Download Landick R, Yanofsky C. 1987. Transcription

77. Transcription Attenuation
ROBERT LAN DICK
AND
CHARLES YANOFSKY
Department of Biological Sciences, Stanford University, Stanford, California 94305
BRIEF HISTORICAL REVIEW ............................................................................... 1276
ORGANIZATION OF THE LEADER REGIONS OF AMINO ACID
BIOSYNTHETIC OPERONS REGULATED BY TRANSCRIPTION
ATTENUATION ...................................................................................................... 1277
MODEL OF THE MECHANISM OF TRANSCRIPTION ATTENUATION
CONTROL OF THE E. COLI tIp OPERON ........................................................ 1278
EVIDENCE FOR INDIVIDUAL FEATURES OF ATTENUATION IN THE
tIp OPERON ........................................................................................................ 1278
Translation of the Leader Peptide Coding Region .............................................. 1278
Functions of the Terminator and Attenuator ...................................................... 1280
Roles of the Transcript Pause and Antiterminator Secondary Structures ...... 1281
RNA Polymerase Recognition of Pause and Termination Signals .................... 1281
FINE FEATURES OF ATTENUATION IN THE tIp OPERON ........................... 1281
Synchronization of Transcription and Translation ............................................ 1281
Transcription Readthrough in the Presence of Excess Tryptophan ................. 1282
Shutdown of Leader Peptide Synthesis ................................................................ 1282
Starvation for Amino Acids Other than Trp, and Significance of the Two
Trp Codons in the Leader Transcript ............................................................... 1283
TRANSCRIPTION ATTENUATION IN OTHER AMINO ACID
BIOSYNTHETIC OPERONS ............................................................................. 1283
The his Operon ........................................................................................................ 1283
Regulation of the his operon .............................................................................. 1283
Sequence of the his leader region ...................................................................... 1283
Detection of the terminated his leader transcript ........................................... 1283
Demonstration that synthesis of the his leader peptide is required for
attenuation ........................................................................................................ 1283
Mutations altering the leader peptide ............................................................... 1284
Mutations affecting his leader transcript secondary structures .................... 1285
Mutations affecting tRNA Hls synthesis ............................................................. 1285
The leu Operon ........................................................................................................ 1286
The thr Operon ........................................................................................................ 1286
The ilvGMEDA Operon .......................................................................................... 1290
The ilvBN Operon ................................................................................................... 1290
The pheA Operon .................................................................................................... 1291
LEADER PEPTIDE SEQUENCES AND FUNCTIONS ......................................... 1291
TRANSCRIPTION ATTENUATION IN NON-AMINO ACID BIOSYNTHETIC
OPERONS ............................................................................................................. 1292
The pheST Operon .................................................................................................. 1292
Pyrimidine Biosynthetic Operons ......................................................................... 1292
The ampC Operon ................................................................................................... 1294
rRNA Operons ......................................................................................................... 1295
Ribosomal Protein Operons ................................................................................... 1295
The tna and liv Operons ......................................................................................... 1296
CONCLUSIONS .......................................................................................................... 1297
ACKNOWLEDGMENTS ............................................................................................ 1297
LITERATURE CITED ................................................................................................ 1297
BRIEF HISTORICAL REVIEW
cantly increased the rate of synthesis of histidine
biosynthetic enzymes (148). Studies on the mechanism of action of this analog suggested that it was an
inhibitor of tRNAHis charging. At about the same time
it was observed that temperature-sensitive valyl-tRNA
synthetase mutants had elevated levels of valine biosynthetic enzymes (40), similarly implicating tRNA
One of the earliest observations suggesting that
tRNA function was involved in regulating expression
of amino acid biosynthetic operons was the finding
that addition of the histidine analog a-methylhistidine to cultures of Salmonella typhimurium signifi1276
TRANSCRIPTION ATTENUATION
77
charging in operon-specific regulation. Following
these early studies, other investigations with the his
operon established that mutations that altered
tRNAHis structure or function affected his operon
regulation (21). Although the possibility was considered that a repressor protein existed that recognized
either charged or uncharged tRNA His , mutational
studies aimed at detecting such a regulatory protein
were unsuccessful (21, 108). Many subsequent analyses focused on a particular re~ulatory mutant that
produced undermodified tRNA is; observations with
this strain, like those with a-methylhistidine, implicated translation of His co dons in the regulation of his
operon expression (6, 21, 169). Further studies with
this and other mutants culminated in the hypothesis
that translation of the early portion of the transcript
of the his operon was involved in his operon regulation (R. G. Martin, B. N. Ames, and P. E. Hartman,
Int. Congo Biochem. Abstr. 2:261, 1967; R. G. Martin
and B. Ames, personal communication).
During this period, investigations with the trp operon of Escherichia coli were concerned principally with
analyses of tryptophan-mediated repression of trp
operon expression. At the time it was thought that
repression was the sole transcription regulatory mechanism controlling this operon's expression (28, 81);
however, a number of findings were inconsistent with
this view. For example, the rate of trp mRNA synthesis
markedly increased when a mutant strain lacking a
functional trp repressor was subjected to severe
tryptophan starvation (9). This would not be expected
if repression were the sole transcription control mechanism specifically responsive to tryptophan. It also
was noted that expression of the operon was elevated
in strains with a defective trY,frtophanyl-tRNA synthetase (119), implicating tRNA rp or its cognate synthetase in trp operon regulation. An additional unexplained finding was that transcription in progress
over the initial segment of the trp operon appeared to
terminate abruptly when tryptophan was added to
tryptophan-starved, repressor-deficient cultures (74,
77).
These and related findings with the trp and his
operons--observations that were not readily explicable by repression-assumed greater significance when
genetic evidence established the existence of regulatory sites within the initially transcribed regions of
these operons that limited their expression. With both
operons it was shown that expression was significantly increased by deletions that removed the initial
segment of the operon and left the respective promoters intact (80, 90). mRNA measurements proved that
this increase was transcriptional and suggested that a
transcription termination site was located in the distal segment of the leader regions of both operons; the
deletions that increased operon expression were presumed to do so by removing this termination site (16,
17,80,90).
A unifying hypothesis was rapidly evolving, namely,
that specific tRNA charging, mRNA translation, or
both provided the basis for the decision whether or
not to terminate transcription in the leader regions of
these operons. This view was solidified when the
leader regions of the trp, his, and other amino acid
biosynthetic operons were sequenced and each was
shown to contain a short peptide coding region rich in
1277
co dons for the regulating amino acid(s). Two Trp
co dons were found in the coding region for the trp
leader peptide (106), seven Phe codons were present in
the pheA leader peptide coding region (186), and seven
contiguous His codons were located in the coding
region for the his leader peptide (10, 38). In addition,
in the ilvGMEDA operon, which is regulated by isoleucine, valine, and leucine, there were several codons
for each of these amino acids in the leader peptide
coding region (103, 121).
The existence of coding regions rich in codons for
the regulating amino acid(s) constituted indisputable
evidence implicating specific tRNAs and translation
in transcription regulation. Additional support was
provided by the key observations that ribosomes could
bind to the presumed translation initiation site for the
trp leader peptide (134) and that transcription terminated in the trp leader region in vivo and in vitro (15,
16, 105).
The case for translational control of transcription
termination in the leader regions of amino acid biosynthetic operons rapidly gained overwhelming experimental support. In the ensuing years a substantial
body of information has been gathered establishing
that transcription attenuation is commonly used to
regulate amino acid biosynthetic operons (13, 132,
175). In addition, operons concerned with a variety of
metabolic functions have been found to be regulated
by transcription attenuation. Mechanistic studies with
amino acid biosynthetic operons have elucidated the
major events involved in transcription attenuation,
providing us with a thorough understanding of this
regulatory phenomenon.
ORGANIZATION OF THE LEADER REGIONS OF
AMINO ACID BIOSYNTHETIC OPERONS
REGULATED BY TRANSCRIPTIO:\[
ATTENUATION
The structural features of the leader regions of
amino acid biosynthetic operons regulated by attenuation are surprisingly similar. These features include
sites of transcription pausing and Rho-independent
transcription termination, a coding region for a short
leader peptide containing codons for the regulating
amino acid(s), and segments that specify alternative
RNA secondary structures (Fig. 1). Transcription
pausing occurs early in the leader region; pausing is
due to the formation of an RNA structure, namely, the
transcription pause signal. Distally located in the
leader region is the attenuator, the DNA site of regulated transcription termination. The attenuator encodes an RNA secondary structure, i.e., the transcription termination signal or terminator. Overlapping
ATTENUATION CONTROL REGION
PROMOTER
transcri pt
ATTENUATOR
pause structure
1
2
FIRST
STRUCTURAL GENE
tenn; nator
3
4
t ant; term; nator
pept; de codi n9
r-eg" on
FIG. I. Structure and relevant features of the leader
regions and leader transcripts of amino acid biosynthetic
operons regulated by transcription attenuation.
1278
LAN DICK AND YANOFSKY
the terminator and the pause structure is an alternative RNA secondary structure termed the antiterminator. Formation of this structure during transcription of the leader region precludes formation of the
terminator, thereby permitting transcription readthrough. The peptide coding region usually overlaps
the promoter-proximal RNA segment that is part of
the RNA pause signal. Translation of the peptide
coding region versus ribosome stalling within it selects between terminator and antiterminator formation, causing either transcription termination or transcription readthrough.
In this article we shall begin by describing attenuation in the trp operon in detail, to illustrate the
individual features of transcription attenuation as
they are currently understood. We shall then consider
other examples of transcription attenuation in amino
acid biosynthetic operons, highlighting features that
are unique to each. Finally, we shall discuss examples
of transcription attenuation in E. coli and S. typhimurium that involve mechanisms substantially different
from those operating in amino acid biosynthetic operons.
MODEL OF THE MECHANISM OF
TRANSCRIPTION ATTENUATION CONTROL OF
THE E. COLI trp OPERON
Attenuation in the trp operon is a dynamic process
involving both transcription and translation. In E. coli
cultures growing at 3rC, transcription initiation at
the trp promoter can occur as often as once every 6 s
(9,16). At this temperature, each transcribing polymerase molecule theoretically can reach the attenuator
in 2 to 3 s. It follows, therefore, that for attenuation to
be an effective regulatory mechanism, the individual
events crucial to attenuation must be rapid and must
be precisely timed. In this section we shall describe
the sequence of events, as we believe they occur, in
attenuation control of trp operon expression. A schematic representation of these events is depicted in Fig.
2.
In the trp operons of E. coli and S. typhimurium,
repression and attenuation function as independent
transcription control mechanisms (see chapter 90).
Tryptophan activates the trp aporepressor; the resulting repressor can bind at the trp operator and inhibit
transcription initiation. Under conditions of maximum repression, the rate of transcription initiation at
the trp promoter is about 1/80 the rate observed in the
absence of tryptophan (79, 179). All polymerase molecules that initiate transcription on the trp operon are
subject to a transcription termination decision at the
attenuator. This decision is based on the cellular level
of charged tRNA Trp. Under starvation conditions, in
which all tRNATrp essentially is uncharged, the rate of
read through transcription at the attenuator increases
about eightfold over the rate observed when tRNATrp
is fully charged. We do not believe that the rate of
transcription initiation at the trp promoter influences
attenuation control, or vice versa, but these possibilities have not been excluded.
The first event crucial to attenuation is formation of
RNA secondary structure 1:2 (Fig. 2). This structure
functions as a transcription pause signal and presumably forms rapidly and spontaneously as the tran-
scribing polymerase molecule moves over the initial
segment of the leader region. While the polymerase
pauses in the leader region, the opportunity is provided for loading of a ribosome at the leader peptide
ribosome-binding site of the leader transcript. After a
ribosome loads and begins translation, it approaches
the paused polymerase. As it nears the paused polymerase it could disrupt the RNA pause structure,
releasing the polymerase and restoring transcription.
Polymerase and ribosome then move in unison over
the leader region and leader transcript, respectively.
At this stage there are two options (176). If there is
a deficiency of charged tRNA Trp, the translating ri bosome would stall at one of the Trp codons in the
peptide coding region. If this occurred, then RNA
segment 1 would be masked by the stalled ribosome.
This would allow RNA segment 2 to pair with RNA
segment 3 as soon as that RNA segment was synthesized. (Paired RNA segments 2 and 3 constitute the
anti terminator.) As transcription continues and RNA
segment 4 is formed, the antiterminator will persist,
preventing formation of the terminator. Since the
terminator does not form, the transcribing polymerase molecule will read through the attenuator and
transcribe the remainder of the operon. Alternatively,
if there is an adequate level of charged tRNATrp, the
ribosome translating the leader peptide coding region
would continue beyond the Trp codons to the end of
the coding region and then either remain at the
translation stop codon (as shown in Fig. 2) or dissociate from the leader transcript. If the ribosome remains at the stop codon while RNA segment 4 is
synthesized, the ribosome would block antiterminator
(2:3) formation and promote terminator (3:4) formation, leading to transcription termination.
For reasons that will become evident, it is thought
that if the ribosome releases quickly at the stop codon
the transcript will usually refold to form structure 1 :2.
When 1:2 forms, then structure 3:4, the terminator,
subsequently will form, and transcription will terminate at the attenuator. However, after ribosome release at the stop codon, structure 2:3 occasionally may
form if segment 3 has been synthesized. If 2:3 does
form, it would prevent formation of the terminator, as
it does during tryptophan starvation. Attenuation in
the trp operon therefore permits the cell to exploit the
position of the ribosome engaged in synthesis of the
trp leader peptide as the basis for the decision whether
or not to terminate transcription at the attenuator.
Whenever the terminator is formed during transcription of the leader region, it serves as a termination
signal, and RNA polymerase terminates transcription
at the attenuator. Whenever the anti terminator forms,
transcription proceeds into the operon.
EVIDENCE FOR INDIVIDUAL FEATURES OF
ATTENUATION IN THE trp OPERON
Translation of the Leader Peptide Coding Region
Attenuation allows the bacterium to regulate transcription and ultimately translation of the trp operon
structural genes as a function of charged tRNA Trp
availability. Conditions and events that reduce tRNATrp
charging increase operon expression by reducing termination at the attenuator. The extent of tRNATrp charging
TRANSCRIPTION ATTENUATION
77
1279
INITIAL STAGES OF TRANSCRIPTION
~
-
POLYMERASE
PAUSES
+
ppp/AUG
1:2
RIBOSOME BINDS
TO TRANSCRIPT
I ~, =r:f=""
-
MOVING
RIBOSOME
RELEASES
THE
PAUSED POLYMERASE
AAA
PPP--AUG
/
--
TRYPTOPHAN-STARVED CULTURE
RIBOSOME STALLS
AT A TRP CODON,
ANTITERMINATOR FORMS
~
GROWTH WITH EXCESS TRYPTOPHAN
RIBOSOME
MOVES TO
THE STOP CODON
l
1
TERMINATOR
CANNOT FORM,
TRANSCRIPTION
CONTINUES
TERMINATOR
FORMS
READ- THROUGH
TERMINATION
FIG. 2. Model depicting the events involved in regulation of the trp operon by transcription attenuation. The ribosome
remains attached to the nascent RNA after it reaches the leader peptide stop codon, although this feature of the attenuator
model remains unclear (see text).
is dependent on the availability of tryptophan, the
rate of tRNATrp charging, and the overall rate of
protein synthesis. Conditions that deplete the pool of
charged tRNA Trp, such as tryptophan starvation or
rapid protein synthesis, increase readthrough at the
attenuator (17, 120). Similarly, all changes that reduce the efficiency of Trp codon translation increase
operon expression. These include alterations in the
structural gene for tRNATrp or tryptophanyl-tRNA
synthetase (119,180) or alterations in the modifying
enzyme that adds the isopentenyl group to the A
residue adjacent to the anticodon of tRNATrp (41, 42,
170, 180). The discovery of a ribosome-binding site in
vitro (134) and its correspondence to a potential translation initiation site in the leader segment of the trp
transcript (104) provided the initial basis for the
suggestion that the leader peptide coding region was
translated. The first proof of this hypothesis was
provided by gene fusion analyses. The trp leader peptide coding region was fused in phase to the lac!
coding region (155) and to the distal segment of trpE
(117). In both instances a fusion polypeptide was
synthesized, and amino acid sequence analyses verified that translation had begun at the leader peptide
start codon. These observations supported the idea
that the signal for attenuation control must include
the ability to translate the tandem Trp codons in the
leader transcript. The importance of translation of the
leader peptide coding region to regulation by attenuation is also shown by the effects of alterations in the
leader region that reduce or eliminate its translation.
Replacement of the AUG start codon for toe leader
peptide by AUA (trpL29; Fig. 3A) essentially eliminates
the relief of termination at the attenuator that normally accompanies tryptophan starvation (32, 187).
Similarly, deletions that replace the Shine-Dalgarno
sequence preceding the leader peptide start codon, or
replace this sequence and the start codon, prevent the
typical tryptophan starvation response (163, 164).
Therefore, both the capacity to translate the leader
peptide coding region and, more specifically, the ability or inability to translate the tandem Trp codons in
this message segment are essential to regulation by
attenuation.
Attempts to detect synthesis of the trp leader peptide in vivo were unsuccessful until recently (37).
However, the elusive peptide was detected in vitro in
analyses using a coupled transcription-translation system (33). These studies also provided explanations for
the lack of success in early attempts to find the
peptide. Turnover measurements in vitro demonstrated that the trp leader peptide is extremely labile. It
also was shown that synthesis of the leader peptide
was depressed when the template contained the segment of the leader region just beyond the peptide
coding region. The corresponding segment of the transcript can fold back and form a base-paired structure
that blocks the translation start site (33). Experiments
exploiting these facts have demonstrated synthesis of
the trp leader peptide in vivo (37).
1280
LANDICK AND YANOFSKY
1:2
A
A
A
UI
St op
IA
C
G ~ A trpL75,76,77
trpL67,6B U ~C=G~
Ser
C=G
AAGUUCACG
U
U
A
A
A
A
Arg
G
trpL29
A
~
Trp
t
Trp
A trpLBO
C
G
3:4
A
U
60 C
G
G
U
A
U-A . /
c=G---8o
A-U
C=G
Thr
A
20
A
G
U
A
U
C
A
U
A
G
U
120 C=G
G
U
G=C
G=C trpLIIB A.- C=G-A trpL130
g:g=:;
~:~
C
Met~YSAlaIlePheValLeULYSGlyG=C
GACAAUGAAAGCAAUUUUCGUACUGAAAGGUU-A
40
trpL131
trpL132
G=C
AUCAGAUACCCA-UUUUUUU
U-A
140
P""""G=C
C=G 100
G
A
U A
A
2:3
A
B
U A
G
AAGUUC
A
C=G 100
A
C
trpL93
A~G=C
U-A
G
U
A
A
A
A
A
A
U
C
A
A
C=G
U-A
U
A-U
U-A
G=C
G
G
G
U
20 A
A
C
C
U
AI
C
GA
AC
U
trpL75,76,77 A "
C=G--A trpLl15
C
A
~ G=C
G
G=C
A MetLysAlaIlePheValLeuLysGlyTrpTrpArgThrSerStop G=C
CAAUGAAAGCAAUUUUCGUACUGAAAGGUUGGUGGCGCACUUCCUGAAAC=GCCUAAUGAGCGGGCUUUUUUU
40
60
120
140
FIG. 3. Alternative secondary structures of the E. coli trp leader transcript. The 1 :2,2:3, and 3:4 secondary structures were
predicted by a computer algorithm (185) and are consistent with the observed pattern of RNase TJ partial digestion (129). The
positions of key mutations are indicated. (A) Termination configuration. Site of transcription pausing (49) is indicated by the
boldface arrow. (B) Antitermination configuration.
Functions of the Terminator and Attenuator
The distal portion of the trp leader region (the
attenuator) specifies a transcript segment that can
fold to form the key structure, the terminator (3:4; Fig.
3A). The base-paired segment of this RNA structure is
followed by a run of U residues that constitute the 3'
end of the terminated transcript. The importance of
this segment of the leader region to transcription
termination is demonstrated by mutational analyses
which have shown that alteration of specific base
pairs in this region (trpL118, trpLJ 3D, trpLJ 31,
trpLJ 32; Fig. 3A) or deletion of any of its segments
eliminates transcription termination at the attenuator in vivo and in vitro (159, 162, 164, 188). These
mutational changes undoubtedly exert their effect by
altering the structure of the corresponding transcript
segment. It is thought that the terminator is the
transcription stop signal recognized by the transcribing polymerase molecule (45, 133, 144, 146). This
conclusion is based on several experiments that distinguish between template and transcript as the termination signal. Analyses involving incorporation of
base analogs (that decrease or increase base-pair stability) into the transcript or DNA template suggested
that the termination signal resided within the RNA
itself rather than within the corresponding DNA segment (45, 47,106). The final compelling evidence was
obtained with DNA heteroduplexes prepared from the
separated strands of the leader regions of wild type
77
and a mutant with an attenuator alteration that
reduces termination (146). One of the heteroduplex
templates specified a wild-type transcript while the
other specified a mutant transcript. Transcription
studies with these templates showed that only when
the mutant transcript was produced was termination
relieved (146).
When restriction fragments containing the trp leader region were transcribed in vitro with purified RNA
polymerase, two major transcripts were detected (105,
106, 129). Both were initiated at the trp promoter.
One, the so-called leader transcript, terminated at
about base pair 140 in the leader region, the position
of in vivo termination, while the other, the runoff
transcript, extended to the end of the restriction
fragment (15,106). The predominant product was the
leader transcript; at 32°C and above, approximately
95% of transcripts initiated at the trp promoter terminated in the leader region (45, 50, 174), whereas at
lower temperatures, the percentage readthrough increased to about 25% (45, 174).
Experiments performed in vitro showed that the
transcription termination complex dissociated spontaneously at the trp attenuator, releasing transcript,
template, and polymerase (14). Accessory complexbinding factors therefore do not appear to be essential
for this dissociation. However, it seems likely that in
vivo such factors interact with the complex or one of
its components and facilitate transcript release. One
candidate for such a protein is the NusA protein (59).
However, this protein did not influence transcription
termination at the trp attenuator when added to transcription reaction mixtures containing trp operon templates (44, 50, 174). A second transcription accessory
factor, the Tau protein, appears to influence termination
complex dissociation with other systems (23).
Roles of the Transcript Pause and Antitermim.tor
Secondary Structures
The segment of the leader transcript just beyond the
translation start codon can form the stable hairpin
designated 1:2 (Fig. 3A). When this transcript segment
is synthesized by the transcribing polymerase molecule, polymerase pauses on the template and forms a
paused transcription complex (46, 91,174). This complex never terminates transcription (49). The longevity of the paused complex is influenced markedly by
the concentration of the next nucleotide to be added,
as the paused polymerase resumes transcription, and
by the presence of the NusA protein (44, 46, 49-52,
101,174). Paused complex release is promoted by high
concentrations of this nucleotide and inhibited by the
presence of the NusA protein. It is thought that RNA
hairpin 1:2 serves as the pause signal. This conclusion
is based on analyses with mutant templates which
specify transcripts with pause structures with reduced stability (trpL67,68, trpL75,76,77, trpL80; Fig.
3A), and on studies employing nucleotide analogs as
transcription substrates, and is supported by the demonstration in vitro that the paused complex is released
by the addition of an oligonucleotide complementary
to the trailing strand of the RNA hairpin (50,52, 101,
173).
RNA segments 2 and 3, which form parts of the
pause and terminator hairpins, also may form the
TRANSCRIPTION ATTENUATION
1281
antiterminator. This structure is not observed in vitro
in the intact leader transcript, but it is readily detected in transcripts of deletion mutants in which the
regions corresponding to the competing segments (1
and 4) of the pause and terminator structures have
been removed (98). Studies in vivo and in vitro with
leader point mutants (trpL75,76,77, trpL93, trpL115;
Fig. 3B) and deletions in which the stability of the
anti terminator is reduced establish that this structure
causes termination relief. Deletions which remove
only RNA segment 1 and thereby prevent 1:2 formation show little or no termination in vivo or in vitro
(162-164). However, if the deletion removes part of
segment 2 as well, thus preventing the antiterminator
from forming, then efficient termination at the attenuator is observed (162-164). When RNA segments
1,2, and 3 are deleted, there is no termination at the
attenuator either in vivo or in vitro (162-164). These
findings establish that termination and termination
relief are consequences of the formation of different
structures in the leader transcript and reinforce the
conclusion that the role of the translating ribosome is
to determine which of the two structures forms.
RNA Polymerase Recognition of Pause and
Termination Signals
Mutants have been isolated that produce altered
RNA polymerases that behave aberrantly in the recognition of transcription pause and termination signals (51, 178). The mutants studied to date were
isolated as rifampin resistant, and all are altered in
rpoB, the structural gene for the J) subunit of RNA
polymerase. The mutants were recognized by their
increased or decreased ability to terminate transcription at the trp attenuator in vivo. When RNA polymerase was purified from these mutants and studied in
vitro, the altered termination behavior seen in vivo
was reproduced (51). These mutant polymerases also
behave aberrantly in transcription pause analyses
(51). Mutant polymerases that terminated transcription at the attenuator more efficiently formed more
stable paused complexes. Conversely, mutant polymerases that terminated less efficiently produced more
short-lived paused complexes. These observations
indicate that the polymerase alterations studied
comparably affect the response to both pause and
termination structures. The participation of the
other polymerase subunits in the response to pause
and termination structures has not been examined.
FINE FEATURES OF ATTENUATION IN THE trp
OPERON
Synchronization of Transcription and Translation
The model of attenuation in amino acid biosynthetic operons suggests that each leader transcript must
load a ribosome and that this ribosome must synthesize all or part of the leader peptide while the transcribing RNA polymerase molecule is within the
leader segment of the operon, i.e., the segment that
specifies alternative RNA secondary structures. Comparative mRNA measurements with wild-type strains
and with deletion mutants that lack the attenuator
have shown that during severe tryptophan starvation
1282
LAN DICK AND YANOFSKY
there is little or no termination at the attenuator (17,
164). Thus, if the attenuation model is correct, bacteria starved of tryptophan must have a ribosome stalled
over one of the Trp co dons of every leader transcript.
This could be accomplished if ribosome loading at the
leader peptide ribosome-binding site were extremely
rapid, or if transcription were delayed at the pause
site in the leader region until the paused polymerase
was released by the translating ribosome. The available evidence favors the latter possibility (100). As
mentioned earlier, the first RNA hairpin structure to
form in the leader transcript functions as a transcription pause signal (46, 49, 101, 174). Experiments
performed using an in vitro coupled transcriptiontranslation system indicate that the paused transcription complex is released by the moving ribosome as
the leader peptide coding region is being translated
(loa). Pause release occurs much more slowly when
translation of the leader peptide coding region is
prevented. Release is observed under tryptophan
starvation and nonstarvation conditions (100). This
is expected, since the Trp codons are within the
segment of the transcript that forms the RNA pause
structure. Thus, transcription pausing, and pause
release by the translating ribosome, may be the
events that synchronize transcription of the leader
region with translation of the coding region for the
leader peptide.
Transcription Readthrough in the Presence of
Excess Tryptophan
When E. coli cultures are grown in the presence of
excess tryptophan, 1 of every 5 to 10 polymerase
molecules that transcribe the leader region does not
terminate at the attenuator (16, 179). Similarly,
during growth with excess tryptophan, repression
permits a transcription initiation frequency 1/S0 the
rate obtained in the absence of repression (179).
Conceivably the low level of trp operon transcription maintained in the presence of excess tryptophan equips the cell with sufficient levels of the trp
enzymes so that small amounts of tryptophan could
be synthesized if a culture growing in tryptophan
excess were shifted abruptly to a tryptophan-free
environment. How is the low level of transcription
read through at the trp attenuator maintained in
cultures growing with excess tryptophan? The available evidence suggests that in tryptophan excess,
read through is influenced both by translation of the
leader peptide coding region and by the relative
stabilities of the RNA pause, anti terminator, and
terminator structures (97,101,163). Base-pair
changes and deletions in the leader region that
reduce or eliminate translation of the leader peptide
coding region result in fivefold greater termination
at the attenuator in vivo in excess tryptophan than
is observed in strains with the wild-type operon
(163, 187). This phenomenon has been termed
superattenuation (163). Since in some of these mutants the transcript secondary structures are unaltered, increased termination must be a direct consequence of the translational defect. These findings
suggest that in the absence of translation the RNA
terminator must form more frequently. We explain
this by assuming that, in the absence of translation,
the pause structure forms normally and causes
pausing, but when the paused polymerase resumes
transcription, reformation of the 1:2 pause structure
prevents formation of the anti terminator. If the
anti terminator does not form, the terminator willand cause termination.
The fivefold higher level of readthrough observed in
wild type in vivo when there is translation probably is
due to the more frequent formation of the antiterminator. There are two ways at least that this could be
accomplished (97, 101). After the moving ribosome
disrupts the pause structure, it could occasionally
remain near the Trp co dons on segment 1 long enough
to favor formation of the anti terminator and allow
RNA polymerase to transcribe past the attenuator.
Alternatively, the translating ribosome might move
rapidly to the leader peptide stop codon. If so, the
basal level of read through would be determined by
how fast the ribosome releases at the stop codon: a key
question to which there is no answer at present. If the
ribosome releases before segment 3 is synthesized,
1:2 reformation would lead to termination. If segment 3 is synthesized before release, 1:2 and 2:3
would compete for formation. Thus, the probability
of read through might be determined by the location
of the transcribing polymerase when the ribosome
dissociated and by the relative probabilities of formation of the competing RNA structures. Analyses
with mutants that form altered pause or antiterminator structures have shown that the stabilities of
these structures do influence the extent of readthrough transcription in vivo in the presence of
excess tryptophan (97, 101). Since the relative stabilities of the trp pause and anti terminator structures vary greatly in different enterobacteria, we
suspect that there may be a wide range of basal
read through levels in these species (177). This could
reflect the different environmental experiences each
species has had to adjust to during its evolution.
Shutdown of Leader Peptide Synthesis
The terminated trp leader transcripts of the several
enterobacterial species that have been studied have a
segment near their 3' end that theoretically can base
pair with the ribosome-binding site used in the synthesis of the leader peptide and thus block its utilization in translation initiation (177). Structural analyses with isolated leader RNAs of S. typhimurium and
Serratia marcescens have shown that the leader ribosome-binding site can in fact base pair with this distal
segment of the transcript (98; K. Brown and C. Yanofsky, unpublished data). Experiments designed to test
whether this pairing blocks synthesis of the leader
peptide have been performed using a highly purified
DNA-dependent dipeptide-synthesizing system and S.
typhimurium trp leader templates that do or do not
contain the segment complementary to the leader
ribosome-binding site. It was observed that synthesis
of the leader initiation dipeptide, fMet-Ala, was reduced about lO-fold when the template contained the
downstream complementary region (33). We assume
from these findings that leader peptide synthesis is
shut down in vivo soon after the decision is made
whether or not to terminate transcription at the attenuator.
77
Starvation for Amino Acids Other than Trp, and
Significance of the Two Trp Codons in the Leader
Transcript
There are two Trp co dons in the coding region for
the 14-residue trp leader peptide. Does ribosome stalling at other co dons in this coding region also relieve
attenuation? This question has been addressed in
amino acid starvation experiments performed with E.
coli cultures containing the E. coli or S. marcescens trp
leader region (164, 187). It was observed that with few
exceptions, starvation for amino acids corresponding
to other co dons in the trp leader transcript did not
relieve attenuation. The interesting exceptions involved starvation for arginine, the amino acid specified by the codon immediately following the Trp
codons, and starvation for histidine in studies with
the S. marcescens leader region. In the trp leader
peptide coding region of the latter organism there is a
single His codon two co dons before the Trp codons.
These findings indicate that the position of the stalled
ribosome on the transcript is crucial to attenuation
relief and that the critical segment of the trp transcript extends beyond the two Trp codons. If one
assumes that a stalled ribosome masks about 13
nucleotides on either side of the codon which is being
read (175, 177), then stalling at the Arg, His, or Trp
codons of the leader transcript would be expected to
promote formation of the anti terminator. Therefore
the results of these experiments illustrate the importance of codon location, as well as the number of the
various leader regulatory codons, to regulation by
attenuation.
TRANSCRIPTION ATTENUATION IN OTHER
AMINO ACID BIOSYNTHETIC OPERONS
The his Operon
Regulation of the his operon. The E. coli and S.
typhimurium his operons each are composed of nine
genes that encode the enzymes required for the synthesis of histidine from ATP and phosphoribosylpyrophosphate (7,18,21; see also chapter 25). In the 1960s
it was recognized that the availability of histidine
regulated the synthesis of the histidine biosynthetic
enzymes in these organisms (145; see Brief Historical
Review, above). By the early 1970s it was recognized
that translation was involved in his operon regulation
(Martin et aI., Int. Congo Biochem. Abstr. 2:261,1967;
Martin and Ames, personal communication), and histidine-specific transcription attenuation was postulated (90).
In the late 1970s and early 1980s, DNA sequence
analyses and mutational studies with the E. coli and
s. typhimurium his operon leader regions led to a
detailed, well-supported model for the regulation of
the his operon by attenuation. Because the large body
of mutational data on regulation of the his operon
provided important underpinnings for the current
general model of attenuation in amino acid biosynthetic operons, we shall describe studies with this
operon in greater detail than other examples of attenuation.
Sequence of the his leader region. The sequences of
the E. coli (38) and S. typhimurium (10) his operon
leader regions were reported in 1978. The sequence
TRANSCRIPTION ATTENUATION
1283
data immediately suggested a model for his operon
attenuation that was compatible with previous regulatory findings. The following significant features were
identified: (i) the his leader regions of both species
encode an identical 16-residue leader peptide that
contains 7 contiguous histidine residues, at positions
8 through 14; (ii) segments of the leader transcript
theoretically can fold into two alternative secondary
structures: one has a potential Rho-independent terminator at its 3' end, and the other structure could
function as an anti terminator, precluding formation
of the terminator; and (iii) the location of the seven
His co dons in the leader peptide coding region would
allow a ribosome stalled at these codons to promote
formation of the anti terminator and hence prevent
formation of the terminator. If the model based on
these features is correct, mutational changes in the
leader peptide coding region or the base-paired segments that form alternative RNA secondary structures should have profound effects on regulation of the
his operon. The alternative secondary structures proposed to regulate attenuation in the his leader region
(Fig. 4A and B) have features that differ only slightly
from those formed by the trp leader transcript. The his
leader transcript can form three hairpin structures in
the termination configuration, 1:2, 3:4, and 5:6 (Fig.
4A), whereas in the alternative, anti termination configuration (Fig. 4B), segment 2 (available when a
ribosome stalls on segment 1) can pair with segment
3, thereby allowing structure 4:5 to form and block
formation of the terminator (5:6).
Detection of the terminated his leader transcript.
Both the E. coli and S. typhimurium his leader transcripts have been identified as products of in vitro
transcription (53, 60). These transcripts originate at
homologous positions, and the promoter sequences
for the operons are nearly identical. Termination of
the his leader transcript in vitro is extremely efficient;
significant readthrough at the S. typhimurium attenuator occurs only when ITP replaces GTP in the
reaction mixture, presumably reducing the stability
of the 5:6 hairpin (53). A transcription pause after
synthesis of the 1:2 hairpin structure has not been
reported, but seems likely.
Demonstration that synthesis of the his leader peptide is required for attenuation. Constitutive mutations that affect the rate of initiation of leader RNA
synthesis have never been obtained. This is in accordance with the attenuation model, since His regulation
only affects termination at the attenuator. To isolate
leader peptide mutants incapable of responding to His
starvation (termed hisO P mutations), 10hnston and
Roth (87) devised a selection for decreased his operon
expression. This selection was based on the adenine
auxotrophy induced by ATP consumption in a strain
with elevated his enzyme levels (hisT -) and a feedback-resistant hisG protein. Mutations causing adenine prototrophy and decreased his operon expression
result in His auxotrophy (strong phenotype) or permit
strains to remain His+ but become sensitive to
aminotriazole (AT', weak phenotype). This selection
was used to obtain a large number of mutations that
limited expression of the his operon (88, 89).
10hnston and Roth (89) described five different
mutations that reduce his operon expression by affecting translation initiation of the leader peptide coding
LANDICK AND YANOFSKY
1284
A
AUCAAAUGA
1:2
A
U
G U
Stop
A
A
A
G
ASp
U A
A-U
A
Pro
U
A
A
His
C
C~G
his09675
UGACACGC
His
~+.~
U-A
A-U
HiS:::
MetrhrAr~alGlnpheLYSHiSHiSHiSHiSA-U
A-U
A-U
U C
U
A
C~G
C~G
G~C
G~C
G
C~G
C
A
A-U
C~G
C~G
G~C
A-U
U
U
G~C
3:4
U-A
U
C
G
G
U
C 160
U
G~C
20 A
~
U
C
C
A
U
U
C
5:6
C
80 U
A C
C~G
A-U
A-U
A~:~G ~:~
g:g
U-A
UUUAUGACACGCGUUCAAUUUAAACACCACCAUCAUC~G--------C~GUGGUGC~GAGA-UUUUUU
~l
40\
~U
hiS09654
60
100
180
+C his09876
A his09856
ilhis09613
C his09709
4:5
B
A C
A
AUCAAAUGA
G
G~C
U-A
A-U 140
A
U
A
A
C~G
G
C
A
U
U
C
20 A
G
G
U
U
A
G
U
G
U
G~C
U
A
A-U
G~C~G
A~U-A
C~G
MetrhrArgValGlnPheLysBisHisBisHisBisBisBisProAsp
U-A
C
C~G
his09864
U"G
C~U-A
C
G~C
G
his09609
his09863
A
A
GA
G~C
his09693
AG
G~C
C~G
U
C
G
G
A
A
U
U
2:3
A~C~~C
his09897
U-A
his09896
his09713
G~U-A
A~C~G~U
his09712
U-A
A-U
UUUAUGACACGCGUUCAAUUUAAACACCACCAUCAUCACCAUCAUCCUGACUAG~CAUU----------CAG=CAUCUUCCGGGGGCUUUUUUUU
40
60
80
160
180
FIG. 4. Alternative secondary structures of the S ..typhimurium his leader transcript. Secondary structures 1:2,2:3,3:4,4:5,
and 5:6 are those reported by Johnston and Roth (89). The positions of key mutations are indicated. (A) Termination
configuration. (B) Antitermination configuration.
region. Changing the AUG initiation codon to AUA
(his09856; Fig. 4A) or ACG (his09709; Fig. 4A) caused
a moderate reduction in his operon expression (weak
phenotype; His+ AT'), whereas a deletion replacing
the AUG with AG (his09613; Fig. 4A) blocked expression sufficiently to cause histidine auxotrophy. Not
only do these results establish that leader peptide
synthesis is required for readthrough at the his attenuator, they also suggest that the AUA and ACG
codons may allow inefficient translation initiation
since they only partially reduced his operon expression. Mutational changes in the sequence immediately
preceding the his leader peptide start codon, which
apparently sequester the ribosome-binding site in a
hairpin secondary structure, can cause either a slight
or large decrease in his operon expression, depending
on the stability of the hairpin created.
Mutations altering the leader peptide. Several non·
sense mutations in the S. typhimurium his leader
peptide coding region have been isolated (89). Analysis of these mutations suggests that the level of his
operon expression is correlated with the position at
which translation terminates in the leader peptide
coding region. Mutations causing translation termination at codon positions 4 (his09675; Fig. 4A) or 5
(his09654; Fig. 4A) of the leader peptide coding region
result in a strong His- phenotype, whereas an insertion mutation (his09876) causing termination at
codon 7, immediately preceding the seven His codons,
allows a modest increase in expression, leading to a
weak His+ AT' phenotype. This result suggests that
when the translating ribosome reaches codon position
7, but not codon position 5, it can occasionally disrupt
structure 1 :2. Thus, the his attenuator has evolved so
r
, that the translating ribosome does not completely
block formation of RNA structure 1:2 until it reaches
the His codons beginning with codon 8 (Fig. 4A).
When appropriate nonsense suppressors are combined with the his09654 (C ~ V, ochre) or his09675
(8-base-pair insertion, opal) alleles, the His- phenotype is suppressed (89). This result verifies that translation of the leader peptide coding region is required
for normal his operon expression and also suggests
that the exact amino acid sequence of the leader
peptide is unimportant for some termination relief.
When the his09675 allele is suppressed and translation continues out of frame to a VAA codon 30 bases
past the normal leader peptide termination codon,
transcription of the his operon is sufficient to give a
His+ phenotype. This suggests that the translating
ribosome closely follows RNA polymerase and disrupts structure 5:6 often enough to allow near-wildtype levels of transcription of the his operon structural genes.
Finally, all hisO-type mutations that affect leader
peptide synthesis are cis dominant. Taken together
with the fact that the variant leader peptides produced by the his09876 allele and the suppressed
his09675 allele allow at least some transcription to
proceed through the attenuator region (His+ AT' phenotype), this result also suggests that the leader peptide per se has no function. Rather, it is the act of
translation of the leader peptide coding region that is
responsible for read through at the attenuator.
Mutations affecting his leader transcript secondary
structures. Analyses by Johnston and Roth (88, 89) of
a set of mutants with alterations in the his leader
region provided convincing evidence for the role of
RNA secondary structures in governing attenuation.
Changes that would weaken the predicted stabilities
of either hairpin 2:3 (his09693, his09609, his09863;
Fig. 4B) or hairpin 4:5 (his09864, his09896, his09713,
his09897, his09712; Fig. 4B) cause greater termination. In general, those changes that would appreciably
destabilize these hairpins (his09693, his09609; Fig.
4B) cause His auxotrophy, whereas those that would
only slightly reduce their stability (his09863,
his09864, his09896, his09713, his09897, his09712;
Fig. 4B) produce the His+ AT' phenotype. These findings established that both hairpins 2:3 and 4:5 are
required for anti terminator function in the his leader
region. Johnston and Roth also characterized five
deletions that removed portions of the antiterminator
sequence (~108-118, ~109-122, ~116-120, ~116-124,
~156-159; see Fig. 4B). All five deletions cause a Hisphenotype. That changes in his anti terminator function are due to alterations in base pairing and not to
primary sequence alterations was established by constructing the double mutant his09712 his09713 (M.
Johnston and J. Roth, personal communication). The
single mutant parents have a His+ AT' phenotype,
indicating that hairpin 4:5 is weakened and hairpin
5:6 forms more readily. However, in the double mutant, the wild-type GC base pair is replaced by an AV
base pair that partially restores the predicted stability
of hairpin 4:5. The double mutant expresses wild-type
levels of his enzymes. As predicted by the attenuation
model, formation of a stable RNA secondary structure
governs termination at the attenuator.
77
TRANSCRIPTION ATTENUATION
1285
Another interesting finding was that his mutations
that affect anti terminator structure were suppressible
by amber suppressors (86, 88, 89). All of these mutations are past the stop codon for the leader peptide
and do not generate amber codons.1t is likely that the
effect of these mutations is reversed by translation of
the normal VAG leader peptide stop codon and continued ribosome movement to an in-frame VGA stop
codon at the base of hairpin 5:6 (nucleotide 140, Fig.
4A). A ribosome approaching or positioned at the VGA
stop codon apparently can block formation of 5:6 and
allow sufficient transcription read through to overcome the His- phenotype.
Mutations affecting tRNAHls synthesis. An interesting aspect of work on the histidine attenuator is that
many of the mutations that increase expression of the
his operon do so by affecting tRNA His or its synthesis
(see chapter 25 for a more complete discussion of
these mutations). In initial investigations with the his
operon it was considered possible that a his repressor
recognized tRNA His as its corepressor. Since no evidence for such a repressor protein ever emerged,
alternative explanations were sought. Ames and coworkers (153) showed that the hisT mutation affects
pseudouridine formation in several tRNAs, including
tRNA His . Whereas hisT mutants possess wild-type
levels of charged tRNA His (108) and synthesize 13galactosidase at a normal rate (86), they utilize supE
(glutamine-inserting amber suppressor) to suppress
the lacU281(Am) mutations less than 5% as well as
wild type (86). These results suggest that increased
expression of the his operon in hisT mutants is caused
by inefficient translation of the seven His codons in
the leader peptide coding region, thus allowing more
frequent formation of the anti terminator RNA structure. Mutations affecting hisR, the tRNA His structural
gene, can have a similar effect, or, like mutations that
affect His-tRNA synthetase (hisS), they can affect
leader peptide synthesis by reducing the efficiency of
tRNA His charging, which would decrease the intracellular concentration of histidyl-tRNA His .
Recently, Ames and co-workers (4) have pointed out
the existence of a high degree of sequence (45 of 75
nucleotides) and structural homology between the his
leader transcript and tRNAHis. They suggest that proteins known to interact with tRNA His , such as tRNA
modifying and processing enzymes, histidyl-tRNA
synthetase, the hisG enzyme, ribosomal proteins, and
elongation factors, may interact with similar sites in
his leader RNA. Such RNA-protein interactions could
provide regulatory input and affect expression of the
his operon by altering the efficiency of termination at
the his attenuator. If such interactions occur, they not
only would provide alternative explanations for the
phenotypes discussed above but also would suggest
that attenuation control of the his operon is considerably more complex than currently believed. Evidence
for these possibilities, such as demonstrating modified
bases in the his leader transcript or documenting a
leader RNA-protein interaction, has not yet appeared.
If this tRNA-leader RNA homology is indicative of
additional interactions and events, it could provide
fertile material for future studies on attenuation.
Alternatively, the tRNAHiS-his leader RNA homology
may reflect an evolutionary relationship, and leader
1286
LANDICK AND YANOFSKY
RNA may be incapable of interacting with proteins
that recognize tRNA (see Conclusions, below).
The leu Operon
Attenuation control of the leu operons of E. coli and
S. typhimurium has been elucidated by Calvo and
co-workers (26, 64, 92,150,172). In these organisms,
leu enzyme levels are elevated as much as 50- and
40-fold, respectively, under conditions of leucine-limited growth (24). In both organisms, the levels of total
leu operon mRNA parallel leu enzyme levels; in S.
typhimurium it has been shown that leu leader RNA is
synthesized constitutively, whereas the level of leu
structural gene mRNA is regulated by exogenous
leucine (150). All transcription regulation of the leu
operon appears to be accomplished by attenuation; no
evidence for a leu repressor has been obtained. The
constitutivity of leu leader RNA synthesis (150) argues
against repression. DNA sequence analyses and in
vitro transcription studies suggest that leu leader
RNA and trp leader RNA fold in similar secondary
structures (Fig. SA); both the E. coli and S. typhimurium leu leader transcripts encode 28-residue peptides
that contain 4 contiguous leucine residues. Comparison of the S. typhimurium and E. coli leu leader
sequences reveals considerable conservation in the
regions thought to be involved in RNA secondary
structure formation, as well as the Leu codon regions.
Regions of the leader thought not to be directly
involved in attenuation show greater divergence. It is
particularly interesting that, as in the case of the trp
leader peptide, some leu leader peptide residues in the
two organisms differCat codons 2, 8, 16, 17, 18,22, and
24; Fig. SA), in agreement with the notion that the
leader peptide amino acid sequence per se (other than
the Leu residues) is unimportant.
Two different G ~ A changes in the 3:4 (terminator)
hairpin of the S. typhimurium leu leader have been
obtained (leu-2007 and leu-2009; Fig. SA; 24,25, 150).
leu-2007 reduces the predicted stability of the hairpin
from -24 to -14 Kcallmol (ca. -100 to -59 kJ/mol)
and increases read through at the attenuator from 2 to
25% in an in vitro transcription system. Strains carrying the leu-200l allele have 30-fold higher levels of
Jj-isopropylmalate dehydrogenase (leuB gene product)
in excess Leu, but near wild-type levels under Leu
starvation conditions, as would be expected on the
basis of the attenuation model. Mutation leu-2009
reduces the predicted stability of the hairpin to -12
kcallmol (ca. 50 kJ/mol); its effect on leu operon
expression is identical to that of leu-2007 (24, 25).
Recently, Calvo and co-workers used site-specific
mutagenesis to replace the four leader peptide Leu
codons with four Thr codons (26). The regulation of
this altered leu operon has some interesting implications for the role of control co dons in attenuation. As
expected, the mutant strain did not show the 40-fold
wild-type response to leucine limitation; however, it
did exhibit a 3-fold increase in the Jj-isopropylmalate
dehydrogenase level upon leucine limitation. More
importantly, growth of a temperature-sensitive threonyl-tRNA synthetase mutant (containing the Leu ~
Thr changes) at the nonpermissive temperature
caused a lO-fold increase in Jj-isopropylmalate dehydrogenase (26). These results argue that ribosomes
stall less frequently at the Thr codons of the mutant
leader transcript during threonine limitation than at
the wild-type Leu codons during leucine limitation.
CUA, the Leu codon used predominantly, is a rarely
used Leu codon (66) whose cognate tRNA is present at
a low concentration (29), whereas the ACU Thr co dons
that replace the Leu codons are translated efficiently
because their cognate tRNA is abundant (29). Thus
leucine limitation may stall ribosomes more efficiently at CUA codons than threonine limitation reduces ribosome movement over ACU codons. Calvo
and co-workers suggested that accumulation of undermodified tRNAs may cause the residual response of
the mutant to leucine limitation. Undermodified
tRNAs do accumulate during leucine starvation (95).
As suggested for the effect of hisT on his operon
attenuation (86), undermodified tRNAs may slow
translation, allowing some ribosomes to reside in
critical regions of the leader peptide coding region
long enough to promote antiterminator formation.
The response of the wild-type leu operon to threonine
limitation may have the same explanation. Both of
these arguments (codon translatability and tRNA
undermodification) suggest that attenuation could be
fine-tuned by adjusting codon usage in a leader peptide coding region (see Leader Peptide Sequences and
Functions, below).
The thr Operon
The threonine (thr) operon of E. coli encodes three
polypeptides; these polypeptides are responsible for
four of the five enzymatic activities required for synthesis of threonine from aspartic acid (54, 166). The
first gene in the operon, thrA, specifies a bifunctional
enzyme, aspartokinase I-homoserine dehydrogenase I,
which catalyzes reactions 1 and 3 of the common
pathway that leads to the formation of threonine,
diaminopimelic acid, lysine, methionine, and isoleucine. The other two genes of the operon, thrB and thrC,
encode polypeptides that catalyze steps 4 and 5 of the
specific pathway leading to threonine. In both S.
typhimurium and E. coli, limiting the growth of appropriate auxotrophs by withholding either threonine
or isoleucine causes a 20-fold increase in the level of
the thrA polypeptide; thus the thr operon is regulated
by both threonine and isoleucine. In this respect, the
thr operon differs from the three operons discussed so
far, the trp, his, and leu operons. It initially was
thought to be regulated by "multivalent repression"
(54); however, much as in the history of his operon
regulation, what was initially considered to be regulation by repression turned out to be regulation by
transcription attenuation.
Gardner (61) reported the DNA sequence of the
region immediately preceding thrA. This region contained both a coding region for a 21-amino acid leader
peptide, with 4 lIe and 8 Thr residues, and segments
that could specify potential alternative RNA secondary structures (Fig. 5B) similar to those observed in
the trp and leu operons. Transcription begins 189
nucleotides preceding the AUG start codon of thrA
(61). During in vitro transcription, RNA polymerase
pauses after synthesis of the 1:2 RNA hairpin (61), and
90% of transcripts terminate immediately after the
3:4 hairpin structure (114; Fig. 5B). These findings are
77
1287
TRANSCRIPTION ATTENUATION
1:2
A
U A U
Phe
U
I1.
U
U
G
A-U
Ala
C=G
Asn
G=C
C=G
Val
Arg
CAA
Leu
2:3
CU
AU
G C
Leu
GAU
Leu
C=G
A
Gly
Stop
Arg
G=C
U
G
G
A
Ph.
Arg
G
U
G
A
A
3:4
Val
C
Gil}
U'G
U-A
U-A
U
C=G
G=C
140 C"'G
G=C
C=G_A leuL2007
U
Val
C
Ile
G U
C=G
20 C
AA
CA
AA
Gln
U'G
AUAACGCAUUCGC
,I.e
His
Pro
C=G
A-U
C=G
U'G
U
U'G
G=C
G
U
C
U
G
The
AA
A
A
G=C
Gil}
120
CG
AG
U-A
C=G
Leu
U
uC
A-U
A
A
C
C
Gl Y
G=C
Gly
G=C
C=G
G=C
G=C
G=C
C=G
Gly
G=C
Ile
Tie C
NetSerHisU-A GlnBisStop
140
G
C=G - - A leuL2009
A-U
Val
Pro
Leu LeuLe uAsnAl a PheI 1 eVal ArgGl yArg
A-U
A-U
U"G
G- U
G' U
C U AC U AC UC MC UCAU U UAUUG UGGGCGGUAGACG =GC GCGGGU UU UU U U
60
80
160
UUAAU GUCAC A UA- U UCAAC AU U AAGUCAGC UCGAAG UCAAACA- UU UU
120
160
B
GCAUAGCGCACAG
A
C
20
U
A
2:3
A
U
U
A
3:4
A
120 G A
AG
AC
"C
AC
G A
C
U
C
A
C
C=G
A-U
thrL140 A_C=G __ A.U thrLISl
thrLIJ9 A +--G=C
1:2
Thr
C
A
Thr
C=G
C=G
A-U
A
A
U
G
GG
G
AA
CA
Stop
AA
G
A
U-A
G1 Y
C:=G-+-U thrL135
G1 Y
G=C
G=C
G::C--+-A thrLl]8
Ala
C::G--....A thrLIJ9
G=C____.._A thrL140
G1!l U-A
ThrThrGl yA.nG"C
ACC ACA GGU AAC G =C UGAC AG UGG GGGC U U U UU U U U
160
consistent with the general attenuation model discussed earlier.
Segments of the thr leader RNA resistant to RNase
T, have been identified (61); the data obtained agree
with the structures shown in Fig. SB. However, the
exact structure assumed by folded thr leader RNA is
as yet unknown. Secondary structure predictions us-
ing the method of Zuker (185) suggest more extensive
1:2 and 2:3 structures than pictured in Fig. SB (see
reference 96). Lynn et al. (113) have proposed two
alternative forms of the 1:2 hairpin structure: the one
shown in Fig. SB, and a second that pairs RNA segments
78 to 87 with RNA segment 2. Interestingly, RNA segments 60 to 69 and 78 to 87, termed lA and IB by
1288
LAN DICK AND YANOFSKY
2:3
G
C
A
A
A
C
G
Stop
AI.
1:2
A
Val
CCUG
Gl Y
3:4
A G A
G
stop
AGCAUCUU
G
Leu
C=G Gly
U
CA
ACe
U-A
U-A
I 120
GA
Uc
CA
G=C
Ala
G=C
C
G:::C
160 C=G
'thr
~
A
CG
Ala
A-U
AG
Arg
Val
20
A
C
C:G
U
A
G
A A
Leu C:::G
Ala C=G
C",G
Ala C=G AI.
G:::C 140
C=G
G=C Ala
LlJs G=C
C:G
"et A-U
LeuGIYArgGl yA-U
C=G
AUUCCUUCGAACAAGAUGCAAGAAA AGAC AAAAUGAC =GC AC U UGGA GAGGAA- UAACGAAC UAAGA- UUUU UUU U
IIe
CG
G
Uu
Leu
G
A
C:G
GUCGU GAUUA
UG
A
AC
G=C
Ser G
A
Uu
A
GG
Ser G·U
Ile C::G CIj_
Leu
140
G"
Arg
G=C
G=C Pro
U-A
G=C
G=C Pro
U·G
V.l
A.
Gl Y
IIo
U
Val
Val
Ly.
U
U-A
A-U
G=C
V.l
IIo
U
U
IIo
U
U-A
U-A
C-G
G=C
G-C
A-U
A-U
Gl!l GaC
G=C 160
C-G
eys G=C
Pro U-A
Pro G"'C
valValValIleIleIle
C
C
GUGGU GG UGA U U AUU A UCCC A C = GA A AGGUC C GGGGGU U UU U U UU
80
180
180
P
o
1:2
U
A C
C
Pro
Ser
C
C:G 80
G=C
AI.
C
CoG
G=C
U-A
CoG
AI.
Sor
Ala
v.,
A
A-U
CoG
CoG
Pro
A-lJ
Leu
U
Val
C
CoG
A-U
Leu
ell
LYB
Val
c
_p
A
II"
G
A-U
C::<G
AI.
Val
G=C
C:G
Arg
A-U
Asn
100
UCA
Leu
C
v.,
GU
Met
Ser
A-U
CoG
C=G
Thr
Val
CoG
A-U
U·G
C-G
v.,
A-U
Val
U'G
A-U
Val
Gl!l
Pro
stop
Aro
G
U Val
G
U
A-U
A-U
G=C
G
G=C
U-A
G
CoG
U·G
G=C
160
StopG=C
GoU Val
G-C
GU
AC
AA
CA
G-C
C=G
U·G
v.l
C
A
GoU Val
v.l
A
G
U
C
G-C
G
ProCzG
U Val
CaG
G-C
G-C
Ala C .. G Gly AlaeaG
Ly_LeuLeuproSerAlaProSerC"'G A_n
Gee
AAACU A CUACC AAC UGC GCC A UCCG' C A - - - - - - AU- AAACCGGGCGGGGU UUU UCGU U U
60
140
G
U Val
v.l
AI.
C'G
A-U
A-U
U-A
A- U
AU UGU UAA"C AC II A A ACe II ACAAGG =C UGGA A C"AC "C"eGA UUC CA- U UCG UU U
20
V.l
A
G
C
C A
A
A
G=C
G=C
G
CoG
CoG
G=C
CoG
160 CoG 180
CoG
G
C
A.sn
A
100
U
3:4
CGGCAAU
GCCGCG
U
Ala
C=G
CoG
C-G
C
A
G'C
Ifet
C
A
C-G
U
G
40 U
C
80
180
FIG. 5. Continued
Gardner, are direct repeats. Each contains two of the
eight Thr codons and one of the four lIe codons for the
leader peptide. The presence of these repeats probably
increases the regulatory capacity of the thr leader region. It is unclear which, if either, of the two potential
1:2 hairpins actually forms in the thr leader transcript.
The regulatory mutation thr-79-20, isolated on the
basis of increased thr operon expression (63), inserts a
C residue in segment 4 of the terminator structure (61;
Fig. 5B) and reduces the efficiency of in vitro transcription termination from 90 to 75% (114). A complete deletion of the 3:4 hairpin, thr01028 (nucleotides 107 to ISS, Fig. 5B), was isolated using a protein
fusion of thrA to lacZ (147). This deletion caused a
lO-fold increase in r3-galactosidase activity as compared with lacZ fusions that retain the entire leader
region (130). Recently, Gardner and co-workers have
characterized 10 additional mutational changes in the
77
TRANSCRIPTION ATTENUATION
1289
1:2
E
~
Stop
A
G
U
U
G
C=G
G~AUG
C=G
Pro
A
CA
C=G
C=G
GA
U
GU 80
Phe
C
GU
Uu
AG
GC
U
U·G
3:4
U-A
U-A
ph.
U
C
U-A
C=G
Phe
U-~
U-A
~-U
C=G
G=C
C=G
Ala
100
120
~
U
~
C
C=G
C=G
C=G
U·G
C=G
C=G
G=C
U-A
U-A
G:::C
A-U
8etLysBl_IleProPhe C=G
A-U
Phe
Phe
40
A
A
A
A-U
Stop
G=C
U·G
140
AAGUCACUUAAGGAAACAA~CAUGAAACACAUACCGUUUUU-ACGAACAAUA-UUUU
20
U
U-A
U-A
80 U-A
G
G
C=G 120
G=C
G=C
A-U
G=C
G=C
G=C
U-A
C=G
U-~
AA
C
GC
~-U
ph.
CA
UG
C
UG
C
Thr
A~
GA
C
60
CG
AA
Pro C=G
Phe . C=G
8etLysHisIlePcoPhePhePheAlaPhePbePherbr C=G
AUGAAACACAUACCGUUUUUCUUCGCAUUCUUUUUUAACUUC=GGCCUUUUUU
40
60
140
1:2
F
80 A U
A
C
G=C
U-A
C=G
C=G
Stop
Thr
A
Ser
Phe
A
C=GG
G=C
A-U
U-A
U·G
2:3
C
U·G
C
Tyr
phe
UCUUUU
Phe
C=G
A-U
U·G
U-A
U·G
U-A
U-A
U·G
U-A
C=G
U-A
100
40
C
G
G
G
A
G
A
A
A
A
100 A
G
A
A
Phe
G
A
G
Arg
G=C
AG
U
C=G
C
U
A
C=G
C=G
A
Ph.
U-A
C=G 140
C
U-A
U-A
C
U-A
C=G
A
IIe
U-A
C=G
A
A
C
G=C
A
AI. U-A
A-U
A
Neta.nAl.C=G
A-U
GGUAACGCAAGCAAUGAAUGCUG=CGCCUGA-UUUUU
CU-\
20
A A
G
A
A
G=C
U-A
G
C=G
G-C
C=G
G=C
3:4
120
Cu
A
G
AA
U-A
CoG
G=C
G-C
A-U
PheArgPbePhePheTyrPheSerrhr
G=C
uUCCGCUUCUUUUUUUACUUUAGCACCUGAAUCCAG=CCAGUGGAGGCUUUUUUU
120
60
80
140
FIG. 5. Continued
3:4 hairpin that increase homoserine dehydrogenase
levels from 2- to lO-fold when the corresponding
mutant strains are grown in excess threonine and
isoleucine (113). In general. the predicted stabilities of
the altered RNA terminator structures correlate with
their termination efficiency. However, several exceptions to this conclusion were noted. Strains with
thrL135 U, thrL139 A, and thrL156 U mutations (mutations are designated by the leader position at which
the substitution occurs, followed by the nucleotide
present in the mutant) (Fig. SB) gave only modest
increases in the level of homoserine dehydrogenase
(approximately twofold) even though the calculated
stabilities of the 3:4 RNA secondary structures were
similar to those of mutants showing a 5- to lO-fold
increase in homo serine dehydrogenase. A strain with
the thr-79-20 mutation synthesized high levels of
homoserine dehydrogenase even though the calculated stability of its 3:4 hairpin was only slightly lower
than that of wild type. Lynn et al. (113) suggested that
1290
LAN DICK AND YANOFSKY
a specific hairpin structure may be required for transcription termination. Mutations that cause large increases in homoserine dehydrogenase expression might
alter this structure substantially, whereas mutations
that cause only modest increases in expression might
leave the key portions of this structure unchanged
even though they weaken its predicted stability. Alternatively, the findings of Lynn et al. may reflect our
incomplete understanding of all the features of RNA
secondary structures that influence stability.
Site-specific mutagenesis has been used to replace
the thr leader peptide stop codon by UGG (Trp); this
change extends the leader peptide coding region to a
UGA stop codon in the loop of the 3:4 secondary
structure (Fig. 5B; 141). A thrA-IacZ fusion was used to
monitor the effects of this change. The twofold reduction in f3-galactosidase levels observed in wild-type
cells when excess isoleucine and valine were present
was almost eliminated in the UGG mutant. These
results corroborate the findings of Johnston and Roth
with the his operon (86, 88, 89). In both cases, translation through the terminator leads to increased readthrough at the attenuator.
The ilvGMEDA Operon
The ilvGMEDA operons of E. coli and S. typhimurium encode four enzymes involved in isoleucine and
valine biosynthesis: acetohydroxy acid synthetase II
(valine resistant; ilvG and ilvM), branched-chain amino acid aminotransferase (ilvE), dihydroxy acid dehydratase (ilvD), and threonine deaminase (ilvA). In
conjunction with polypeptides encoded by other ilv
genes, these enzymes catalyze the conversion of threonine to isoleucine via condensation of threonine
deaminase-derived a-ketobutyrate and pyruvate and
the synthesis of valine from pyruvate. The pathways
of isoleucine and valine synthesis are parallel for four
analogous steps beginning with the condensation of
either two pyruvate molecules or one molecule each of
pyruvate and a-ketobutyrate. In E. coli K-12, the ilvG
gene is normally cryptic because of a frameshift mutation within the ilvG reading frame (102). This mutation renders E. coli K-12 susceptible to growth
inhibition by valine owing to the valine sensitivity of
the acetohydroxy acid synthetase I encoded by the
ilvE gene. Other strains of E. coli and S. typhimurium
encode a normal ilvG gene and are valine resistant.
ilvM, the recently discovered fifth gene in the ilvGEDA
operon (M.-F. Lu and H. E. Umbarger, Fed. Proc.
44:1417,1985), apparently encodes the small subunit
of acetohydroxy acid synthetase II (R. M. Kutny and
J. V. Schoss, Fed. Proc. 43:1867, 1984). Deletion of
ilvM resulted in loss of acetohydroxy acid synthetase
II activity in E. coli (Lu and Umbarger, Fed. Proc.
44:1417,1985).
The ilvGMEDA operon is regulated multivalently by
the levels of aminoacylated tRNA Val, tRNA Ilc , and
tRNALeu (20, 154). Multivalent control of ilvGMEDA
transcription is mediated by attenuation and involves
synthesis of a 32-residue peptide containing six valine,
five isoleucine, and five leucine residues (103). The
leader region is 270 nucleotides in length (1), and it
yields a 185-nucleotide terminated transcript that
potentially can fold into secondary structures (Fig. 5C)
similar to those involved in other examples of atten-
uation. A transcription pause site in the ilvGMEDA
leader region has been documented at position 117
(Fig. 5C) shortly after the 1:2 hairpin (73). This site
presumably functions to synchronize transcription
and leader peptide synthesis as postulated for the trp
operon (see Roles of the Transcript Pause and
Antiterminator Secondary Structures, above).
One distinguishing feature of the ilvGMEDA transcript is the bifurcated stem-loop structure that is
postulated to serve as the 1:2 hairpin (Fig. 5C). Interestingly, a complex 1:2 stem-loop also has been predicted for the ilvEN attenuator (see The ilvEN Operon,
below). To explain the participation of these complicated structures in attenuation, it has been suggested
that two ribosomes may occupy segment 1 of ilv
leader RNA during amino acid starvation. The two
leucine co dons thought to be responsible for reduced
termination in response to leucine starvation are
located six (ilvGMEDA) or eight (ilvEN) codons preceding the Val control codons. In a static view of
attenuation, a ribosome stalled at Valor Ile co dons at
the top of structure 1:2 would not prevent base pairing at the bottom of 1 :2. A ribosome stalled at this
location, then, might not promote formation of the
antiterminator and hence would allow the terminator
hairpin to form.
To explain ilv attenuation, Hatfield and colleagues
(71, 103) proposed that when a ribosome stalls at the
top of 1 :2, a second ribosome initiates synthesis of the
leader peptide on the same transcript, encounters the
stalled ribosome, and blocks the bottom of 1 :2. This
so-called "double-ribosome" model has been applied
to regulation of both ilvGMEDA and ilvEN. To date
there has not been any experimental test of this
model. An alternative view is that once the initial
translating ribosome has disrupted the 1:2 hairpin
and stalled at the upper 1:2 portion, the presence of
the newly synthesized distal sequences would favor
formation of the antiterminator rather than the bottom of the 1:2 hairpin. Indeed, this seems to be a more
plausible explanation, given current thinking on the
role of translational release of transcription pausing
in synchronizing leader region transcription and leader transcript translation in operons regulated by at·
tenuation (see Roles of the Transcript Pause and
Antiterminator Secondary Structures, above). If RNA
polymerase pauses after synthesizing the first 117
nucleotides of the ilvGMEDA leader transcript (73;
Fig. 5C) and this paused complex is released when the
translating ribosome disrupts the 1:2 hairpin, it is
difficult to imagine how a second ribosome could
initiate translation and move to the appropriate location before the attenuation decision was made.
The ilvBN Operon
The ilvE gene of E. coli and S. typhimurium encodes
the valine-sensitive acetohydroxy acid synthetase I
described in the previous section. The size of the ilvB
transcription unit is unknown; however, it apparently
contains ilvN, the gene for the small subunit of this
enzyme (56 171). In addition to regulation by ppGpp
(55) and cyclic AMP (55,56, 165), transcription of the
ilvEN gene is controlled by a transcription attenuator
responsive to starvation for leucine and valine, the
end products of the acetohydroxy acid synthetase I
77
pathway (56, 71). Thus ilvBN regulation is another
example of multivalent attenuation. There is no control by isoleucine, consistent with the fact that
acetohydroxy acid synthetase I is feedback inhibited
only by valine.
In vitro transcription of the ilvBN leader region
gives a 188-nucleotide terminated leader transcript
that encodes a 32-amino acid peptide containing nine
valine and three leucine residues (71). The leader
transcript potentially can form alternative secondary
structures (Fig. 5D) that are consistent with an attenuation mechanism.
Hauser and Hatfield (72) have proposed that ilvBN
attenuation is controlled not only by leucine and
valine, the pathway end products, but also by threonine and alanine, amino acids that are substrates in
these pathways. Threonine is deaminated to o.-ketobutyrate by threonine deaminase, the ilvA gene product,
and alanine is deaminated to pyruvate by alanine
transaminase; o.-ketobutyrate and pyruvate are the
substrates of acetohydroxy acid synthetase. This form
of control has been termed substrate attenuation.
Using hybridization probes to detect leader versus
distal RNA, Hauser and Hatfield (72) observed attenuation relief in the ilvBN operon in response to Leu,
Val, Thr, and Ala starvation. However, in a separate
study (167), no significant elevation of acetohydroxy
acid synthetase I levels was observed in response to
Thr or Ala starvation. The explanation for this apparent disagreement is unknown.
The pheA Operon
The gene pheA encodes chorismate mutase-prephenate dehydratase, the enzyme that catalyzes the first
two of the three steps in phenylalanine synthesis from
chorismate. In E. coli, pheA is found in a monocistronic operon at map position 57 next to the oppositely
transcribed aroF-tyrA operon (8, 76a). DNA sequence
and in vitro transcription studies have revealed that
the 170-base-pair pheA leader region has the features
of a classic attenuation region (76a, 186). A 145-base
terminated in vitro transcript has been identified
(186) which contains a coding region for a IS-residue
leader peptide with 7 phenylalanine residues; the
leader transcript can fold into both terminator and
anti terminator configurations (Fig. 5E). An alternative folding scheme in which the leader peptide coding region pairs with the loop region of an alternate
1:2 hairpin has also been described (186). Mutations
in E. coli that alter RNA polymerase (rpoB rifampin
resistance mutations) or block the modification of
tRNA Phe (miaA) or affect a gene of unknown function
that maps at min 93 (pheR) all lead to elevated
expression of pheA (65). The rpoB and miaA effects
agree with the proposed transcription attenuation
mechanism, whereas the effect of pheR suggests that a
separate mechanism also may control pheA expression. The features of the pheA leader region otherwise
are unremarkable.
LEADER PEPTIDE SEQUENCES AND
FUNCTIONS
The leader peptides of amino acid biosynthetic
operons regulated by attenuation generally contain
TRANSCRIPTION ATTENUATION
1291
multiple residues of the regulating amino acid(s). As
we have seen, the ability or inability to incorporate
these amino acids during leader peptide synthesis
provides the basis for the decision whether or not to
terminate transcription at the attenuator. Comparison of leader peptide coding regions reveals considerable variation in the number and positions of regulatory codons. Codon usage varies as well. In addition,
each peptide coding region contains codons for amino
acids that have no apparent relationship to regulating
amino acids. These features raise fundamental questions about the regulatory significance of leader peptides and their coding regions. The leader peptides
themselves appear to play no regulatory role; all tests
for a trans-acting function have been negative. In
addition, the existing data suggest that leader peptides are degraded rapidly. These observations are
consistent with the conclusion from the many studies
on attenuation, namely, that it is the act of leader
peptide synthesis that has regulatory significance.
The number and distribution of regulatory codons
varies from operon to operon and from species to
species. We suspect that this variation reflects fundamental differences in the extent to which the different
operons are regulated by attenuation. Thus in the trp
operon, where attenuation allows only modest regulation (179) and then only under conditions of severe
tryptophan starvation, there are only two Trp codons
in the leader peptide coding region. By contrast, in the
His operon, where attenuation has greater regulatory
importance, there are seven His codons in the leader
peptide coding region. A significant difference in regulatory codon number also is seen for Leu codons in
the ilvGMEDA leader regions of E. coli versus S.
marcescens: there are four Leu codons in the former
but only one in the latter (70). Either the single Leu
codon of S. marcescens is functionally equivalent to
the four Leu codons of E. coli (76), or leucine regulation of the ilvGMEDA operon is of lesser importance in
S. marcescens.
Rare synonymous codons are present in the leader
peptide coding regions of several operons regulated by
transcription attenuation. For example, in the leu
leader peptide coding region of S. typhimurium three
of the four Leu codons are CVA, whereas in the
ilvGMEDA leader peptide coding region of S. marcescens the sole Leu codon used is CVA. The use of rare
co dons in leader peptide coding regions could increase the cell's sensitivity to amino acid starvation.
The minor isoaccepting tRNAs that correspond to rare
co dons may be charged poorly or may translate inefficiently under starvation conditions; thus, attenuation would be relieved more readily upon amino acid
starvation.
Regulatory co dons often are interspersed with other
codons, as in the leader regions of the phe and ilv
operons. There may be several explanations for such
arrangements, but certainly location and spacing of
regulatory co dons are most relevant to the locations of
the RNA segments that form regulatory secondary
structures.
The significance of co dons for apparently unrelated
amino acids in leader peptide coding regions is not
obvious. Starvation for these amino acids often can
relieve termination when the corresponding codons
are located adjacent to a regulatory codon. In one
1292
LANDICK AND YANOFSKY
such instance, in which Ala limitation relieves termination in the ilvBN operon, there is a relationship
between the Ala and Ilv metabolic pathways (72).
However, as mentioned, this finding has been questioned (167). With regard to the trp operon there are
no obvious relationships between the Arg, His, and
Trp biosynthetic pathways that would account for the
relief of attenuation that accompanies Arg or His
starvation (164). One possible explanation for the
existence of presumably unrelated codons in leader
peptide coding regions is their use to signal whether
the cell has many of the amino acids needed for
protein synthesis. Thus it has been shown that mutations that limit initiation of synthesis of the trp or his
leader peptides increase transcription termination at
the respective attenuator (88, 89, 163). Conceivably,
ribosome stalling over one of the initial co dons in the
corresponding peptide coding region can have the
same effect.
TRANSCRIPTION ATTENUATION IN
NON-AMINO ACID BIOSYNTHETIC OPERONS
Following elucidation of the mechanism of attenuation control of amino acid biosynthetic operons,
many workers expected that variations on this regulatory mechanism would be found to control
expression of genes for other cellular functions (e.g.,
catabolism of amino acids, amino acid transport,
and synthesis of other metabolic products). In fact,
transcription attenuators have been demonstrated or
implicated for many such operons, but the diversity of
attenuation mechanisms that has been observed was
unanticipated. In contrast to the uniformity that is
apparent from studies of attenuation in amino acid
biosynthetic operons, these mechanisms appear to
reflect accommodation to the regulatory peculiarities
of each function or operon. Next we shall discuss
several examples demonstrating this diversity.
The pheST Operon
The pheST operon of E. coli encodes the small (pheS)
and large (pheT) subunits of phenylalanyl-tRNA synthetase (30, 116). Unlike expression of the genes for
alanyl-tRNA synthetase (alaS), which is subject to
autogenous transcriptional repression (137), and the
gene for threonyl-tRNA synthetase (thrS) , which is
con trolled by translational repression (157), pheST is
regulated by transcription attenuation (156, 158). In
vitro transcription of the pheST leader region produces transcripts initiating 351 base pairs preceding
the pheS AUG codon (48). Most of these transcripts
terminate in the leader region at a site 150 base pairs
downstream from the initiation site; termination at
this site was relieved by substituting ITP for GTP in
the transcription reaction (48). The nucleotide sequence of the pheST leader region revealed a classic
arrangement of potential alternative secondary structures in the first 150 bases of the pheST leader transcript and an open reading frame for a 14-residue
leader peptide containing five phenylalanine residues
(Fig. SF). Recent studies have shown that deletion of
the terminator structure (3:4, Fig. SF) leads to a
lO-fold increase in transcription of downstream sequences and that undermodification of phenylalanyl-
tRNA caused by the miaA mutation increases expression of the operon as well (156). These results indicate
that ph eST is regulated much like the amino acid
biosynthetic operons regulated by attenuation.
In contrast to attenuation in amino acid biosynthetic operons, however, more than 200 base pairs separate the attenuator from the pheS start codon. The
function of this segment, which includes a 31-codon
open reading frame with no phenylalanine codons, is
unknown. Translation of this reading frame might
well be required to prevent Rho-dependent termination in the 200-base-pair spacer region. Possible roles
of the spacer region and the second leader peptide are
important unsolved problems.
Pyrimidine Biosynthetic Operons
In E. coli and S. typhimurium, UMP is synthesized
from aspartate and carbamoylphosphate in five enzymatically catalyzed steps (26). The enzymes for these
conversions are encoded in five unlinked operons:
pyrB!, for the catalytic and regulatory subunits of
aspartate transcarbamoylase; pyrc, for dihydroorotase; pyrD, for dihydroorotate oxidase; pyrE, for
oro tate phosphoribosyltransferase; and pyrF, for orotidine-5' -monophosphate decarboxylase. Carbamoylphosphate is synthesized from ATP, CO 2 , and glutamine by carbamoylphosphate synthetase, the product
of the carAB operons (designated pyrA in S. typhimurium). Pyrimidine starvation results in increased rates
of synthesis of all six of these enzymes (93, 131, 149).
The response of the pyr genes is noncoordinate, suggesting that individualized regulatory controls exist
for each operon.
The most thoroughly characterized pyr regulatory
system is that of the pyrB! operon. Expression of this
operon is regulated transcriptionally by the intracellular concentration of UTP and involves UTP-dependent transcription attenuation (168). The proposed regulatory mechanism is substantially different from the attenuation mechanism used to control
amino acid biosynthetic operons. In vivo, most pyrB!
transcripts originate approximately 160 nucleotides
preceding the pyrB start codon (122). In vitro, a
promoter 350 nucleotides upstream from this start
codon also is utilized; transcripts initiating at both
promoters usually terminate in a region of dyad symmetry centered 40 base pairs before the start of pyrB
(3:4 in Fig. 6). The RNA terminator encoded by this
symmetrical region is typical of those found in amino
acid biosynthetic operons; it contains a GC-rich hairpin followed by a run of U residues. However, there is
no apparent alternative RNA secondary structure that
could prevent formation of the pyrB! terminator.
There is a second hairpin that can form in the leader
transcript; it is centered about 100 bases preceding
the pyrB AUG codon (1:2 in Fig. 6A). RNA polymerase
transcribing the pyrB! leader region in vitro pauses
after synthesis of this hairpin 0:2 in Fig. 6) in a
UTP-dependent process (168). The pyrB! leader transcript encodes a 44-residue leader peptide whose coding region spans both the 1:2 and 3:4 RNA hairpins
(Fig. 6).
These features have been incorporated into a model
for pyrB! attenuation that can account for the increased expression associated with uridine starvation
77
TRANSCRIPTION ATTENUATION
1293
ACAAU
U
U
G
Lys
C
C
60 A
A A
G
G
G
A
G
G
A
Leu
Arg
U
G
A
G
A
U-A
C=G
U-A
G=C
C=G
G=C
Pro
Ne~ValGlnCysValArgBisPheValLeuC=G
Asn
A U
Lys
A
C 120
C=G
Arg
Leu
U-A
C=G
C=G
Gly
Pro C=G
GI n C =G Al a
Asp
Ala
U GI y
LeuProPhePhePheProLeuIleThrHisSer G=CPhePhe
UAUGGUUCAGUGUGUUCGACAUUUUGUCUUAC=GCCUGCCGUUUUUCUUCCCGUUGAUCACCCAUUCCCA-UUUUUUUU
20
40
~
100
P
FIG. 6. Secondary structures in the E. coli pyrBlleader transcript. Region of transcription pausing (168) is indicated by the
bracket.
(122, 143, 168; Fig. 7). According to this model, transcription attenuation in the pyrB! leader region is
controlled by the rate of release of the transcription
pause complex, formed after synthesis of RNA structure 1 :2. It is assumed that when the cell has an
adequate pyrimidine supply, the resulting high UTP
pool allows the paused transcription complex to release before a ribosome can initiate leader peptide
synthesis and catch up with the transcribing RNA
polymerase molecule (Fig. 7). Once polymerase escapes the pause site and it is not closely followed by a
translating ribosome, it can rapidly elongate the message and synthesize the terminator (3:4 hairpin, Fig.
6), which will cause transcription termination. However, when a cell is starved for pyrimidines and has a
low UTP pool, the paused transcription complex may
remain at the pause site until it is approached by the
ribosome synthesizing the leader peptide (Fig. 7). The
ribosome could release the paused transcription complex in a manner analogous to pause complex release
in the trp operon leader region, allowing the transcribing polymerase molecule and the translating ribosome to proceed in a tightly coupled fashion. As RNA
segments 3 and 4 are synthesized, the translating
ribosome could prevent segment 3 from base pairing
with segment 4. If the RNA terminator does not form,
transcription will proceed into the pyrB! structural
gene region. The overall result is that pyrimidine
starvation promotes transcription readthrough at the
pyrB! attenuator.
Thus, transcription pausing, exaggerated and controlled by pyrimidine limitation, and translation of a
LOW UTP
POLYMERASE
PAUSES
~
PPI'
HIGH UTP
POLYMERASE DOESN'T
PAUSE
I2
~"
ppp/AUG
:
1·2
j
RIBOSOME INITIATES
TRANSLATION AND
RELEASES PAUSED
POL YMERASE
~
",
RIBOSOME LAGS
BEHIND POLYMERASE
GAC
PPP--AUG
RIBOSOME DISRUPTS
TERMINATOR
TERMINATOR FORMS
READ - THROUGH
TERMINATION
FIG. 7. Model for pyrBl attenuation (168). A ribosome is able to initiate translation and cause transcription readthrough
only when RNA polymerase pauses in response to UTP limitation.
1294
LAN DICK AND YANOFSKY
leader peptide coding region allow efficient regulation
of transcription termination in the pyrBI operon. This
model has the intriguing consequence that termination would occur whenever the cell is unable to
synthesize the pyrBI leader peptide efficiently. These
effects would protect the cell from wasting energy and
nucleotides on pyrBI mRNA synthesis when it is
incapable of synthesizing proteins. This result is reminiscent of superattenuation in amino acid biosynthetic operons (see Leader Peptide Sequences and Functions, above).
There is much experimental support for transcription attenuation control of pyrBI expression. An S.
typhimurium mutant with an altered RNA polymerase
constitutively expresses elevated levels of aspartate
transcarbamoylase and orotate phosphoribosyltransferase (84). In this mutant, RNA polymerase pausing
at the pyrBI pause site may not be relieved by elevated
UTP levels. Levin and Schachman (107) and Roland et
al. (142) have coupled the pyrBI leader region to galK
and lacZ, respectively, and have found that galactokinase and ~-galactosidase levels are increased in
response to uracil starvation. When deletions removing the 3:4 hairpin region (Fig. 6) were introduced,
uracil-dependent regulation was reduced appreciably
(107, 142). Translation initiation at the pyrBI leader
peptide initiation codon did occur when the leader
peptide coding region was fused to lacZ (27, 142).
When the pyrBI leader peptide initiation codon was
changed from AUG to ACG, expression and regulation
of the pyrBI operon was reduced greatly (142). Using a
pyrB-IacZ fusion, uracil-dependent regulation was observed regardless of the reading frame in which ribosomes translated the 3:4 hairpin region, but was lost
when translation terminated prematurely at a stop
codon 49 nucleotides before the 3:4 hairpin (27).
The pyrBI system offers perhaps the best opportunity to examine the in vivo role of pausing in attenuation. It would be particularly interesting to isolate
pause RNA from UTP-starved cells and demonstrate
the expected decrease in pause RNA stability when
the UTP supply was adequate. Another objective
should be to replace the run of U residues immediately following the 1:2 hairpin. If the model is correct,
this should abolish UTP-sensitive pausing and prevent
termination relief.
A similar but less well substantiated model has
been proposed for attenuation control of transcription of pyrE, the structural gene for orotate phosphoribosyltransferase (19, 136). The pyrE "leader
transcript" originates over 800 nucleotides preceding pyrE and contains an open reading frame (designated orfE) that encodes a polypeptide 238 residues in length. orfE is separated from the pyrE
coding region by sequences capable of forming an
RNA hairpin typical of Rho-independent terminators. A 238-residue leader peptide is unprecedented;
it is likely that orfE encodes a protein with a
catalytic or regulatory function. Although it was
shown in vitro that transcription of a DNA fragment
containing pyrE terminated at a site corresponding
to the potential RNA hairpin between orfE and the
pyrE start codon (136), no UTP-dependent pause site
has been identified.
Bonekamp et al. (19) have identified elements in the
pyrE leader region essential for what appears to be
attenuation control. They constructed a plasmid that
contains the lac promoter-operator and the first few
co dons of lacZ, fused to the last 14 codons of orfE
preceding the presumptive pyrE attenuator. This plasmid also has an in-frame fusion of pyrE to lacZ. By
changing the lacZ-orfE fusion, they varied the position
of the translation stop for orfE. UTP-sensitive regulation occurred when translation could proceed to the
terminator or end 8 nucleotides before it (natural
arrangement), but was lost when translation stopped
31 or 62 nucleotides before the terminator. These
results suggest that UTP-dependent regulation of pyrE
attenuation requires that translation of the upstream
open reading frame proceed to the terminator segment of the transcript. However, they do not address
the mechanism of translation-transcription coupling,
nor do they suggest how UTP depletion might relieve
attenuation. While the evidence for a pyrE attenuator
is persuasive, key questions remain unanswered. In
particular, experiments to determine the function of
the 238-residue protein encoded by orfE and to detect
UTP-dependent transcription pausing in the leader
region are desirable. Analyses of pyrA, pyrc, pyrD, and
pyrF regulation also are attractive goals for future
investigations.
The ampC Operon
The ampC gene of E. coli encodes a ~-lactamase that
is normally produced in small amounts and secreted
into the periplasm, where it hydrolyzes penicillin and
related antibiotics (125). Expression of ampC is not
induced by members of this family of antibiotics;
however, its expression is growth rate regulated (83).
The transcript of the ampC operon begins 41 base
pairs preceding the ampC structural gene. Transcription of the ampC leader region in vitro generates a
terminated transcript ending at a typical Rho-independent terminator. Mutational analyses have shown
that a mutation that introduces a base-pair mismatch
in the terminator eliminates growth rate regulation
(82). This mutation also increases readthrough transcription in vitro. Interestingly, the 41-nucleotide
ampC leader RNA does not contain sequences that
might form an alternative secondary structure that
could preclude terminator formation, nor does it contain a peptide coding region. However, it does contain
tandem translation start (AUG) and stop (UAA) co dons
beginning at position 8 of the leader transcript (82),
but no discernible Shine-Dalgarno sequence. To explain growth rate regulation, Normark and co-workers proposed that the first 10 residues of the leader
transcript serve as a weak ribosome-binding site and
that a ribosome bound at this site prevents formation
of the terminator (82). Since rapidly growing cells
have a higher ribosome content than slowly growing
cells (78), rapid growth would increase ribosome binding at this site and promote read through into the
ampC gene. While this ingenious model can account
for growth rate-dependent attenuation of ampC transcription, it raises several unanswered questions. Can
a ribosome bind at the (apparently) weak ampC leader
ribosome-binding site fast enough to prevent terminator formation? Also, is there a significant increase in
the number of free ribosomes in rapidly growing
cells?
TRANSCRIPTION ATTENUATION
77
rRNA Operons
A
Pl
In E. coli and S. typhimurium the 16S, 23S, and 5S
rRNAs are encoded in seven unlinked operons approximately 5,000 base pairs in length (43). All seven
operons are organized similarly and have the order
165, 23S, 5S. Genes for tRNA are located between the
165 and 23S rRNA genes in these operons and after
the 55 gene in some of the operons. In polypeptidespecifying operons, transcription generally terminates
prematurely when translation is prevented, apparently because the absence of translation triggers Rhodependent transcription termination (2). Thus transcription of rRNA operons present a paradox: how
does the transcribing RNA polymerase escape activation of Rho-dependent transcription termination
when synthesizing an untranslated transcript over
5,000 nucleotides in length?
It has been found in fact that Rho-dependent termination signals present in transposons Tn9 and Tnl0
and the insertion element lSI do not cause termination when these signals are inserted in rRNA operons
(22, 118, 152). Also, when rrnB (94) and rrnC (182)
promoter-leader regions are transcribed in vitro, premature transcription termination is observed, even in
the absence of Rho protein. In addition, when the
normal rrnG promoter-leader region and the first 80
nucleotides of the 16S gene are replaced by the araBAD
promoter, Rho-dependent transcription termination
is observed within the 16S gene (3).
In 1984, a model was proposed that accounts for
these observations (75, 109). It was hypothesized that
RNA polymerase is modified at a site in the rrn leader
region, allowing it to read through downstream transcription termination signals. The model is similar to
the well-documented anti termination mechanism
used during lytic growth of bacteriophage lambda
(57). When lambda is switched from lysogeny to lytic
growth, two phage anti termination proteins, Nand Q,
are formed. Several host proteins (termed Nus factors)
and the phage nut sites participate in this antitermination. The E. coli NusA protein is thought to bind
transiently to RNA polymerase (69) and to render it
susceptible to modification. Apparently, the NusAassociated RNA polymerase recognizes the nut site,
where the N protein, and perhaps other Nus factors,
interact with the transcription complex and convert it
to the anti termination state. Friedman and colleagues
(58, 128) have suggested that a nucleotide sequence,
termed BoxA and located shortly before the nut site, is
required for NusA participation. The Q antitermination system seems to involve a similar but simpler
antitermination mechanism. Q anti termination can
occur in a purified in vitro system containing only
RNA polymerase, an appropriate DNA, and the NusA
and Q proteins (67, 68).
The nucleotide sequences of six rrn operon leader
regions have been reported (rrnD and rrnX [183]; rrnA
and rrnE [34]; rrnB [31]; rrnG [151]). The leader
sequences are highly conserved, in particular the P2
promoter, a BoxA sequence following it, and the TL
region found to cause transcription termination in
vitro (Fig. 8A). The role of these sequences in antitermination has been tested by Li et al. (109). A 67-basepair restriction fragment immediately following the
rrnG P2 promoter contains the BoxA sequence. When
1295
P2
BoxA
rrs
in vitro ••- _ _ _ _ _--<.
• ._-------in vivo ••- - - - - - - - <.
• . _ - - - - - - - - - -_..
_
B
P
LP,lnaC BoxA
InaA
~Z··~····~·~.~--------------~c==
•
•
•
+TRP'••-------------------~.~
c
P
LP
livJ
~
•
-LEU.
..
FIG. 8. Schematic representations of leader regions for
genes controlled by unconventional transcription attenuation mechanisms. P, Promoter regions; LP, leader peptide
coding regions; T L , a NusA-dependent terminator (94). Lines
below the leader region diagrams represent terminated and
read through transcripts produced under the indicated conditions. (A) E. coli rrn operon leader regions. (B) E. coli
tryptophanase operon (tna) leader region. (e) Leader region
of the gene (ilvI) for the E. coli LIV-binding protein.
this fragment was placed in front of a known terminator sequence, it caused a 50% decrease in transcription termination (109). Translation through the 67base-pair fragment or reversal of its orientation resulted in a complete loss of antitermination activity.
Thus, it is possible that RNA polymerase is modified
when it reaches a site early in rRNA operons so that it
is resistant to subsequent Rho-dependent and Rhoindependent transcription termination signals. The
cellular factors responsible for this modification have
not been identified. However, the existence of an
anti termination mechanism that participates in rRNA
synthesis raises the possibility that there are additional mechanisms for regulation by transcription attenuation. If modification of RNA polymerase could be
controlled at specific sites, the cell could modulate the
frequency of read through transcription in any operon.
Ribosomal Protein Operons
Regulation by transcription attenuation has been
demonstrated in two ribosomal protein operons. The
SlO ribosomal protein operon encodes ribosomal protein components of both the 50S and 30S ribosomal
subunits (110, 184). The protein product of the third
gene in the operon, protein L4, regulates transcription
termination at a site in the leader region of the operon
(Ill). The second example of attenuation involves the
rplJL-rpoBC operon. This operon contains the structural genes for two 70S ribosomal protein components
and the two large subunits of E. coli RNA polymerase.
Transcripts that originate upstream of rplK can be
terminated at a site between rplL and rpoB (12).
1296
LANDICK AND YANOFSKY
In the SlO operon, attenuation shuts off expression
of the operon when the level of free L4 protein exceeds
that necessary for ribosome assembly. Excess L4 protein also acts as a translational repressor of ribosomal
protein synthesis (181), a function also attributed to
ribosomal proteins Ll and Ll0l11 (85). The transcript
of the SlO operon contains a 172-nucleotide leader
region preceding the first gene, rpsJ, encoding SlO
(127). Within this region there is an open reading
frame potentially coding for a 28-amino acid residue
le<>.der peptide. A possible Rho-independent terminator can be formed from a dyad symmetry within the
leader peptide open reading frame. Interestingly, the
potential RNA hairpin not only is followed by a run of
V's consistent with the observed length of an in vivo
attenuated transcript (127), but it also contains a
nine-base homology to the sequence in the 23S rRNA
that can be cross-linked to protein L4 by irradiation
with VV light (115). Additional sequence homologies
between this region of 23S rRNA and the segment of
the SlO transcript past the putative termination site
also exist (127).
No model has been proposed to describe how protein L4 might interact with these target sequences in
the SlO leader region and cause termination at the
putative attenuator.
Ribosomal protein LlO (rplJ gene product) and the
two large subunits of RNA polymerase, f3 and W
(products of the rpoB and rpoC genes, respectively),
are cotranscribed from one major promoter situated
before the rplJ gene (135). Minor promoters also exist
preceding rplJ and rpoB (11). The possibility of attenuation between the rplL and rpoB genes was first
suspected because ribosomal proteins are present in
greater quantities than RNA polymerase subunits
(112). Termination occurs in an untranslated segment
of RNA that could fold into two successive hairpins
followed by four V residues (12). No model explaining
attenuation at this site has been proposed.
The tna and liv Operons
In E. coli and S. typhimurium there are two distinct
mechanisms of transcription termination: Rho-dependent and Rho-independent termination (2, 132,
133). Although most of the examples we have described involve Rho-independent termination, it
seems reasonable to expect that Rho-dependent attenuation also exists. While anti termination in rRNA
operons might be an example of this, the metabolic
basis for regulation of these operons has not been
established. Recently, two examples of Rho-dependent transcription attenuation have been uncovered.
The best characterized of these, tryptophan-mediated
induction of expression of the E. coli tryptophanase
(Ina) operon, appears to involve an anti termination
override of Rho-dependent termination in a leader
region (Fig. 8B). The other example, leucine-regulated
expression of the gene for the E. coli leucine-, isoleucine-, and valine (LIV)-binding protein that is part of
the high-affinity branched-chain amino acid transport
system, may involve coupling of the translation of a
leader peptide coding region to Rho-dependent transcription termination (Fig. 8e).
The tna operon contains at least two genes: tnaA,
which encodes tryptophanase, a catabolic enzyme
that allows E. coli to grow with tryptophan as the sole
source of carbon and energy (35, 124); and tnaB,
which codes for a low-affinity tryptophan permease
(35, 39). Expression of the tna operon is induced by
tryptophan and is subject to catabolic repression. In
vitro transcription experiments have located a cyclic
AMP-cyclic AMP receptor protein-dependent promoter 319 base pairs upstream from the tnaA initiation
codon (36). This 319-nucleotide leader region contains
an open reading frame that encodes a 24-amino acid
residue leader peptide, designated tnac. The remainder of the leader RNA can base pair to form a number
of secondary structures (160), but none resembling the
conventional attenuator arrangement has been identified. In vivo, transcription from the tna promoter
terminates at a set of discrete sites in the tna leader
region (160, 161). Addition of tryptophan to cultures
greatly reduces this termination (161). The in vivo
termination phenomenon can be closely mimicked in
an in vitro transcription system by addition of Rho
protein (160). E. coli strains bearing the strong mutant
allele rho-IS (161) exhibit semiconstitutive tna operon
expression. Furthermore, a lacZ fusion to the early
part of the tnaA gene shows normal tryptophan-dependent induction, whereas a lacZ fusion to tnaC is
expressed constitutively (161). Taken together, these
results suggest that tryptophan induction of tna operon expression is accomplished by suppression of Rhomediated transcription termination in the tna leader
region.
This hypothesis is supported by the additional finding that all isolated tna constitutive mutations but
one have nucleotide changes within a 9-base segment
of the tna leader at the end of the tnaC gene. Interestingly, this sequence is homologous to the BoxA sequence thought to be responsible for NusA protein
interaction in transcription antitermination (58, 69,
128). It seems likely that transcription attenuation in
the tna operon is controlled by an anti termination
mechanism that, when triggered by a signal involving
tryptophan, overrides Rho-dependent transcription
termination in the tna leader region. Antitermination
may involve modification of RNA polymerase at the
BoxA-like sequence, as has been postulated in the rrn
and A tRI systems (57, 75, 109). According to this
model, a cellular protein must exist that causes modification only when it is bound to tryptophan. No
trans-acting factors have as yet been identified (161; V.
Stewart, personal communication). Further understanding of the attenuation mechanism used in tna
operon regulation requires elucidation of the basis of
tryptophan-media ted anti termina tion.
The high-affinity branched-chain amino acid transport system of E. coli is composed of three inner
membrane proteins and two periplasmic amino acidbinding proteins with slightly different specificities
(123). The system designated LIV-I is induced by
leucine limitation and scavenges branched-chain
amino acids from the growth medium. The gene for
the LIV-binding protein, livJ, is in a transcriptional
unit that is separate from the other genes, which
apparently form a single operon (123). Early work on
the regulation of liv gene expression revealed that
mutations in rho and leuS (encoding leucyl-tRNA
synthetase) caused elevated levels of LIV-I transport
activity (138, 139). Mutations in a previously unde-
77
fined locus, livR, also increased transport (5). The
results obtained suggested that expression of the liv
genes might be controlled by both repression and
attenuation (140).
Recent studies have shown that the livi gene and the
liv operon contain 124 and 250 nucleotide leader
regions, respectively (R. Landick, unpublished results). Both leader regions contain potential short
peptide coding regions that contain leucine codons,
and long stretches of unstructured, CA-rich sequences
that are similar to known Rho-dependent termination
sequences. It is possible that translation of the leader
peptide is somehow coupled to Rho protein entry on
the nascent transcript (99). In such a scenario, stalled
translation in response to leucine limitation could
block Rho-dependent termination. Clearly this mechanism of regulation is not well defined. Isolation of
leader region mutations that result in increased liv
gene expression will be required to understand how
the postulated Rho-dependent attenuators function.
Further analysis of regulation of the tna and liv
operons may reveal different ways that Rho-dependent termination is utilized in attenuation. The tna
system appears to involve an anti termination function that overcomes Rho-dependent termination,
whereas the liv system may couple the efficiency of
translation of a leader peptide coding region to Rho-dependent termination. Although both systems require
more work, the findings obtained already suggest new
variations on the attenuation theme.
CONCLUSIONS
Transcription attenuation is an attractive regulatory mechanism because it provides regulatory options
to the growing cell that are not appropriate to regulation by repression. During the process of transcription, specific RNA sequences and structures are synthesized that present novel opportunities for modulating the course of transcription. These RNA segments
may serve as regulatory signals, they may act as sites
of interaction with accessory proteins, or, if they
contain coding regions, they may allow the cell's
translation machinery to influence the course of transcription. In analyses of attenuation in biosynthetic
operons we have seen how RNA transcription pause
and termination signals are altered by translation to
effect a specific regulatory response. One expects that
RNA sequences and structures also serve as recognition sites for specific RNA-binding proteins which,
when bound, can have regulatory consequences. Some
examples of this already exist. In addition, accessory
transcription factors such as the NusA, Rho, and Tau
proteins, which interact with transcription complexes
and modify readthrough or termination, introduce
other targets for regulation. Similarly, the subunits of
the transcribing polymerase molecule are targets for
interaction with or modification by regulatory molecules. Thus the participants and events involved in
transcription elongation and transcription termination provide a variety of regulatory opportunities that
do not exist in repression.
A second feature of attenuation that adds to its
attractiveness as a specific regulatory mechanism is
its dependence on minimal genetic information. In the
biosynthetic operons regulated by attenuation, speci-
TRANSCRIPTION ATTENUATION
1297
ficity is imparted by the unique sequence of 100 or so
base pairs in the leader region. The normal components of transcription and translation do the rest. By
contrast, repressors require a protein coding region
and flanking expression-control sequences. In addition, repressor binding must be highly specific so as
not to affect expression of genes for unrelated functions. In evolutionary terms, then, relatively few genetic events may be needed to create the leader region
of an operon regulated by attenuation. As we have
mentioned, extensive sequence and structural homology between tRNA His and the transcript of the his
leader region has been noted (4). This homology may
result from common ancestry, suggesting a likely
source of genetic material for the evolution of regulatory leader regions.
A major conclusion that can be drawn from the
many regulatory studies performed with E. coli and S.
typhimurium is that multiple regulatory mechanisms
are commonly used in the control of gene expression.
Perhaps this is so because expression of many bacterial genes is influenced by both intracellular and
extracellular events and because the functions of these
genes are crucial to cell growth. The overall picture
that has emerged is that virtually every event involved in gene expression in bacteria has been optimized through the use of some control feature or
regulatory mechanism.
ACKNOWLEDGMENTS
We are greatly indebted to Terry Platt, Irving Crawford, Charles
Turnbough, and Valley Stewart for their helpful comments on this
review.
R.L. was a postdoctoral fellow of the U.S. Public Health Service
when this chapter was written, and c.Y. is a Career Investigator of the
American Heart Association.
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