next upstream codon is
GTG, which is used as the start codon in
50% of the M. luteus genes (5) , and it is preceded by an excellent
Shine-Dalgarno sequence (Fig. 2). We thus conclude that
translation of the M. luteus rho gene starts at the GTG codon
at position 1 and that the initiating Met residue is removed
post-transcriptionally. The amino acid composition of the predicted
protein from the completed gene sequence is in excellent agreement with
the amino acid composition of the purified protein (Table 1). The
molecular mass of the M. luteus Rho protein is 74,957 Da;
therefore, the protein runs anomalously (
95 kDa) in the Laemmli
gel system (8) (Fig. 1).
Substrate Specificity
It has previously been
demonstrated that the E. coli Rho protein is capable of
catalyzing the hydrolytic conversion of any one of the four
ribonucleoside triphosphates to the corresponding nucleoside
diphosphate and P in the presence of an RNA cofactor such
as poly(C)(21) . This aspect of M. luteus Rho was
investigated by assaying for P release with each of the
four different NTPs (Table 2). M. luteus Rho was able to
utilize all four rNTPs as well as dATP as substrates, but only when RNA
was present. These results indicate that M. luteus Rho, like E. coli Rho, is an RNA-dependent NTPase. However, M.
luteus Rho differs from E. coli Rho in having a
significantly lower activity with CTP. The results also show that, on a
molecular basis, M. luteus Rho catalyzed ATP hydrolysis at the
same rate as E. coli Rho.
M. luteus Rho Has a Broader RNA Cofactor Specificity than
E. coli Rho
To determine whether M. luteus Rho is
significantly different from E. coli Rho with respect to its
RNA-dependent ATPase activity, the rate of ATP hydrolysis of the two
proteins was determined in the presence of various synthetic
polynucleotide homopolymers (Table 3). Both proteins were
dependent on the presence of an RNA for ATP hydrolysis, and neither
hydrolyzed ATP in the presence of poly(dC). Of the RNA polymers tested,
poly(C) was the most effective activator for both proteins. M.
luteus Rho, however, had appreciable activity with poly(A) as well
as relatively high activity with poly(U) and measurable activity with
poly(I). This was significantly different from E. coli Rho.
Thus, the spectrum of RNA molecules that can activate ATP hydrolysis is
greater for M. luteus Rho than for E. coli Rho.
30% of that with ATP
(data not shown). Thus, the lower activity of M. luteus Rho
with CTP was not a consequence of using poly(C) as an activator.
Bicyclomycin Inhibits the ATPase Activity of M. luteus
Rho
Bicyclomycin is an antibiotic that has recently been shown
to specifically inhibit E. coli Rho function(22) .
Mutants that exhibit bicyclomycin resistance contain mutations in the
ATPase domain of the Rho protein(22) . Because M. luteus Rho contains strong sequence homology in that conserved region, it
was hypothesized that bicyclomycin would be an inhibitor of the M.
luteus Rho protein. To investigate this, the standard ATPase assay
was performed in the presence of increasing concentrations of
bicyclomycin (Fig. 3). The results reveal that bicyclomycin is a
potent inhibitor of M. luteus Rho. In the presence of 25
µM bicyclomycin, the lowest concentration tested, both the E. coli and M. luteus Rho proteins retain <30% of
their poly(C)-dependent ATPase activity. Activity continues to decrease
with increasing bicyclomycin concentrations and is nearly abolished at
200 µM.
) or E. coli (
)), 10
µg/ml poly(C), and bicyclomycin as indicated. Reactions were
initiated by the addition of ATP (final concentration of 1 mM)
to prewarmed solutions, and P release was detected
colorimetrically. 100% activity is 11.5 units (M. luteus) and
11.5 units (E. coli).
M. luteus Rho Terminates Transcription
To
determine whether the RNA-dependent ATPase from M. luteus is a
transcription termination factor, the purified protein was assayed for
its effect on transcription of a cro template with E. coli RNA polymerase. This template has the well
characterized Rho-dependent terminator tR
(23) . The
control experiments show that starting with isolated complexes,
incubation for 3 or 6 min in the absence of factors yielded a
372-nucleotide RNA (Fig. 4, lanes 4 and 5),
the readthrough transcript from the promoter (P) to the end
of the template. When E. coli Rho factor was present at 28
nM, a 3-min incubation yielded RNA molecules with 290,
312, and
345 nt arising from termination at subsites I, II,
and III(23) , respectively, as well as the 372-nucleotide
readthrough transcript (Fig. 4, lane 6). The overall
termination efficiency was
50% for this condition. The addition of
the E. coli termination cofactor NusG, which has been shown to
cause Rho-dependent termination at sites upstream of tR
in vitro(24) , yielded the expected pattern (Fig. 4, lane 7). When M. luteus Rho was used
at the same concentration (28 nM), a new set of discrete RNA
molecules was formed with sizes in the range of 90-280
nucleotides (Fig. 4, lane 9), indicating that it caused
termination at points well upstream from those used by E. coli Rho. The addition of E. coli NusG had only a small effect
in enhancing the yield of smaller transcripts with M. luteus Rho (Fig. 4, lane 10). This effect of M.
luteus Rho was not specific to tR. Similar results
were obtained with another E. coli Rho-dependent terminator, tiZ1, an intragenic terminator in the lacZ gene. M. luteus Rho terminated transcription at points earlier than E. coli Rho or E. coli Rho assayed in combination
with NusG. This ability of M. luteus Rho to give
rise to smaller RNA molecules during transcription of the cro template was completely blocked when 200 µM bicyclomycin was present in the reaction mixture (Fig. 4, lane 12), showing that this inhibitor of the ATPase activity
of M. luteus Rho also inhibits its termination function.
cro template were prepared and elongated as described under
``Experimental Procedures.'' Lanes 1-5 show
the distribution of RNA transcripts after elongation for 2 s, 5 s, 8 s,
3 min, and 6 min, respectively. Samples in lanes 6-13 were incubated for 3 min with the following additions: lane
6, 28 nME. coli Rho; lane 7, 28 nME. coli Rho and 25 nM NusG; lane 8,
none, then for an additional 3 min with 28 nME. coli Rho; lane 9, 28 nMM. luteus (Mlu) Rho; lane 10, 28 nMM. luteus Rho and 25 nM NusG; lane 11, none, then for an
additional 3 min with 28 nMM. luteus Rho; lane
12, 28 nMM. luteus Rho and 200 µM bicyclomycin; lane 13, none, then for an additional 3 min
with 28 nMM. luteus Rho and 200 µM bicyclomycin. The nucleotide lengths of the RNAs indicated at the
right were determined by transcribing the cro template using
RNA chain-terminating analogs (data not shown). RT,
readthrough.
74%(5) . In
contrast, the G + C content of E. coli DNA is only 50%.
In its function, the Rho factor of E. coli acts by binding to
the nascent transcript at regions of the RNA called rut (rho utilization site). Although rut sequences lack a consensus(1) , they do have certain
specific, defining characteristics; they have little base-paired
secondary structure (1) and usually have a compositional bias
that is high in C residues and low in G residues(29) . Because
of their high G + C content, the RNA molecules in M. luteus are likely to have more extensive base pairing than the RNA
molecules in E. coli. Thus, M. luteus Rho has likely
been adapted to use a rut site that has more extensive base
pairing than is typical for a rut site in E. coli.
Evidence in support of this hypothesis is our finding that M.
luteus Rho caused termination of transcription at a site located
well before the rut site used by E. coli Rho on the
cro gene template. The RNA encoded by the upstream
region of
cro forms extended base-paired secondary
structures (30) , thus making it unavailable as a rut site for E. coli Rho. This interpretation is supported by
the finding that E. coli Rho will cause termination at
upstream sites when transcription is performed with ITP in place of GTP
because the resulting inosine-substituted RNA has less stable
base-paired secondary structure than the normal cro transcript(31) . M. luteus Rho, in contrast, was
able to use these segments in the first 100 nucleotides of a normal,
guanosine-containing cro transcript as its rut site
to cause termination.
and Gly
(Fig. 5). In Rho factors from most
organisms, these two phylogenetically conserved landmark residues are
usually separated by 14 amino acids with very little phylogenetic
conservation. With its insert, M. luteus Rho has 263 residues
instead of 14 in this putative loop region. The first part of the
insertion sequence is rich in Ala residues, while the C-terminal part
is rich in Arg, Asp, Gly, and Asn residues. Also, in a stretch of 238
residues, there are no amino acids with a hydrophobic side chain
(excluding Pro and Ala residues). Since patterns of polar and nonpolar
residues are important in the formation of ordered
-stranded and
-helical secondary structures (32) and since hydrophobic
residues have a major role in the formation of ordered tertiary
structures for globular domains(33) , we predict that this very
hydrophilic segment of the protein will be randomly coiled, lacking a
defined secondary structure. Indeed, when the insert sequence was
analyzed for secondary structure (PHDsec Secondary Structure Prediction
Program, EMBL, Heidelberg,
Germany)(34, 35, 36) , 80% was predicted
to exist as a loop. However, this segment has approximately an equal
number of positively and negatively charged residues and might form an
unprecedented, ordered structure consisting of many salt bridges.
and Gly
, and the gray area the 10-amino acid segment between Lys
and Gln
. The E. coli RNA-binding and
ATP-binding domains are indicated by arrows below. The amino
acids are numbered from the open reading
frame.
)and Mycobacterium
leprae (GenBank accession number U15186). The open
reading frames of these genes predict Rho proteins with 706 and 610
residues, respectively. With both, the major part of the additional
residues over the
420 that are typical of Rho homologs from other
phylogenetic groups start after Ile
in S. lividans Rho and after Ile
in M. leprae Rho and end
before Gly
and Gly
, respectively. Thus, S. lividans Rho has 263 and M. leprae Rho has 162
residues between these landmark residues. Like M. luteus Rho, S. lividans Rho has a major part that is very rich in Arg,
Asp, Gly, and Glu residues, but is different in having many Gln
residues instead of many Asn residues. The M. leprae sequence
is also rich in polar residues. Like the M. luteus Rho insert,
these sequences are very deficient in hydrophobic residues. These
observations suggest that the presence of a polar, random-coiled
structure insert is a conserved feature of the Rho proteins in these
organisms that have a very high G + C content. However, in spite
of the similar features, the three known Rho RNA-binding domain
insertion sequences did not reveal any obvious phylogenetic
relatedness. It will be of great interest to learn how the presence of
a structurally unordered subdomain can help these Rho factors contend
with their nascent transcripts to cause termination.
to Gln
(Fig. 5; see (5) ). It is between two phylogenetically conserved residues in
the RNA-binding domain corresponding to Glu
and
Arg
in E. coli Rho. The S. lividans and M. leprae Rho homologs have insertions of three and six amino
acids in that position, respectively. Like the large upstream insertion
sequence, these lack amino acids with hydrophobic side chains.
)))
We thank Barbara Stitt and Lislott Richardson for
generously providing NusG and E. coli Rho, respectively. We
also thank Fujisawa Pharmaceutical Co. Ltd. for providing bicyclomycin.
We appreciate the insightful advice offered by Christopher Burns and
thank Colin Ingham and Iain Hunter (University of Glasgow) for
communicating the sequence of S. lividans rho prior to
publication.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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