(Received for publication, May 2, 1995; and in revised form, June 13, 1995)
From the
Previous work has shown that, in the bacterium Escherichia
coli, the aat gene is essential for the degradation of
proteins bearing amino-terminal Arg and Lys residues via the N-end rule
pathway of protein degradation. We now show that the aat gene
encodes directly the leucyl/phenylalanyl-tRNA-protein transferase
(L/F-transferase). This enzyme catalyzes the transfer of Leu, Phe, and,
less efficiently, Met and Trp, from aminoacyl-tRNAs, to the amino
terminus of acceptor proteins. We have used the cloned aat gene to overexpress and purify an affinity tagged L/F-transferase.
The recombinant L/F-transferase is as active as the previously purified
wild type enzyme and contains no detectable RNA component. We have used
the recombinant enzyme to demonstrate that both the solubility and
substrate specificity, for aminoacyl-tRNA substrates, of the
L/F-transferase are dependent on ionic strength conditions and that the
modified nucleotides found in natural tRNAs are not essential for
recognition by the enzyme. Limited digestion of the L/F-transferase
with trypsin removes the proline rich NH terminus of the
enzyme identifying a globular core, and circular dichroism demonstrates
that the L/F-transferase is predominantly
-helical. Finally, a
region of sequence conservation between the L/F-transferase and the
NH
-terminal protein acetylases has been identified.
The leucyl/phenylalanyl-tRNA-protein transferase
(L/F-transferase) ()catalyzes a peptidyltransferase reaction
that is very similar to the reaction catalyzed on the large subunit of
the ribosome. The L/F-transferase catalyzes the transfer of Leu, Phe,
and, less efficiently, Met and Trp, from aminoacyl-tRNAs to the amino
terminus of acceptor proteins (Kaji et al., 1965; Leibowitz
and Soffer, 1971a). All known acceptor proteins or peptides contain the
basic amino-terminal residues Arg or Lys and are predominantly
unstructured at their NH
termini (Soffer, 1973). The
L/F-transferase catalyzes a true peptidyltransferase reaction, making
it unique among those enzymes that catalyze non-ribosomal peptide bond
synthesis in Escherichia coli cells. Previously characterized
enzymes activate the transferred amino acid as an aminoacyl-enzyme
thioester (Lipmann, 1971) or an aminoacyl-phosphate (Wright and Walsh,
1992) prior to peptide bond formation. The L/F-transferase contains
only 234 amino acid residues (Shrader et al., 1993), and the
purified enzyme lacks any detectable RNA component (see below), making
the L/F-transferase a tractable system for in-depth mechanistic
dissection of aminoacyl-tRNA recognition and peptidyltransferase
reactions.
Substrate modification by the L/F-transferase serves as a
powerful degradation signal, identifying the enzyme as a component of
the N-end rule pathway (Varshavsky, 1992). It has been shown previously
that E. coli cells degrade a -galactosidase variant,
engineered to expose amino-terminal Arg (Arg-
-gal), only after
modification to Leu-Arg-
-galactosidase (Shrader et al.,
1993; Tobias et al., 1991). The addition of an amino-terminal
degradation signal is a simple yet unusually powerful signal for
protein turnover. Mutant cells that lack L/F-transferase activity
degrade unmodified Arg-
-gal 100-1,000 times more slowly than
wild type cells. The ATP-dependent Clp (Ti) protease (Hwang et
al., 1988; Katayama et al., 1988) mediates degradation of
these proteins (Tobias et al., 1991). Inclusion of
L/F-transferase in the N-end rule pathway provided a functional assay
for L/F-transferase activity that allowed the cloning of a gene, termed aat (aminoacyl transferase), essential for
L/F-transferase-dependent degradation (Shrader et al., 1993).
The aat gene was presumed to encode directly the
L/F-transferase, but the lack of significant sequence similarity with
any known enzyme left viable other possibilities. In this paper we
demonstrate that the aat gene directly encodes the
L/F-transferase.
The aat gene lies at the end of a three gene operon whose other members are homologous to the P-glycoproteins responsible for multidrug resistance in mammalian cells (Shrader et al., 1993). These multidrug resistance homologs have been identified as essential for E. coli cells to exit from the stationary phase (Siegele and Kolter, 1993) and are also required for the proper expression of cytochrome d, although they do not encode subunits of this enzyme (Poole et al., 1993). Cytochrome d is a terminal oxidase in the E. coli respiration chain. Correspondingly, these genes have been named surB/cydD and cydC. The chromosomal position of the aat gene remains an intriguing clue to the cellular function of the L/F-transferase.
Previous attempts to purify L/F-transferase from cells expressing natural abundance levels of the enzyme suggest that L/F-transferase is present at very low levels (Leibowitz and Soffer, 1970). To prepare quantities of the enzyme for subsequent characterization, we have constructed overexpression systems for two different affinity-tagged L/F-transferases. The overexpressed enzymes show moderate solubility in vivo and under most conditions in vitro. However, preparations of the soluble fraction of hexa-His-L/F-transferase yield milligram quantities of active enzyme. We present an initial characterization of this recombinant L/F-transferase and demonstrate that the introduced changes do not change the important qualities of the enzyme. In addition, we have demonstrated that unmodified tRNAs are substrates for the enzyme and determined the domain structure and approximate secondary structural content of the L/F-transferase.
The affinity
modified L/F-transferase enzymes containing the NH-terminal
glutathione S-transferase (encoded by the GST-aat allele) or hexa-His (His
-aat)
affinity domains were constructed from a single PCR-derived DNA
fragment encoding the entire L/F-transferase. This DNA fragment results
in in-frame fusions, with the DNA sequences encoding either affinity
domain, when inserted into the BamHI restriction site of
commercially available expression vectors (see ``Experimental
Procedures''). We reasoned that the
His
-aat-encoded enzyme, which is less drastically
modified, would be more suitable for subsequent characterization.
However, we also reasoned that if the GST-aat encoded enzyme
retained L/F-transferase activity, it would provide confidence that
N-terminal modifications to the enzyme do not alter its important
characteristics. Data demonstrating that the GST-aat encoded
enzyme retains L/F-transferase activity and thereby compliments TS351
cells (MC1061 aat::minitet) (Shrader et al., 1993)
for their inability to degrade Arg-
-gal are shown in Fig. 1. Expression of stable X-
-gals in E.
coli cells results in higher steady state levels of
-galactosidase than expression of short-lived X-
-gals (Tobias et al., 1991). Wild type E.
coli cells are unable to degrade Met-
-gal and aat,
or ClpA mutants are unable to degrade Met-
-gal or
Arg-
-gal. Complementation of TS351 cells for their defect in
Arg-
-gal degradation by the plasmid borne GST-aat allele
results in an approximate 10-fold reduction in the steady state level
of Arg-
-gal, as measured using ONPG assays (see
``Experimental Procedures''). Complimenting L/F-transferase
activity is seen in the absence of full induction of the GST-aat
expression system and probably results from incomplete repression of
the powerful hybrid promoter upstream of the GST open reading frame.
Complementation is more complete from the GST-aat fusion construct than
from an alternate L/F-transferase construct (pAat) that expresses the
wild type enzyme from cryptic promoter elements located internal to the surB gene immediately upstream of the aat gene
(Shrader et al., 1993). These results demonstrate than the
addition of large NH
-terminal extensions to the
L/F-transferase result in, at most, partial loss of activity.
Figure 1:
In vivo activity of a
GST-L/F-transferase fusion protein. ONPG assays were used to monitor
the steady state levels of X--gal enzymes in wild type
and mutant E. coli cells. X-
-gals are
-galactosidase enzymes engineered to contain the
NH
-terminal residue X. Met-
-gal is stable,
irrespective of cellular L/F-transferase activity, whereas
Arg-
-gal is degraded only in cells expressing the L/F-transferase
and the ClpAP protease. The steady state value of Met-
-gal is
normalized to 250 units. Samples: TS357, MC1061 clpA::minitet
cells expressing Arg-
-gal; TS351, MC1061 aat::minitet
cells expressing Arg-
-gal; M-
gal, MC1061 cells
expressing stable Met-
-gal; pAat, MC1061 aat::minitet (pAS45 (aat
)) cells expressing Arg-
-gal; Gst-Aat, MC1061 aat::minitet (pGST-aat (see methods))
cells expressing Arg-
-gal; R-
gal, MC1061 cells
expressing unstable Arg-
-gal. Assay results from four isolates of
each clone are shown.
Induction of expression of both the GST-aat and His-aat alleles results in the synthesis
of the fusion proteins to levels constituting a substantial fraction of
total cellular protein as shown in Fig. 2. Induction is only
moderately deleterious to cells as wild type growth rates are observed
for two to three generation times after the addition of IPTG.
Subsequent fractionation of the induced cells demonstrates that
50-90% of the synthesized enzyme is not soluble. This lack of in vivo solubility of the L/F-transferase in its natural host
is curious. Both fusions show the same characteristics implicating the
L/F-transferase moiety as responsible for fusion protein insolubility.
Based on previous reports that the L/F-transferase purifies over
several chromatographic steps with a bound tRNA, we interpret our
solubility results to suggest that the L/F-transferase exists in cells
predominantly as a complex with aminoacyl-tRNAs and that the nucleic
acid is partially responsible for the in vivo solubility of
the complex. Interestingly, the fraction of soluble protein is
substantially greater for the GST-aat expression system than for the
His
-aat expression system. This suggests that the soluble
26 K
GST domain aids in the
solubilization of the L/F-transferase enzyme in vivo and
indicates that fusions to very large, soluble domains may further
increase the yield of active enzyme. We argue below that the
insolubility of the L/F-transferase results from a disordered
NH
-terminal domain and that conditions that increase enzyme
stability in vitro result in substantial increases in the
enzyme's solubility.
Figure 2:
Cellular fractionation of L/F-transferase
fusion proteins. Coomassie-stained 12% SDS gel of cells and cell
fractions of MC1061 cells expressing the His-aat and GST-aat genes.
Lanes: U, total cellular protein from uninduced cells; I, total cellular protein from induced cells; S,
soluble protein from induced cells; P, insoluble pellet
protein from induced cells.
Figure 3:
Purification of the L/F-transferase.
Coomassie-stained 10% SDS gel monitoring the purification of the
L/F-transferase. Lanes: C, total cellular protein; P,
insoluble proteins from cell pellet; S, soluble proteins; F, nonbinding flow-through from Ni-NTA-agarose affinity
purification step; Ni, protein eluted from Ni-NTA-agarose; NC, concentrated Ni sample used for gel filtration; GF, final L/F-transferase enzyme after gel filtration and
concentration to 4 mg/ml.
Activity data for the
purified L/F-transferase is shown in Table 1. The recombinant
enzyme transfers both Leu and Phe from their cognate aminoacyl-tRNAs to
the acceptor protein -casein. In each case, the level of
radiolabeled amino acid incorporated into protein (hot trichloroacetic
acid-precipitable counts) is >5-fold higher than the steady state
level of amino acid available as aminoacyl-tRNA (cold trichloroacetic
acid-hot trichloroacetic acid), demonstrating multiple rounds of tRNA
charging and transfer of the amino acid to
-casein. This result
also demonstrates that the aat gene directly encodes the
L/F-transferase. We had previously shown that the aat gene is
required for L/F-transferase activity, so this is an expected result.
However the lack of sequence homology between the gene encoding the
L/F-transferase and the yeast gene encoding the physiologically
analogous R-transferase (Balz et al., 1990) left possible more
indirect roles of the aat gene in L/F-transferase expression.
The lack of homology between the two NH
-terminal aminoacyl
transferases remains a surprise.
As expected from the work of
previous laboratories, the transfer of both Ala and Arg by the
L/F-transferase is barely detectable (Table 1). However,
significant levels of both Thr and Val are incorporated into a hot
trichloroacetic acid-stable form, and this incorporation is dependent
on the presence of -casein. This suggests that the L/F-transferase
is somewhat less specific than previously reported with respect to its
choice of aminoacyl-tRNA substrates. Interestingly, raising the ionic
strength of the reaction to either 0.5 M KCl or 0.1 M
(NH
)
SO
increases the specificity of
the enzyme with respect to aminoacyl-tRNA recognition. Ionic strength
changes do not significantly alter the incorporation of Leu into
-casein, suggesting that the specificity of the enzyme is being
altered rather than overall enzyme activity. Increased ionic strength
also prevents the purified enzyme from aggregating (see above). We
attribute the increase in substrate specificity and decreased
aggregation of the L/F-transferase at relatively high ionic strengths
to an increase in the order of the NH
-terminal domain of
the enzyme (see below).
The assays described above to determine the
substrate specificity of the His-L/F-transferase fusion
protein are qualitative in nature. We have also compared the specific
activity of our preparation with that of the purified wild type enzyme
(Leibowitz and Soffer, 1970). The Soffer laboratory defined one unit of
L/F-transferase activity as the amount of enzyme that transfers 1 nmol
of amino acid to the acceptor protein
-casein in 1 min under
defined reaction conditions (see ``Experimental
Procedures''). Using this assay, the purified wild type enzyme
contains
60 units of activity/mg of protein for the transfer of
both Leu and Phe (Leibowitz and Soffer, 1970). Using these assay
conditions, our purified enzyme displays 330 units of activity/mg of
protein for the transfer of Leu from natural tRNA
(CAG)
and 250 units of activity for the transfer of Leu from T7 RNA
polymerase-transcribed tRNA
(CAG). The small difference
in specific activity, between natural and unmodified tRNAs, in this
L/F-transferase assay may be misleading. Unmodified tRNAs have lower
stability than the fully modified tRNAs produced in vivo (Peterson et al., 1993). This may result in some
unfolding of the T7-transcribed tRNAs with concomitant loss of activity
as a substrate for the L/F-transferase.
The most abundant
co-purifying polypeptide in our partially purified L/F-transferase
preparation is a polypeptide with an approximate molecular mass of 65
kDa visible in lane NC of Fig. 3. This protein binds to
the Ni-NTA-agarose column only in the presence of the L/F-transferase
(data not shown). Because this 65-kDa polypeptide might have
represented a L/F-transferase substrate or interacting polypeptide, we
have determined its NH-terminal sequence. The
NH
-terminal sequence of this polypeptide:
A-A-K-D-(V/K)-(L/F)-I-(H/K/G)-N-D identifies it as GroEL
(NH
-terminal AAKDVKFGND). The interaction of the
L/F-transferase with this molecular chaperonin is provocative in light
of the enzyme's preference for unstructured substrates (Soffer,
1973). The possibility that natural substrates of the L/F-transferase
are presented to the enzyme, in extended form, by the cellular folding
machinery must now be considered. A role for the molecular chaperones
in protein degradation has been demonstrated previously in both
bacterial and eukaryotic cells (Straus et al., 1988, Sherman
and Goldberg, 1992). A second potential role of the GroEL chaperonin in
the cellular activity of the L/F-transferase is suggested by the
enzyme's in vivo insolubility in the absence of bound
tRNA (see above). The chaperonin may be required to maintain solubility
of the enzyme during the period of tRNA
aminoacyl-tRNA exchange
after substrate modification.
Figure 4:
Limited proteolysis of the
L/F-transferase. Silver-stained 15% SDS gels were used to monitor the
time course of trypsin digestion of the purified L/F-transferase. An arrow indicates the stable core of the enzyme. The
NH-terminal sequence of this fragment corresponds to Phe-81
of the native L/F-transferase. Lane markers: numeric values correspond
to the duration of incubation of L/F-transferase with trypsin: L, lysozyme (M
= 14,300); I, soybean trypsin inhibitor (M
=
21,500); A, bovine serum albumin; T, trypsin enzyme
used in digestion reactions; U, undigested
L/F-transferase.
The far-UV circular dichroism spectrum of the L/F-transferase
is shown in Fig. 5. Visual inspection of the CD spectrum suggest
an overall similarity to the spectrum of an -helix, however the
characteristic inflection at 215 nm is not observed. Secondary
structure calculations suggest values of 45-55%
-helical
residues and approximately 25% each of residues in random coil and
-turn conformations. No
-sheet structure is detected. Based
on the high concentration of Pro residues in the
NH
-terminal 80 amino acids and the protease sensitivity of
this region, this suggests that the majority of the residues that
comprise the protease sensitive NH
-terminal domain are in
random coil conformation. Correspondingly, this data indicates that the
protease resistant COOH-terminal domain is predominantly
-helical.
Overexpression and purification of the globular domain of the
L/F-transferase are in progress. A comparison of the CD spectrum of the
isolated globular domain with the spectrum of the intact protein should
allow definitive identification of the
-helical regions of this
enzyme.
Figure 5:
Circular dichroism spectrum of purified
L/F-transferase. The circular dichroism spectrum of the purified
L/F-transferase suggests that the enzyme is strongly -helical.
Quantitative curve fitting yields values of 47%
-helix, 25% random
coil, and 28% turn. The observed spectrum (solid line), the
spectrum calculated based on the above percentages (dashed
line), and the difference spectrum (dotted line) are
shown.
The ability to generate milligram quantities of active
L/F-transferase has allowed us to employ biochemical methods in
conjunction with genetic approaches to separate the regions of the
enzyme responsible for substrate recognition and catalysis. A schematic
of the primary sequence of the L/F-transferase, depicting our current
understanding of the enzyme, is shown in Fig. 6. A striking
aspect of this figure is the strong correlation between protease
sensitivity and the distribution of Pro residues. The
protease-sensitive NH-terminal region of the enzyme
contains 10 proline residues, whereas inspection of the primary
sequence of the globular COOH-terminal region reveals a continuous
stretch of 116 residues completely lacking proline. The proline
distribution is consistent with our assignment of the
-helical
residues, identified using circular dichroism, with the COOH-terminal
globular region of the L/F-transferase.
Figure 6:
Schematic of the L/F-transferase enzyme.
The complete amino acid sequence of the His-L/F-transferase
is shown. Previous genetic data characterizing the non-essential
portion of the enzyme and the NH
terminus of the
trypsin-resistant core are indicated. The non-uniform proline
distribution is highlighted by small circles. Also indicated
are the abundant Cys residues (underlined) and a
histidine-rich cluster (double underlined). Experiments are in
progress to determine whether these residues are involved in metal
binding.
Our working model for the
L/F-transferase invokes the protease-sensitive N-terminal domain in
substrate recognition. This is suggested by the increase in substrate
specificity by the L/F-transferase under conditions that reduce
aggregation (see above). The requirement for a flexible substrate
recognition domain is also suggested by the wide range of acceptor
peptide and aminoacyl-tRNA substrates utilized by this enzyme. Acceptor
protein recognition by the L/F-transferase is degenerate with
amino-terminal Arg and Lys residues being recognized (Leibowitz and
Soffer, 1971b). Arg and Lys each contain basic side chains but are not
isosteric, and it is difficult to envision a tight binding pocket with
this degenerate specificity. In addition, peptide binding experiments
demonstrate that residues beyond the NH terminus further
modulate binding (Soffer, 1973).
We have demonstrated that
recognition of aminoacyl-tRNAs by the L/F-transferase does not strongly
depend on the modified nucleotides found in cellular tRNAs. This result
is important for two reasons. First, the lack of crucial contacts
between the L/F-transferase and the modified nucleotides found in tRNAs
supports previous results implicating the tRNA acceptor helix in
L/F-transferase aminoacyl-tRNA recognition (Scarpulla et
al., 1976). There are no modified nucleotides in the acceptor
helix (Clark et al., 1995). Second, our ability to utilize T7
RNA polymerase to generate mutant tRNAs in vitro will allow us
to determine the importance of individual nucleotides for recognition
by the L/F-transferase. The L/F-transferase uses a subset of cellular
aminoacyl-tRNAs as substrates. This level of specificity is
intermediate between enzymes that recognize a single tRNA (the
aminoacyl-tRNA synthetases) and enzymes that recognize many or all
aminoacyl-tRNAs (elongation factor TU and the ribosome). This subtle
discrimination is based on both the amino acid and tRNA moieties
(Leibowitz and Soffer, 1971b). In an in vitro reaction using
purified components, the L/F-transferase is
30 times more
efficient catalyzing the transfer of Met from the Met elongator tRNA
(tRNA
) to casein than it is catalyzing the
transfer of Met from the initiator tRNA
(tRNA
) to casein (Scarpulla et al.,
1976). Initiator tRNAs differ from the family of elongator tRNAs by a
unique non-Watson-Crick C:A base pair at the start of the acceptor
helix (RajBhandary, 1994). Elongation factor TU also differentiates
against the initiator tRNA on the basis of this C:A base pair
(RajBhandary, 1994). There are no nucleotides unique to the family of
tRNAs active as L/F-transferase substrates. This suggests that nucleic
acid recognition is more dependent on shape than sequence and
implicates the junction between single-stranded and double-stranded RNA
as a recognition determinant.
The recognition of the aminoacyl
moiety of aminoacyl-tRNAs is intriguing and suggestive of a flexible
enzyme structure. The L/F-transferase differentiates between Leu, Val,
and Ile but accepts Met, Phe, and Trp (Kaji et al., 1965;
Scarpulla et al., 1976). This recognition suggests that a
large hydrophobic side chain is recognized but that an unbranched
-carbon is also required. The corresponding recognition cavity,
hydrophobic with a restricted opening, is difficult to imagine without
invoking a flexible enzyme that becomes more structured in the presence
of substrates or folds around the amino acid side chain. Studies to
monitor increases in secondary structure of the L/F-transferase in
response to aminoacyl-tRNA are in progress. Several authors have
identified
-sheet structures that interact with RNA (Oubridge et al., 1994). The lack of detectable
-structure in the
free L/F-transferase should make detection of induced
-sheet
formation more straightforward. In addition we are purifying truncated
L/F-transferase enzymes lacking their NH
-terminal 33
(active in vivo) and 80 residues (the globular core). A
comparison of the overall enzymatic activity of these enzyme and their
free and induced secondary structure contents should allow definitive
inclusion or exclusion of the disordered NH
-terminal region
of the enzyme in aminoacyl-tRNA recognition.
We have shown that the L/F-transferase catalyzes a peptidyltransferase reaction without a detectable RNA component. In this respect, the L/F-transferase reaction may differ from the peptidyltransferase reaction catalyzed on the large subunit of the ribosome. This central reaction in translation can be isolated from the complex overall process, and a model peptidyltransferase reaction consists of peptide bond formation between the amino group of puromycin and the carbonyl carbon of formyl-Met donated by a CAACCA-(fMet) ester (Monroe and Marker, 1967). This reaction, along with affinity labeling of ribosomes, employing modified Phe-tRNAs, identified proteins L2, L15, and L16 along with the 23 S ribosomal RNA as essential for the reaction (Noller et al., 1992). Interestingly, the minimal complex, active in the model peptidyltransferase reaction, is extremely resistant to proteases and removal of protein by phenol extraction (Noller, 1993). This has resulted in a search for the true catalytic core of the peptidyltransferase reaction. To paraphrase H. Noller, the ``protein-centric'' view of the peptidyltransferase reaction is that the proteins provide the catalytic groups but are protected by the RNA. Conversely, the ``ribo-centric'' view is that the proteins form a scaffold essential for the proper conformation of a catalytic RNA (Noller, 1993). Understanding the L/F-transferase reaction mechanism in detail may clarify the essential features required for enzymes that catalyze peptidyltransferase reactions.
Using the sequence data banks, we have identified a region of
sequence similarity between the L/F-transferase and the RimL
NH-terminal acetylase (Tanaka et al., 1989). We
have tentatively assigned this region of the L/F-transferase enzyme a
role in binding and, perhaps, activating the NH
terminus of
acceptor peptides. This assignment is based on the participation of a
substrate NH
-terminal amino group in each reaction, and the
conserved cluster of acidic residues found in the conserved region of
each enzyme. The conserved region is depicted in Fig. 7and was
identified based on a region of strong sequence similarity between the
L/F-transferase and bacterial enzymes that acetylate the NH
terminus of ribosomal proteins. Interestingly, this same region
has been identified by previous authors as the region most closely
conserved between the NH
-terminal acetylases (Tanaka et
al., 1989). The acetylases catalyze a reaction that is similar to
that catalyzed by the L/F-transferase and are thought to use an acetyl
group activated as acetyl-CoA and the NH
terminus of
ribosomal proteins as substrates. Note: the suggestion of substrate
recognition motifs in the globular domain is not inconsistent with our
model invoking a flexible NH
-terminal region in the
recognition of the family of L/F-transferase substrates, as a free
-amino group is common to all acceptor peptide substrates and,
therefore, its recognition would not require enzyme flexibility.
Finally, the observed sequence conservation between the L/F-transferase
and a family of NH
-terminal acetylases (Tanaka et
al., 1989) provides an additional evolutionary link between the
similar activation schemes that have long been noted for the building
blocks of protein and lipid synthesis (Lipmann, 1971).
Figure 7:
Sequence similarities between the
L/F-transferase and the NH-terminal acetylases.
Translations of the genes encoding three ribosomal protein
NH
-terminal acetylases aligned with the aat-encoded L/F-transferase. The regions of the
NH
-terminal acetylases shown has been identified previously
by Isono and co-workers (Tanaka et al., 1989) as the region of
strongest sequence conservation between the acetylases. The optimal
alignment reported by these authors has been retained. Regions of
sequence similarity between the RimL protein and the L/F-transferase
are underlined. The regions of the proteins shown are: RimI,
residues 78-98; rimJ, 72-92; rimL, 68-92;
L/F-transferase, 120-144.