(Received for publication, May 17, 1995)
From the
The Escherichia coli thr operon attenuator has a structure similar to other Rho-independent terminators. The DNA sequence immediately 5` to the termination site is dG+dC-rich and contains a region of dyad symmetry that, when transcribed into RNA, encodes a hairpin structure in the transcript. It also contains a stretch of 9 consecutive dA-dT residues immediately distal to the region of dyad symmetry which encode uridine residues at the 3` end of the terminated transcript. In addition, the thr attenuator has a stretch of 6 dA-dT residues immediately upstream of the region of dyad symmetry which encode 6 adenines. These adenines could potentially pair with the distal uridines to form a hairpin structure extended by as much as 6 A-U base pairs. In this report we have examined the role of the upstream adenines in transcription termination. We used templates that specify mismatches or create new base pairs in the potential A-U secondary structure of the transcript as well as templates that delete segments of the A residues upstream of the hairpin. We conclude that A-U pairing is not required for efficient transcription termination at the thr attenuator. This conclusion is likely to apply to other Rho-independent terminators that contain hairpin-proximal dA-dT residues.
The control of gene expression in bacteria often occurs at the level of transcription termination. Transcription terminators are found upstream of operons, between genes in an operon, and at the ends of operons (for reviews, see (1, 2, 3) ). In general, transcription terminators have been divided into two classes: Rho-dependent or Rho-independent, depending upon their requirement for Rho factor in vitro(2) .
Rho-independent terminators terminate transcription in vitro in the absence of Rho protein or other factors, and have two common structural characteristics(2) . The first is a dG+dC-rich region of dyad symmetry that encodes a stem-loop or hairpin structure in the nascent mRNA. The second feature is a dA+dT-rich region of 4-9 base pairs immediately distal to the region of dyad symmetry within which the transcript terminates. Thus, when RNA polymerase transcribes through a Rho-independent terminator, the transcript forms a hairpin followed by 3` uridine residues. Evidence from several laboratories has shown that both of these features are required for efficient termination(2, 3) .
The thr attenuator is a Rho-independent terminator that has a dG+dC-rich region of dyad symmetry that encodes a hairpin containing a stem of 8 base pairs and a loop of 6 bases. The hairpin is followed by a tract of 9 consecutive dA-dT residues and termination predominately occurs at the 7th or 8th uridine in the transcript(4) . The importance of the uridine residues was established by Lynn et al.(5) , who made deletions that varied the length of the dA-dT tract. They found that the deletion of 1 or 3 dA-dT residues had no effect on transcription termination in vivo or in vitro. The deletion of 4, 5, or 6 dA-dT residues showed a linear decrease in termination efficiency. When 7 or 8 dA-dT residues were deleted, termination was abolished.
The thr attenuator also has a dA-dT tract of 6 bases immediately upstream of the region of dyad symmetry(4) . The dA-dT tract encodes 6 A residues in the transcript that could potentially pair with 6 of the hairpin-distal U residues to extend the length and stability of the RNA hairpin. Approximately one-third to one-half of the Rho-independent terminators characterized to date also contain hairpin-proximal dA-dT residues as part of an AANAA sequence(6) , which suggests that pairing of A and U residues could play a functional role in transcription termination. In this report, we describe a systematic analysis of the role of the hairpin-proximal A residues in transcription termination at the thr attenuator.
Figure 1: Construction of the thrA-lacZ protein fusion plasmids. A, the scheme for the construction of pMC1396 derivatives that contain an in-frame fusion of the thrA gene to the 5` end of the lacZ coding sequence of the plasmid pMC1396 is shown. The EcoRI (*) site is only present in pMC1396. B and C, the schemes for the construction of thrA-lacZ protein fusion vectors which are deleted for the sequences involved in the formation of the 1:2 hairpin structure of the thr leader region are shown. See ``Materials and Methods'' for details of the constructions.
The DNA sequence from +44 to +110 of the thr leader region was replaced by two synthetic
oligodeoxyribonucleotides, one 21-mer 5`-CGCATGGAACGCATTAGCTGA-3` and
the other 23-mer 5`-CGCGTCAGCTAATGCGTTCCATG-3`. These two oligomers
were annealed at 80 °C for 1 min and slowly cooled to room
temperature. The hybridized oligomers contained HpaII and MluI half-sites at the ends. The HpaII sites of the BstEII-HpaII fragment of the M13mp9-AT45GG and the
annealed deoxyribonucleotides were ligated. The resulting 129-bp
fragment was purified and ligated with M13mp11-WT thr vector
that had been digested with BstEII and MluI
endonucleases. The resultant recombinant clone that contained 2 extra
base pairs at the ligation junction and deleted the sequences necessary
for the formation of the 1:2-hairpin structure of the thr leader region was isolated and named M13mp11-WT thr 1:2. The HincII-EcoRI fragment of
M13mp11-WT thr
1:2 was then subcloned into the pMC1403
vector to construct pMC1403-WT thr
1:2 by following the
procedure described above. Plasmids pMC1403 and pMC1396 are analogous
to each other, except that pMC1403 contains a unique EcoRI
site (11) .
To construct the pMC1403 attenuator mutant
derivatives with the same deletion in the 1:2 region, the MluI-BamHI fragments from the corresponding pMC1396
derivatives (Fig. 1A) were isolated and ligated with
the EcoRI-MluI fragment of pMC1403-WT thr 1:2. The ligation products were then subcloned between the EcoRI and BamHI sites of the pMC1403 vector (see Fig. 1C). These constructions produced plasmids that
are isogenic with pMC1403-WT thr
1:2.
Approximately 100 µg of plasmid DNA containing the various dA-dT
tract deletions (5) were digested with RsaI and XbaI, and the fragments were separated by electrophoresis in a
10% polyacrlyamide gel. After staining with ethidium bromide and
visualization with ultraviolet light, the appropriate segments of gel
containing the bands were excised and soaked in 0.5 ml of 0.5
TE buffer (5 mM Tris-HCl (pH 7.9) and 0.5 mM EDTA) at
room temperature overnight. The gel particles were removed by
centrifugation and DNA fragments were purified on DE52
columns(5) .
The pUC18 and pUC19 DNA (Fig. 2) were
digested with XbaI and HincII. The RsaI-XbaI fragments were ligated into pUC18 and pUC19
using T4 DNA ligase. The ligation mixtures were transformed into
competent KK2186 cells [ (lac-pro), supE, thi, strA, sbcB-15, endA/F` traD36, lacI
Z
15 (12) by the CaCl
method (13) . The desired
clones were identified as white colonies on LB plates supplemented with
X-gal, and the sequences of the constructs were verified by direct DNA
sequence analysis.
Figure 2: Construction of templates containing deletions of the dA-dT tract upstream and downstream of the hairpin. A, construction of templates containing deletions of the hairpin-distal dA-dT tract. RsaI-XbaI fragments containing varying numbers of dA-dT base pairs in the underlined region of the sequence were cloned into the HincII-XbaI sites of pUC19. The construct shown corresponds to A6-T8(+) (See text and Fig. 5). A6-T6(+), A6-T3(+), and A6-T1(+) contain 6, 3, or 1 dT-dA base pairs in the underlined region, respectively. B, construction of templates containing deletions of the dA-dT tract upstream of the hairpin. RsaI-XbaI fragments, identical to those above, were inserted into the XbaI-HincII sites of pUC18. The construct shown corresponds to A8-T6(-) (see text and Fig. 5). A6-T6(-), A3-T6(-), and A1-T6(-) contain 6, 3, or 1 dA-dT base pairs in the underlined region, respectively.
Figure 5: Transcription termination directed by deletion templates. The values for the frequency of transcription termination (% T) are indicated next to the terminator structures.
Transcription
reactions (50 µl) were terminated after 10-20 min at 37
°C by the addition of an equal volume of phenol, and carrier tRNA
was added to a final concentration of 0.5 mg/ml. After the phenol
extraction, the aqueous phase was adjusted to 0.3 M sodium
acetate and the samples were ethanol-precipitated, desalted, dried in vacuo, and resuspended in formamide dyes (80% formamide, 1
TBE buffer, 0.1% xylene cyanol, and 0.1% bromphenol blue). The
products of the transcription reactions were analyzed on 8%
polyacrylamide, 8 M urea sequencing gels. After
autoradiography, regions of the gel corresponding to the terminated and
read-through transcripts (9) were cut out of the gel and
counted in a Beckman LSI801 scintillation counter. The molar ratios of
the terminated and read-through transcripts were calculated by
correcting for the lengths and base compositions of the
transcripts(9) .
All the lysogens were verified by their
sensitivity to vir and resistance to lysis by
h80del9C(17) . Lysogenic
strains containing only one copy of a
prophage were identified by
a modified method of Sly and Rabideau(18) . Determination of
the copy number of the
prophage was based on the susceptibility
of the lysogen to different dilutions of a
cI 90c17 lysate. MC4100 strains with a single
prophage
were used in assays for
-galactosidase activity.
Several other
Rho-independent terminators including the phe(19) , leu(20) , and ilvB(21) attenuators
also contain upstream adenosines as part of a conserved AANAA sequence
that can also potentially form base pairs with the uridines. In
addition, the bidirectional tonB/P14 terminator and
operon terminator, t
, contain stretches of adenosines
upstream of the hairpin(22, 23) . If A-U base pairing
is important for efficient transcription termination at these
terminators, a simple prediction is that disruption of the base pairs,
by introducing substitutions upstream of the hairpin or in the
hairpin-distal region of the template, should reduce the efficiency of
transcription termination. In addition, compensating mutations that
restore base pairing should direct efficient transcription termination.
We constructed a series of mutant templates that contained single or
double mutations in the thr attenuator and measured their
transcription termination efficiencies in vitro and in
vivo. The mutants specified potential base pair mismatches or new
base pairs in the stretch of A and U residues of the transcript (Fig. 3B). Most of these variants, each containing
single or double base changes, were made by oligonucleotide-directed
site-specific mutagenesis. L131G is a substitution mutant with an A to
G change in the run of A residues immediately 5` to the attenuator.
Mutants L158C, L160A, and L160G disrupt the U tract by substituting C,
A, or G for U at positions +158 and +160, respectively.
Mutants L153A (24) and L160C ()were isolated by a
genetic selection that involved isolating mutants that decrease
transcription termination at the thr attenuator in
vivo. Variants L131U/L160A and L131G/L160C contain different base
substitutions at the same positions: A to U or G at position +131
and U to A or C at position +160, respectively. These two variants
disrupt the sequences of both the A and U stretches, but maintain the
potential complementarity at the base of the stem. Variant L153A/L160C,
which contains two single base changes at positions +153 and
+160, has disruptions in both the G+C-rich region and the U
tract of the thr attenuator.
Figure 3: In vitro transcription template and the RNA secondary structure of the thr attenuator and its variants. A, a BstEII-SstI fragment carrying the thr regulatory region was used for in vitro transcription experiments shown in Fig. 4. The terminated and read-through transcripts are 164 and 300 bases, respectively. B, the secondary structure of the thr attenuator RNA is presented in the conformation that maximizes base pairing interactions. The positions of the nucleotides in the transcript are numbered starting from the transcription initiation site of the thr leader RNA, which is designated as +1. The mutational changes in the thr attenuator that were generated by in vitro mutagenesis are indicated as boldletters. Variant L160C was originally isolated by a genetic method that showed relief of transcription termination at the thr attenuator in vivo.
Figure 4: Transcription termination in vitro.Invitro transcription reactions were performed and subjected to electrophoresis as described under ``Materials and Methods'' using the BstEII-SstI fragment containing the wild-type or mutant thr terminators as templates. The terminated (T) and the read-through (RT) transcripts are 164 and 300 nucleotides in length, respectively. A, reactions were performed in the presence of GTP. B, reactions were performed in the presence of ITP substituted for GTP. Lane1, wild-type template; lane2, L131G; lane3, L160C; lane4, L160A; lane5, L160G; lane6, L131G/L160C; lane7, L131U/L160A; lane 8, L153A/L160C.
The BstEII-SstI restriction fragments bearing the thr operon leader region and different attenuator mutants were isolated from the M13mp10-thr constructs and used for in vitro transcription studies (Fig. 3A). The transcribed RNA was subjected to electrophoresis on 8% polyacrylamide, 8 M urea gels and subjected to autoradiography. The terminated (164 bases) and read-through (300 bases) transcripts were isolated, and the amount of radioactivity in each gel species was measured. The in vitro transcription termination efficiencies of the attenuator variants are presented in Fig. 4and Table 1.
The results showed that variant L153A/L160C, which contains C-A mismatches in both the dG+dC-rich region and the U stretch of the attenuator, significantly decreased the termination frequency. Variants with substitutions at position +160 only (L160C, L160G, and L160A) showed termination frequencies that were only slightly lower than wild type. All of the remaining variants bearing base substitution(s) that disrupted the runs of A or U residues terminated efficiently.
It has been shown previously (9) that substitution of ITP for GTP caused a decrease in termination frequencies for several other thr attenuator variants. Inclusion of ITP decreased termination at the mutant terminators dramatically but had only a slight effect on termination at the wild type site(9) . If ITP caused a similar decrease in the termination frequencies of the mutants constructed in this study, it would be possible to determine the effects of base changes that either disrupted or restored the putative complementarity in the runs of A and U residues of the thr attenuator.
The termination frequencies obtained from ITP-substituted in vitro transcription experiments are shown in Fig. 4and Table 1. As observed previously, incorporation of ITP in the transcripts showed only a slight effect when the wild-type template was used with a termination value of 65%(9) . Variants L160C, L160G, and L160A, which contain disruptions in the U tract by substituting C, G, or A, respectively for U at position +160, showed drastically decreased termination frequencies. Since these three variants have the same G+C-rich sequences and are expected to form hairpin structures identical to the wild type with one potential mismatch in the A-U region, it is likely that the decrease in the termination values for these variants is caused by effects of the substituted bases at the U stretch, possibly by affecting RNA-DNA template interactions or by direct effects of the sequence changes themselves. This interpretation was further supported by comparing the results from two attenuator variants, L131G/L160C and L131U/L160A. These two variants were specifically constructed so that the putative base pairing at the base of the stem of the thr attenuator were restored; i.e. I-C and U-A base pairs in L131G/L160C and L131U/L160A, respectively. If the effect exhibited by the single mutants (L160C or L160A) arose from disruption of the A-U base pairings at the base of the thr attenuator, the two variants would be expected to terminate as efficiently as the wild type. The results show that both of the variants retained termination frequencies similar to the variants with the same change in the U stretch only (L160C or L160A). Taken together, the in vitro transcription studies with ITP suggested that single base changes in the run of A residues have no effect on termination.
To determine the effects of the
variants in vivo, the same mutants were subcloned into the
vector pMC1403 to construct in-frame thrA-lacZ protein fusions (Fig. 1, B and C). These
constructs lack the leader region that encodes the upstream secondary
RNA structures that are involved in regulating the formation of the thr terminator structure encoded by the attenuator. These
plasmids were constructed to avoid possible complications introduced by
the upstream sequences. The fusions were then crossed onto RZ5 by
homologous recombination and single-copy lysogens were constructed. The
level of
-galactosidase expression should only reflect the
termination efficiencies of these attenuator variants in vivo.
Table 2shows the results of -galactosidase assays
performed on cells grown in minimal medium. The results show that
variant L131G exhibited the same level of
-galactosidase activity
as wild type. This result could be explained by the formation of a G-U
base pair in the helix. However, variants which only disrupted the U
tract showed 3.7-4.0-fold (L160C and L158C) and
7.5-7.9-fold (L160A and L160G) higher
-galactosidase
activities than that of wild type. Furthermore, the
-galactosidase
activities of the two variants bearing base changes that could
potentially form base pairs in the transcript at both the runs of A and
U were 3.1 times (L131G/L160C) and 5.9 times (L131U/L160A) the
wild-type value. The results again suggested that the restoration of
complementarity at the base of stem cannot compensate for the effects
of a single substitution mutation in the U stretch. In vivo
-galactosidase assays of the variants that contain an intact thr leader region also gave similar results (data not shown).
PvuII fragments varying in size from 364 to 371 bp were purified from the clones and used as templates in in vitro transcription reactions (Fig. 5). Using templates with the (+) orientation, the termination efficiency decreased from 61% for A6-T8(+) to 7% for A6-Tl(+) as the number of uridine residues in the transcript decreased from 8 to 1 as observed previously(5) . In contrast, with templates of the(-) orientation, in which the number of adenosine residues upstream of the hairpin were varied, termination was efficient and varied from 64% to 78%. Since transcription termination is efficient even on a template that contains a deletion that removes 5 of the 6 adenosine residues upstream of the hairpin, these results also indicate that A-U base pairing is not important for efficient transcription termination.
In summary, we have constructed variants of the thr attenuator to determine if the tract of 6 dA-dT residues upstream of the sequence encoding the dG+dC-rich hairpin are important for transcription termination. Both the in vitro and in vivo results indicate that an intact dA-dT tract is not essential for efficient transcription termination. These results argue against a model that proposes that pairing between the adenosine residues upstream of the hairpin and downstream uridine residues is necessary for transcription termination. In addition, the dA-dT tract upstream of the hairpin does not contribute essential structural or sequence information because deletion of 5 of the 6 A residues from the transcript does not affect the efficiency of termination. The results are compatible with current models that propose that termination is a multi-step process involving active participation of the RNA polymerase (3, 25, 26) or that termination is controlled by the relative stabilities of DNA-DNA, RNA hairpin, and DNA-RNA interactions at the termination site in the transcription bubble(2, 27) .
It is interesting to note that a
study by Wright et al. (23) with the t terminator has shown that a deletion of the tract of 7 adenosines
upstream of the dG+dC-rich element increases the frequency of
read-through at the terminator by a factor of 10 in vivo. They
concluded that pairing between the adenosines in the A-tract and the
uridines in the distal U tract is important for termination. We have no
obvious explanation for the differences observed between the thr and t
terminators. As discussed by Wright et
al. (23) it is possible that, as a consequence of the
deletion of the A tract, the formation of an alternative stem-and-loop
structure could occur. It would contain 4 instead of 5 G-C base pairs
in the stem, and the loop would be 6 rather than 4 bases. It is also
possible that the length of the helix in the stem of the RNA could be
important in determining the requirement for pairing between the A
tract and the distal uridines. The thr and t
stems are 8 and 5 base pairs in length, respectively. Perhaps the
t
terminator requires A-U base pairs in addition to the
G-C base pairs to extend the length of the stem helix in order to act
as an efficient terminator. Additional systematic studies will be
required to determine if either of these explanations is correct.