(Received for publication, January 6, 1997, and in revised form, February 18, 1997)
From the Department of Microbiology and Molecular Genetics, University of Texas Houston Medical School, Houston, Texas 77030-1501
We purified polyhistidine
(His6)-tagged and native Escherichia coli
MiaA tRNA prenyltransferase, which uses dimethylallyl diphosphate
(DMAPP) to isopentenylate A residues adjacent to the anticodons of most
tRNA species that read codons starting with U residues. Kinetic and
binding studies of purified MiaA were performed with several
substrates, including synthetic wild-type tRNAPhe, the
anticodon stem-loop (ACSLPhe) of tRNAPhe, and
bulk tRNA isolated from a miaA mutant. Gel filtration shift and steady-state kinetic determinations showed that affinity-purified MiaA had the same properties as native MiaA and was completely active
for tRNAPhe binding. MiaA had a
Kmapp (tRNA substrates)
3 nM, which is orders of magnitude lower than that of
other purified tRNA modification enzymes, a
Kmapp (DMAPP) = 632 nM, and a
kcatapp = 0.44 s
1. MiaA activity was minimally affected by other
modifications or nonsubstrate tRNA species present in bulk tRNA
isolated from a miaA mutant. MiaA modified
ACSLPhe with a
kcatapp/Kmapp
substrate specificity about 17-fold lower than that for intact tRNAPhe, mostly due to a decrease in apparent substrate
binding affinity. Quantitative Western immunoblotting showed that MiaA
is an abundant protein in exponentially growing bacteria (660 monomers
per cell; 1.0 µM concentration) and is present in a
catalytic excess. However, MiaA activity was strongly competitively
inhibited for DMAPP by ATP and ADP
(Kiapp = 0.06 µM), suggesting that MiaA activity is inhibited
substantially in vivo and that DMAPP may bind to a
conserved P-loop motif in this class of prenyltransferases. Band
shift, filter binding, and gel filtration shift experiments support a
model in which MiaA tRNA substrates are recognized by binding tightly
to MiaA multimers possibly in a positively cooperative way
(Kdapp
0.07
µM).
The tRNA prenyltransferase (EC 2.5.1.8) encoded by the
miaA gene of Escherichia coli catalyzes the
addition of a 2-isopentenyl group from dimethylallyl
diphosphate (DMAPP)1 to the
N6-nitrogen of adenosine adjacent to the
anticodon at position 37 of 10 of 46 E. coli tRNA species
(i6A37, see Fig. 1 and Refs. 1-4). On the basis of a
theoretical model of yeast tRNASer structure, the
N6-nitrogen of A37 in tRNA
substrates of MiaA is recessed and points inward toward the center of
the anticodon loop (5). In E. coli, the i6A37
tRNA modification is further methylthiolated by the action of the MiaB
and MiaC enzyme activities to form ms2i6A37
(Fig. 1), except for tRNASec (6).
ms2i6A37-modified tRNA species read codons
starting with U residues and include tRNAPhe,
tRNATrp, tRNATyr (I and II),
tRNACys, tRNALeu (IV and V), and
tRNASer (II and III) but not tRNASer (I and V)
(7, 8).
The E. coli MiaA prenyltransferase is an excellent model to
study fundamental aspects of the modification process. Unlike many
modifications, the function of ms2i6A37 in
translation has been studied extensively in vivo and
in vitro (reviewed in Refs. 3, 4, and 9). The
ms2i6A37 tRNA modification is thought to
stabilize tRNA-mRNA interactions by improving intrastrand stacking
within tRNA anticodon loops and interstrand stacking between codons and
anticodons (10, 11). ms2i6A37 also seems to
influence the conformation of the tRNA anticodon loop and thereby
affect interstrand stacking interactions between wobble bases at
position 34 in tRNA and bases immediately 3 to codons (4, 10). Lack of
ms2i6A37 in the tRNA of miaA mutants
of E. coli and Salmonella typhimurium results in
multiple defects in translation efficiency, codon context sensitivity,
and fidelity (10-21). These translation defects impart broadly
pleiotropic phenotypes to miaA mutants that contain A37 instead of ms2i6A37 in their tRNA (Fig. 1) (3,
4), including decreased growth rate and yield (12, 13), altered
sensitivity to amino acid analogs (12), increased oxidation of certain
amino acids, and tricarboxylic acid cycle intermediates (22), altered
utilization of primary carbon sources (22), moderately increased GC
TA transversion frequency in nutritionally limited cells (19, 23), suppression of Tet(M)-protein-induced tetracycline resistance (24),
decreased DNA oxidation damage in exponentially growing cells,2 and temperature
sensitivity for colony formation at 45 °C (25). Lack of
ms2i6A37 does not seem to affect the
aminoacylation of tRNA (21, 26, 27) or amino acid-tRNA-EFTu·GTP
selection (4). Many of these miaA phenotypes may be caused
by disruptions in translational control mechanisms, such as the ones
that regulate expression of the tryptophan (trp) (28, 29)
and the tryptophanase (tna) (30, 31) operons.
The E. coli miaA gene was cloned (23, 32), sequenced (19), and found to be a member of a superoperon with unusual structure and complex modes of regulation (22, 25, 33). Homologs of E. coli miaA have also been sequenced in several other organisms (34, 35). Notably, the miaA homolog of yeast, designated MOD5, has been developed into an important system to study subcellular localization of proteins (36). Over 20 years ago, Rosenbaum and Gefter (37) and Söll and co-workers (38) partially purified E. coli MiaA. These pioneering studies demonstrated the substrates and some of the conditions required for MiaA activity and established the sequential pathway for ms2i6A37 biosynthesis in tRNA (Fig. 1; reviewed in Refs. 3 and 4). In this paper, we report rapid methods of MiaA purification and analyses of MiaA steady-state kinetics and tRNA substrate utilization and binding. We also present direct quantitation of the cellular amount of the MiaA tRNA modification enzyme. Our results show that MiaA substrate selection is complicated and likely regulated by several mechanisms.
[-32P]CTP (10 mCi per ml, >400
Ci per mmol) was purchased from Amersham Corp.
[1-3H]DMAPP (0.5 mCi per ml; 15 Ci per mmol) and cold
DMAPP were from American Radiolabeled Chemicals. Restriction enzymes,
polynucleotide kinase, T4 DNA ligase, and Riboprobe and Ribomax RNA
synthesis systems were purchased from Promega. BstN1 and
strain JM105 were obtained from New England Biolabs. Plasmid pET-15b,
E. coli strain HMS174, and biotinylated thrombin were
obtained from Novagen. Plasmid pKK223-3, a Superose 12 HR 10/30 column,
and PD-10 columns were from Pharmacia Biotech Inc. W-POREX DEAE
columns (250 × 4.6 mm) were from Phenomenex. DEAE-cellulose
(DE-52) was from Whatman. ATP, ADP, and other biochemicals were
purchased from Sigma. A protein isolation kit for sorbent
identification (PIKSI) was purchased from American International
Chemical. Bradford and DC protein assay reagents were
bought from Bio-Rad.
Cloning and other molecular biological procedures were done by standard methods (39), unless indicated otherwise. A his6-tag-miaA+ gene fusion under the control of the T7-phage promoter was constructed in vector pET-15b by ligating together the following three fragments: (i) a 5.7-kilobase fragment of pET-15b digested with NdeI and BamHI; (ii) a 519-bp NdeI-MslI fragment generated by polymerase chain reaction with primers UMiaA and LMiaA (Table I) containing the amino-terminal segment of miaA with an NdeI site added at the miaA start codon; and (iii) a 579-bp MslI-BstYI fragment containing the carboxyl-terminal segment of miaA+ obtained from plasmid pTX312 (22). This strategy limited the length of polymerase chain reaction-amplified DNA fragments used in the construction. Ligation mixtures were transformed into strain JM105, and the desired construct, designated pTX439, was identified by restriction digestion patterns of purified plasmid DNA, DNA sequencing of the miaA segment generated by polymerase chain reaction amplification, and complementation of a miaA::Tn10 null mutation in E. coli strain DEV15 (14, 19). pTX439 was transformed into E. coli strain HMS174 to give strain TX3371, in which the His6-MiaA protein was overexpressed (see Ref. 40).
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The native MiaA protein was overexpressed from plasmid pTX440, which was constructed by ligating a 1.1-kilobase FspI-ScaI fragment containing the intact miaA+ gene from pTX312 (22) downstream of the Ptac promoter in plasmid pKK223-3. Ligation mixtures were transformed into strain JM105 to give strain TX3367. The orientation of the miaA+ fragment in purified pTX440 plasmid was confirmed by restriction digestion patterns and DNA sequencing.
Overexpression and Purification of His6-MiaA960-ml cultures of strain TX3371 were grown
in LB medium containing 50 µg of carbenicillin per ml and induced
with 1 mM IPTG as described before (40).
His6-MiaA protein was purified by one-step batchwise
Ni2+-chelation affinity chromatography as described
previously (40), except that the wash buffer contained 40 mM imidazole instead of 60 mM imidazole. Eluted
His6-MiaA was desalted on PD-10 columns into 2 × TMD
buffer (60 mM Tris-HCl (pH 7.5 at 24 °C), 20 mM MgCl2, and 2 mM dithiothreitol
(DTT)). Similar MiaA concentrations were found using the Bradford and
DC(Lowry) protein assays with bovine serum albumin (BSA) as
the standard. After desalting, an equal volume of 72% (v/v) glycerol
was added to the His6-MiaA preparation, and the protein
solutions were divided into small volumes and stored at 70 °C.
His6-MiaA samples were thawed only once and used
immediately.
The His6-tag was cleaved from 50 µg of His6-MiaA by digesting at room temperature for 3 h with 1 unit of biotinylated thrombin in the buffer provided by the manufacturer. The resulting protein mixture was used immediately for enzyme assays and binding studies without further purification. His6-MiaA purity and the extent of thrombin cleavage were determined by SDS, 15% PAGE (5.6% stacking) as described before (41).
Purification of Native MiaA Protein by Mimetic Dye ChromatographySix 400-ml cultures of E. coli strain
TX3367 were grown in LB medium containing 50 µg of ampicillin per ml
at 37 °C with shaking (300 rpm) until they reached a turbidity of 50 Klett (660 nm) units, upon which IPTG was added to a final
concentration of 1 mM. Cultures were incubated at 37 °C
with shaking for 3 h longer, after which they were chilled on ice.
All subsequent steps were carried out at 4 °C, unless noted
otherwise. Cells were collected by centrifugation (5000 × g) for 15 min, and pellets were suspended in 300 ml of TMD
buffer (30 mM Tris-HCl (pH 7.5 at 24 °C), 10 mM MgCl2, and 1 mM DTT). Cells were
collected again by centrifugation (5000 × g) for 15 min, and pellets were suspended in 18 ml of TMD buffer. The cell
suspension was passed twice through a chilled French pressure cell at
20,000 p.s.i. Cell lysates were centrifuged at 150,000 × g for 60 min, and supernates were filtered through 0.22-µm
acetate filters (Micron Separations). 60 mg of protein extract was
loaded by gravity flow onto each of the 10 different mimetic dye
PIKSI-kit columns, which had been equilibrated with TMD buffer. Each
column was washed by gravity flow with 5 ml of TMD buffer containing
0.05 M KCl. Proteins were eluted by two consecutive 5-ml
washes of TMD buffer containing 0.2 M KCl followed by 0.8 M KCl. 100-µl drops of the eluants from each column were placed on Type VS filter disks (0.025 µm, Millipore), which had been
floated on TMD buffer, and the eluants were dialyzed against TMD buffer
at 4 °C for 30 min. 15 µg and 5 ng of the dialyzed eluants from
each column were analyzed by SDS-PAGE and assayed for MiaA
prenyltransferase activity (below), respectively. The samples eluted
with 0.8 M KCl from the Mimetic RedTM2 A6XL and Mimetic
OrangeTM1 A6XL columns had the most MiaA activity and fewest other contaminating bands (data not shown). The eluant from the Mimetic
Red column was pumped (0.3 ml per min) onto a room temperature Superose
12 HR 10/30 column, which was equilibrated and eluted with TMD buffer.
Fractions were collected into chilled tubes and analyzed by SDS-PAGE
and assayed for MiaA activity. The fractions that had a molecular mass
of about 35 kDa and maximum MiaA enzyme activity were pooled, diluted
with an equal volume of 72% (v/v) glycerol, distributed into small
volumes, and stored at 70 °C.
A synthetic E. coli pheU gene encoding wild-type tRNAPhe
(Fig. 2A, see "Results") was constructed
by ligating together six oligonucleotides (tRNAphe1 to tRNAphe6; Table
I) to form an upstream SalI restriction sequence, a T7-phage
RNA polymerase promoter preceding the 76-base pair (bp) pheU
gene, and downstream BstNI and BamHI restriction sites (42). Positive strand oligomers tRNAphe1, tRNAphe2, and tRNAphe3
and negative strand oligomers tRNAphe4, tRNAphe5, and tRNAphe6 were
phosphorylated with T4-phage polynucleotide kinase and annealed
together in T(0.1)E buffer (10 mM Tris-HCl (pH 8), 0.1 mM EDTA) by heating at 90 °C for 5 min followed by slow
cooling to room temperature. The annealed oligomers were ligated with an equal amount of pUC18 DNA that had been digested with
SalI and BamHI, and the ligation mixture was
transformed into strain JM105. The desired plasmid, designated pTX442,
was identified by restriction digestion patterns, which were confirmed
by DNA sequencing of the entire synthetic pheU gene.
A synthetic mutant pheU gene specifying tRNAPhe(U60C) (Fig. 2A, see "Results") was constructed by replacing tRNAphe3 and tRNAphe4 with U60C-forward and U60C-reverse (Table I) in the above cloning strategy to give plasmid pTX476. A synthetic mutant pheU gene specifying tRNAPhe(A37G) (Fig. 2A, see "Results") was constructed by replacing tRNAphe2 and tRNAphe5 with A37G-forward and A37G-reverse (Table I) to give plasmid pTX475. A synthetic gene specifying wild-type ACSLPhe (Fig. 2B, see "Results") was constructed by annealing together SL1 and SL2, which provided an upstream HindIII site, a T7-phage polymerase promoter abutting the 17-bp ACSLPhe gene, and downstream SmaI and BamHI sites. The two oligomers were phosphorylated and annealed as described above and ligated into pUC18 cut with HindIII and BamHI to give plasmid pTX539. A mutant variant, ACSLPhe(A11G) (Fig. 2B, see "Results"), was constructed in the same way by replacing SL1 and SL2 with SL-A37G1 and SL-A37G2 to give plasmid pTX540.
In Vitro Synthesis and Purification of tRNAPhe, ACSLPhe, and Mutant VariantsPlasmids pTX442, pTX475,
pTX476, pTX539, and pTX540 were purified using a MidiPrep kit (Qiagen).
Purified plasmids pTX442, pTX475, and pTX476 or pTX539 and pTX540 were
digested to completion with BstNI or SmaI,
respectively, which ultimately give the correct 3-ends in the
transcribed products (Fig. 2, A and B,
respectively) (43). Digestion mixtures were extracted once with an
equal volume of TM (30 mM Tris-HCl (pH 7.5 at 24 °C) 10 mM MgCl2)-saturated phenol:chloroform (1:1) and
four times with ethyl ether before being precipitated with ethanol
(39). Linearized DNA templates were transcribed in vitro by
T7-phage RNA polymerase provided in the Riboprobe or Ribomax kits
according to the manufacturer's instructions. Transcripts were labeled
with 32P by adding 5 µl of [
-32P]CTP per
100-µl transcription reaction mixture. Transcription reaction
mixtures were incubated at 37 °C overnight. DNA templates were
removed by digestion with RQ1 DNase (1 unit per 1 µg of DNA) at
37 °C for 1 h. Digestion mixtures were extracted once with an
equal volume of TM-saturated phenol:chloroform (1:1) and four times
with ethyl ether before precipitation with ethanol. Pellets were
collected by centrifugation in a microcentrifuge, dried, and suspended
in T(0.1)E buffer.
tRNAPhe, ACSLPhe, and mutant variants were
further purified from nucleotides and aborted transcripts by DEAE high
performance liquid chromatography. Resuspended mixtures were applied at
a flow rate of 1 ml per min to a W-POREX DEAE column equilibrated with
20 mM sodium phosphate buffer (pH 6.5). RNA molecules were
eluted with a linear 0.2 to 1 M NaCl gradient (ramp = 60 min) in 20 mM sodium phosphate buffer (pH 6.5) (no urea)
at room temperature. Intact tRNAPhe or ACSLPhe,
which eluted at about 44 or 36 min, respectively, into the gradient, were pooled, precipitated with ethanol, and stored as dried pellets at
20 °C. Before use, the purified tRNAPhe and
ACSLPhe preparations were suspended in T(0.1)E buffer,
heated at 90 °C for 2 min, and cooled slowly to room temperature.
Concentrations of the tRNAPhe and ACSLPhe
molecules were determined by using A260
extinction coefficients calculated from base compositions by the Oligo
4.0 program (National Biosciences). The yield from the Riboprobe
or Ribomax kit was 7.5 µg of tRNAPhe from 5 µg of
linearized DNA in a 100-µl reaction mixture or 400 µg of
tRNAPhe from 20 µg of linearized DNA in a 200-µl
reaction mixture, respectively. tRNAPhe(wt) preparations
analyzed by urea-20%-PAGE lacked detectable contamination by an
(n + 1) tRNAPhe product, which contains an
extra 3
-nucleotide (data not shown (43)).
Bulk tRNA was
purified from 4 liters overnight LB cultures of strains NU398 (DEV15
miaA::Tn10) and NU394 (DEV15 miaA+) as
described previously (23) with some modifications. Briefly, cultures
were chilled and cells were collected by centrifugation (5000 × g) for 10 min and suspended in 20 ml of cold TMD buffer. All
remaining steps were performed at 4 °C, unless noted otherwise. 20 ml of TM-saturated phenol was added to the suspended cells, and the
mixture was agitated vigorously on a wrist shaker for 1 h. Lysed
cells and phenol were removed by centrifugation (14,000 × g) for 30 min. The aqueous phases were loaded by gravity
flow onto separate low pressure DEAE-cellulose columns (2.5 × 3 cm) equilibrated with TM buffer containing 0.02 M NaCl. The
columns were washed with TM buffer + 0.02 M NaCl at a flow
rate of 2 ml per min for 110 min and then eluted with a linear 0.02 M to 1 M NaCl gradient (ramp = 110 min) in
TM buffer. Samples with A260 > 1 were pooled,
precipitated with ethanol, and stored as dry pellets at 20 °C. For
kinetic experiments, bulk tRNA was suspended in T(0.1)E buffer, heated
at 90 °C for 2 min, and cooled slowly to room temperature.
Concentrations of bulk tRNA were determined from
A260 (extinction coefficient = 40 µg per
A260 unit). The yield of bulk tRNA was 15-25 mg
per 10 g of wet cells.
Binding reaction mixtures (200 µl) contained TMD buffer and 100 µg of BSA per ml, usually 3.3 to
16.4 µM of His6-MiaA protein, and up to 3.3 µM of tRNAPhe(wt) or
tRNAPhe(A37G). Binding reaction mixtures were incubated at
24 °C for 30 min before being injected onto a Superose 12 HR 10/30
sizing column equilibrated with TMD buffer (flow rate = 0.3 ml per
min at room temperature). Molecules were resolved in TMD buffer at the
same flow rate and detected by A280. The column
was calibrated with size standards (thyroglobulin, 669 kDa;
-amylase, 200 kDa; BSA, 66 kDa; chicken bovine albumin, 35 kDa;
carbonic anhydrase, 29 kDa; and cytochrome c, 12.4 kDa)
between runs of different samples.
Reactions were
optimized by checking several conditions (see below). Standard reaction
mixtures (50 µl) contained TMD buffer and 100 µg of BSA per ml.
DMAPP excess reactions contained 4.0 µM (0.105 Ci/mmol)
[1-3H]DMAPP (7 × Kmapp), and the
tRNAPhe, bulk tRNA from a miaA mutant, or
ACSLPhe concentration was varied from 0.004 to 0.16 µM (24 or 37 °C), 0.04 to 0.4 µM
(37 °C), and 0.04 to 0.4 µM (24 °C), respectively. tRNAPhe excess reactions contained 0.08 µM of
wild-type tRNAPhe (
35 × Kmapp), and
[1-3H]DMAPP (0.105 Ci/mmol) was varied from 0.1 to 10 µM. Reactions were started by adding 5 ng of MiaA enzyme
preparations, which had been diluted from storage buffer into cold TMD
buffer immediately before use. Reactions were stopped at various times
by adding 0.5 ml of cold 10% (w/v) trichloroacetic acid. Precipitates
were collected onto 25-mm Whatman GF/C filters, which were washed with 10 ml of cold 10% trichloroacetic acid and then 10 ml of cold 100%
ethanol. The filters were dried for 10 min under an infrared heat lamp
and counted in 3.5 ml of Ultima Gold XR scintillation mixture
(Packard). Product formation was linear with time for at least 8 min at
the highest and lowest concentrations of substrates used, and initial
velocities were usually determined from 2-min reactions for
intermediate substrate concentrations. ATP or ADP concentrations
between 30 nM and 10 µM were added to
tRNAPhe excess reactions to test inhibition of MiaA for
DMAPP. Kinetic parameters were calculated by using the Enzfitter
nonlinear regression data analysis program (Biosoft).
As noted previously for partially purified preparations (37), the activity of purified His6-MiaA was strongly reduced in reaction mixtures at pH values below 6 and above 10, containing 50 mM NaPO4 buffer (pH 7.5) instead of Tris-HCl (pH 7.5), and lacking reducing agent, such as DTT (data not shown) (37, 38). Omission of BSA from reaction mixtures reduced His6-MiaA activity by 42%, and plots of (product formation) versus (enzyme concentration) × (assay time) (44) indicated that BSA stabilized His6-MiaA in reaction mixtures at 37 °C (data not shown). His6-MiaA activity was maximal in reaction mixtures containing DTT, BSA, and 10 mM MgCl2 or 1 mM MnCl2, but was reduced to 53, 43, 6, or <1% when 10 mM MgCl2 was replaced by 20 mM MgCl2, 10 mM MgSO4, 10 mM MnCl2, or 10 mM ZnSO4 or 1 mM EDTA (data not shown). His6-MiaA (5 ng) diluted into TMD containing 100 µg of BSA per ml was thermally stable at 24 to 37 °C for at least 15 min but was rapidly inactivated by incubation at temperatures above 45 °C (data not shown).
Quantitative Western Immunoblotting BlottingWe used
purified His6-MiaA as an antigen for the production of
anti-MiaA polyclonal antibodies in rabbits (see Ref. 41). E. coli strains TX2494 (CC104 miaA+) and
TX2590 (CC104 miaA::Kmr) were grown
in 400 ml of Vogel-Bonner (1 × E) minimal salts medium supplemented with 0.4% glucose and enriched with 0.5% acid casein hydrolysate (Difco) (25). Subsequent steps was carried out as described previously (45), except that His6-MiaA or
thrombin-treated His6-MiaA were used as standards.
Air-dried immunoblots were scanned on a Hewlett-Packard ScanJet 4C
scanner, and bands were quantitated by using SigmaScan software
(Jandel).
The binding reaction mixture (50 µl) contained TMD and 100 µg of BSA per ml. In protein excess titrations (46-48), the concentration of 32P-labeled tRNAPhe or ACSLPhe molecules was fixed at 0.8 or 3.6 nM, respectively, and the concentration of His6-MiaA protein was varied between 5.8 nM and 14.2 µM. In ligand excess titrations (46-48), the concentration of His6-MiaA protein was fixed in the range of 0.5 to 1.3 µM, and the concentration of 32P-labeled tRNAPhe or ACSLPhe molecules was varied from 0.1 to 1.6 µM or 0.1 to 10 µM, respectively. Binding reaction mixtures were incubated for 30 min at room temperature. RNA-protein complexes were resolved from free RNA molecules by electrophoresis through native 6% (w/v) polyacrylamide gels (29:1 acrylamide:bisacrylamide) containing 10 mM Tris acetate (pH 8.0), 0.1 mM EDTA, and 1 mM DTT. Gels were prerun at 200 V at 4 °C for 2 h immediately before use. 30 µl of 40% sucrose was added to samples before loading them onto gels. Gels were run at 200 V at 4 °C for 2 h. Radioactive bands were visualized by autoradiography of dried gels and were quantitated by direct counting in an Instant Imager (Packard).
Nitrocellulose Filter Retention AssaysTriplicate binding reactions were carried out in 100 µl of TMD containing 100 µg of BSA per ml. In protein excess titrations, the concentration of 32P-labeled tRNAPhe or ACSLPhe molecules was fixed at 20 pM or 1.8 nM, respectively, and the concentration of His6-MiaA protein was varied between 0.56 nM to 15.9 µM. In some protein excess titrations, 100 µg of BSA per ml was added to filter soaking and washing solutions (48). In ligand excess titrations, the concentration of His6-MiaA protein was fixed in the range from 1.8 to 2.2 µM, and the concentration of 32P-labeled tRNAPhe or ACSLPhe molecules was varied from 10 nM to 5.0 µM. BSA was omitted from the filter soaking and washing solutions used for ligand excess titrations, because we found that the saturation level for MiaA·tRNAPhe complex formation was about 2-fold higher when BSA was omitted. Binding reaction mixtures were incubated for 30 min at room temperature and passed through soaked (TMD buffer for at least 1 h) nitrocellulose filters (13 diameter, 0.45 µm pore size; Schleicher & Schuell) filters contained in a manifold (Hoefer Scientific) connected to house vacuum (15-20 in Hg). Filters were washed twice with 300 µl of cold TMD buffer, dried briefly on the manifold, and counted in Ultima Gold XR scintillation mixture (Packard). The background dpm of control reactions lacking His6-MiaA was less than 10% of the input radioactivity and was subtracted in all cases. The retention efficiency of MiaA·tRNAPhe(wt) complexes ranged from about 65% to about 85% at saturation in protein excess titrations when BSA was added or omitted, respectively, from soaking and washing solutions. The retention efficiency of MiaA·ACSLPhe(A11G) complexes was about 40% at saturation in protein excess titrations when BSA was omitted from soaking and washing solutions. MiaA·ACSLPhe(wt) complexes were not detected by the filter binding assay.
We set up rapid methods to
purify large amounts of active MiaA protein for kinetic and binding
studies. We constructed an in-frame translation fusion between the
His6-containing leader peptide encoded by vector pET15b and
the presumed translation start codon of MiaA (see "Experimental
Procedures" (19)). IPTG induction of the T7-phage promoter driving
the fusion caused 1,340-fold overexpression of His6-MiaA in
crude extracts as measured by quantitative Western immunoblotting
(Fig. 3, lanes 2 and 3;
"Experimental Procedures"). Single-step, batchwise, metal ion
affinity chromatography on activated Ni2+ resin resulted in
electrophoretically pure His6-MiaA (Fig. 3, lane
4). The yield of His6-MiaA from 960 ml of bacterial
culture was 6 mg with a specific activity of 700 nmol of
i6A formed per min per mg of protein using synthetic
tRNAPhe(wt) as a substrate (see below, and see
"Experimental Procedures"). The His6-affinity tag could
be completely cleaved from the fusion protein by thrombin protease
(Fig. 3, lane 5; "Experimental Procedures"). Gel
filtration analyses indicated that the cleaved
His6-affinity tag was likely released from the MiaA protein
(below).
We also devised a two-step purification of small amounts of native MiaA to allow comparisons with the kinetic properties of His6-MiaA and thrombin-treated MiaA. We overexpressed native MiaA by IPTG induction of the Ptac promoter of plasmid pTX440 (see "Experimental Procedures"). The MiaA protein was purified by step-elution off a mimetic red dye column followed by gel filtration (see "Experimental Procedures"). The purified preparation contained approximately equal amounts of one 46-kDa contaminant band and the 35-kDa MiaA band on Coomassie-stained SDS-polyacrylamide gels (data not shown). The yield of native MiaA from 2.4 liters of bacterial culture was 0.02 mg with a specific activity of 440 nmol of i6A formed per min per mg of protein using synthetic tRNAPhe(wt) as a substrate (see below and see "Experimental Procedures").
Number of MiaA Molecules Per CellWe performed quantitative
Western immunoblot analyses to determine the average number of MiaA
molecules present in an E. coli cell growing exponentially
in enriched minimal salts/glucose medium at 37 °C
((Fig. 4; see "Experimental Procedures") (45). As
standards for these analyses, we added known amounts of purified
thrombin-treated MiaA to crude extracts of a miaA null
mutant (Fig. 4, lanes 4-9). We then loaded amounts of crude
extract from miaA+ cells so that the MiaA
detected was within the linear range of the standards (Fig. 4,
lanes 1-3). By this analysis, we found about 4.0 ng of MiaA
protein was present in 100 µg of extract of E. coli K-12
growing exponentially in enriched minimal salts/glucose medium at
37 °C. This amount corresponds to about 660 monomers of MiaA per
cell and a cellular MiaA concentration of about 1.0 µM,
where the volume of an E. coli cell was taken as 1.0 × 1012 ml (45). The spreading of MiaA standard bands on
gels (Fig. 4, lanes 5-9) was caused by salt from the
thrombin cleavage reaction. Similar quantitative results were obtained
when His6-MiaA instead of thrombin-treated MiaA was used as
a standard, in which case the standard bands were as tight as those
from the wild-type extracts (data not shown). Finally, the cellular
amount of MiaA dropped about 3-fold in cells in stationary phase (Fig.
4, lanes 1 and 2) compared with exponential phase
(Fig. 4, lane 3).
Preparation of RNA Substrates
Seven RNA substrates were
purified for this study of MiaA enzymology. We chose wild-type E. coli tRNAPhe (Fig. 2A) as a model synthetic
substrate for MiaA, because it had been characterized previously by
Uhlenbeck and co-workers (49) in their analyses of
aminoacyl-tRNAPhe synthetase. We synthesized
tRNAPhe(wt) in vitro by using T7-phage RNA
polymerase and purified the tRNAPhe(wt) away from aborted
transcripts and nucleotides by DEAE high performance liquid
chromatography (see "Experimental Procedures"). The synthetic
tRNAPhe(wt) differed from native tRNAPhe in
that it contained a 5-triphosphate instead of a 5
-monophosphate and
was fully unmodified at all positions (Fig. 2A).
We also constructed and synthesized four mutant variants of tRNAPhe(wt) and purified bulk tRNA from an E. coli miaA mutant and its miaA+ parent strain. Mutant tRNAPhe(U60C) (in which U at position 60 is replaced by C; Fig. 2A) is readily cleavable by lead ions if the tRNA molecule folds properly (49). tRNAPhe(U60C) was synthesized in case tRNAPhe(wt) was not fully available as a substrate for MiaA. Mutant tRNAPhe(A37G) (Fig. 2A) was synthesized as a specificity control and was not expected to be isopentenylated by MiaA. The synthetic ACSLPhe(wt) and its corresponding (A11G) mutant (Fig. 2B) were synthesized to test whether the determinants for i6A formation were contained in the ACSLPhe. High performance liquid chromatography analyses indicate that bulk tRNA isolated from miaA mutants seems to contain all RNA base modifications except for ms2i6A37 (23). Bulk tRNA from a miaA mutant also contains the majority of tRNA species that are not substrates for MiaA. tRNA isolated from the miaA+ strain should be fully modified, including with ms2i6A37, and was not expected to be a substrate for purified MiaA.
Binding Activity of His6-MiaA to Synthetic tRNAPhe(wt), ACSLPhe(wt), and tRNAPhe(A37G)We determined whether the purified
His6-MiaA was fully active for binding to
tRNAPhe(wt) and vice versa by using a gel
filtration shift assay (Fig. 5). tRNAPhe(wt)
and bulk tRNA from a miaA mutant ran anomalously with an apparent molecular mass of 78 kDa instead of 25 kDa on a calibrated Superose 12 HR 10/30 sizing column (Fig. 5A). Purified
His6-MiaA and thrombin-treated MiaA (23 µg) had apparent
molecular masses of about 34 kDa (Fig. 5B) and 37 kDa,
respectively, which approximated the 34-kDa monomer molecular mass
predicted for MiaA from its amino acid sequence (19). The slightly
lower mobility of His6-MiaA compared with thrombin-treated
MiaA may have been caused by interaction between the
His6-tag and the column matrix. A relatively large amount
of crude extract (6 mg) from wild-type cells produced a single peak of
MiaA activity with a molecular mass of about 45 kDa on the same column
(data not shown). Thus, His6-MiaA, thrombin-treated MiaA,
and native MiaA behaved as monomers under the experimental conditions
used here.
Addition of an equimolar amount of tRNAPhe(wt) to His6-MiaA caused complete disappearance of the 34-kDa His6-MiaA monomer peak, about a 50% reduction in the free tRNAPhe(wt) peak, and the appearance of a new 110-kDa leading peak containing the His6-MiaA·tRNAPhe(wt) complex (Fig. 5C). This result showed that the purified His6-MiaA was fully active for binding to tRNAPhe(wt). Similar results were obtained for thrombin-treated MiaA (data not shown). Addition of His6-MiaA in a 10-fold molar excess over tRNAPhe(wt) caused the free tRNAPhe(wt) peak to disappear completely with a corresponding increase in the 110-kDa peak containing the His6-MiaA·tRNAPhe(wt) complex (Fig. 5D). This result showed that the tRNAPhe(wt) substrate was fully capable of binding to the His6-MiaA enzyme. The above conclusions were confirmed by kinetic and quantitative binding studies (below).
Gel filtration shift assays were also performed with mixtures of His6-MiaA and the ACSLPhe(wt) microhelix (Fig. 2B) or mutant tRNAPhe(A37G) (Fig. 2A). Free ACSLPhe(wt) ran anomalously with an apparent molecular mass of 25 kDa instead of 5 kDa on the Superose 12 HR 10/30 column (data not shown). All of the ACSLPhe(wt) could be shifted into a His6-MiaA·ACSLPhe(wt) complex with an apparent molecular mass of about 45 kDa (data not shown), which approximated the sum of the predicted monomer molecular masses of His6-MiaA (36 kDa) and ACSLPhe(wt) (5 kDa). The free His6-MiaA peak also disappeared completely from binding mixtures containing equimolar amounts of tRNAPhe(A37G) and His6-MiaA (Fig. 5E). However, the resulting complex had an apparent molecular mass of about 63 kDa, which approximated the sum of the predicted monomer molecular masses of His6-MiaA (36 kDa) and tRNAPhe(A37G) (25 kDa). Thus, on the basis of complex size, His6-MiaA bound to the ACSLPhe(wt) microhelix and mutant tRNAPhe(A37G) in an apparent 1:1 molar ratio, whereas His6-MiaA and thrombin-treated MiaA seemed to form a larger, comparatively stable 110-kDa complex with tRNAPhe(wt). The stoichiometry of tRNAPhe(wt) binding is considered below.
MiaA Enzyme KineticsWe optimized an assay for determining
initial rates of [3H]DMA transfer to unlabeled RNA
substrates at 24 and 37 °C (see "Experimental Procedures").
[3H]i6A-modified RNA was recovered by
precipitation with trichloroacetic acid. Typical Lineweaver-Burk plots
of His6-MiaA with tRNAPhe(wt) and DMAPP are
shown in Fig. 6, A and B, respectively, and steady-state kinetic data for various RNA substrates and MiaA preparations are compiled in Table II. The apparent
substrate inhibition of His6-MiaA by
tRNAPhe(wt) (Fig. 6A) was also
observed for bulk tRNA from a miaA mutant (data not shown).
Since the synthetic tRNAPhe(wt) substrate and bulk tRNA
were prepared by different methods (see "Experimental Procedures"),
it seems unlikely that this substrate inhibition (Fig. 6A)
was caused by a low level contaminant in the synthetic
tRNAPhe(wt) preparations. tRNAPhe(A37G) and
bulk tRNA from a miaA+ strain were not modified
by MiaA, confirming the specificity of the in vitro
i6A37 modification reaction (data not shown).
|
The Kmapp and kcatapp for tRNAPhe(wt) were the same within experimental error for His6-MiaA, thrombin-treated MiaA, and native MiaA (Table II, lines 1, 5, and 6). Thus, the presence of the His6-tag did not appreciably affect the association state (above) or the kinetic properties (Table II) of the MiaA prenyltransferase. Consequently, His6-MiaA was used in most subsequent experiments and will be referred to simply as MiaA hereafter. tRNAPhe(wt) and tRNAPhe(U60C) showed equivalent kinetic properties (Table II, lines 1 and 2), implying that both substrates were folded correctly enough to act as substrates for MiaA. Consistent with this interpretation, prolonged incubation of reaction mixtures containing excess MiaA resulted in complete i6A modification of tRNAPhe(wt) as judged by the molar amount of [3H]DMA incorporated (data not shown). MiaA had nearly the same Kmapp and kcatapp for tRNAPhe(wt) and bulk tRNA isolated from a miaA mutant (Table II, lines 1 and 3). To make this comparison, the concentration of MiaA substrates was taken as 12.9% of all tRNA species in the bulk tRNA isolated from a miaA mutant grown in LB medium at a rate of 2.5 doublings per h (7).
The ACSLPhe(wt) microhelix (Fig. 2B) was also a
substrate for the MiaA enzymes (Table II, line 8). These reactions were
carried out at 24 °C to prevent melting of the GC-rich
ACSLPhe(wt) (predicted Tm 63 °C
from the Oligo 4.0 program (National Biosciences)). Control experiments
showed that prolonged incubation with excess MiaA led to complete
i6A modification of ACSLPhe(wt) and that mutant
ACSLPhe(A11G) was not modified by MiaA (data not shown).
The
kcatapp/Kmapp
substrate specificity constant was about 17-fold lower for
ACSLPhe(wt) than for tRNAPhe(wt) due primarily
to an 8-fold reduction in Kmapp
(lines 7 and 8).
MiaA homologs have
been sequenced from several organisms and share an ATP/GTP P-loop
binding motif (50). Hence, we checked whether ATP, ADP, and other
nucleotide di- and triphosphates (NDPs and NTPs) affected MiaA enzyme
activity in vitro. We found that MiaA activity was strongly
inhibited by ADP (Fig. 7A) and ATP (Fig.
7B). The inhibition was classically competitive with respect to the DMAPP substrate with
Kiapp(ATP) = 0.07 µM and Kiapp(ADP) = 0.05 µM. We tested other NTPs, such as GTP and CTP, and found similar inhibition as with ATP (data not shown). Thrombin-treated MiaA lacking the His6-tag was inhibited by ATP or ADP to
the same extent as His6-MiaA (data not shown). Last, we
found that mutant tRNAPhe(A37G) acts as a strong
competitive inhibitor of i6A-37 modification in
tRNAPhe(wt) (Kiapp = 4.23 nM (Fig. 7C)). This finding is consistent
with the results from gel filtration (Fig. 5E) and binding
studies (below).
Stoichiometry of MiaA Binding to RNA Substrates
We further
investigated the composition of complexes formed between MiaA and
synthetic tRNAPhe or ACSLPhe molecules by
performing band shift and filter binding assays (Figs. 8, 9, 10; see
"Experimental Procedures"). For both kinds of assays, we performed
protein excess titrations, in which the tRNAPhe(wt)
concentration was held constant far below the estimated
Kdapp, and the MiaA concentration
was varied (Figs. 8A and 10). We also performed ligand excess titrations, in which the MiaA concentration was
held constant near its estimated cellular concentration (1.0 µM; see above), and the tRNAPhe and
ACSLPhe concentrations were varied (Figs. 8B and
9).
Unexpectedly, the molar ratio of tRNAPhe(wt) or tRNAPhe(A37G) bound per MiaA at saturation was 0.5 for both types of binding assays (Fig. 9, A and B). Given that the MiaA preparations were completely active for binding (Fig. 5), this result showed that a MiaA dimer, rather than a monomer, bound to each intact tRNA molecule at saturation. Consistent with this interpretation, the predicted molecular mass of a MiaA2·tRNAPhe(wt) complex (100 kDa) matched the 110-kDa molecular mass observed during gel filtration (Fig. 5C). The discrepancy between the predicted molecular mass of a MiaA2·tRNAPhe(A37G) complex (100 kDa) and the 63-kDa complex observed during gel filtration (Fig. 5E) may indicate dissociation of the MiaA2·tRNAPhe(A37G) complex upon dilution during gel filtration. In contrast to tRNAPhe(wt) binding, ACSLPhe(wt) or ACSLPhe(A11G) bound MiaA in a 1:1 molar ratio at saturation (Fig. 9, A and B), suggesting that MiaA bound ACSLPhe microhelices as a monomer. This result confirmed the conclusion that the MiaA enzyme preparations were completely active for binding. The predicted molecular mass of a MiaA·ACSLPhe complex (41 kDa) was near that of the 45-kDa complex observed during gel filtration (above).
Examination of the gels used for the band shift assays further confirmed a difference in the way tRNAPhe(wt) and tRNAPhe(A37G) bound to MiaA. In ligand excess titrations, the MiaA·tRNAPhe(wt) complex formed at lower tRNAPhe(wt) concentrations (Complex 1, Fig. 8B) had a lower mobility and was possibly larger than the complex formed at saturation, which contained a 2:1 molar ratio of MiaA to tRNAPhe(wt) (Complex 2; Fig. 8B and Fig. 9). In contrast, Complex 1 predominated at tRNAPhe(A37G) concentrations as high as 0.5 µM in ligand excess titrations, whereas Complex 2 appeared and predominated at tRNAPhe(A37G) concentration near saturation (data not shown).
Last, we used the concentration of MiaA at half-saturation in protein
excess titrations of filter binding assays (Fig. 10) to
estimate Kdapp 0.07
µM for MiaA binding to tRNAPhe(wt) (46, 47,
51). Because of MiaA's complicated binding behavior (above), this
Kdapp(tRNAPhe(wt)) is
probably not a simple dissociation constant but rather a function of
several binding constants (e.g. see Ref. 51). Protein excess
titrations of band shift assays (Fig. 8A) gave a
Kdapp(tRNAPhe(wt))
1.0 µM, which was about 10-fold greater than that
obtained by filter binding. This higher
Kdapp(tRNAPhe(wt))
may reflect dissociation of complexes during electrophoresis. Since
MiaA bound ACSLPhe(A11G) by an apparently simple (R·P
R + P) mechanism, we estimated Kd(ACSLPhe(A11G)) = 1.1 µM
from ligand excess titrations of filter binding assays (Fig.
9B) (46, 47). The filter binding method failed to detect
binding between MiaA and the ACSLPhe(wt) microhelix,
suggesting that ACSLPhe(wt) bound to MiaA with a lower
affinity than 1.0 µM.
We report here steady-state kinetic and binding studies of the E. coli MiaA tRNA prenyltransferase modification enzyme. Only a limited number of tRNA and rRNA modification enzymes have been purified and studied to date, despite the fact that many different kinds and families of RNA modification enzymes are present in all cells (3, 4, 9). Biochemical studies of these RNA modification enzymes are aimed at understanding the mechanisms of RNA-protein recognition and catalysis and the functions and regulation of RNA modification in cells. The properties of the MiaA enzyme show similarities and noteworthy differences compared with other purified tRNA modification enzymes, including the E. coli TrmA m5U54-methyltransferase (51-53), the E. coli TrmD m1G-methyltransferase (54, 55), the E. coli HisT pseudouridine synthase I (56), and the E. coli and Zymomonas mobilis Tgt tRNA-guanine transglycosylases (57-60).
Similar to the TrmA and Tgt enzymes (51, 58), MiaA can modify a 17-mer
synthetic microhelix stem-loop substrate, in this case corresponding to
the ACSL of tRNAPhe(wt) (Table II). Thus, the minimal
recognition elements for the MiaA tRNA prenyl transfer reside in this
limited ACSL structure. However, the
kcatapp/Kmapp
substrate specificity is reduced significantly by about 17-fold for the
ACSLPhe(wt) microhelix compared with intact tRNA molecules
(Table II) (or 34-fold if MiaA is catalytically active as a dimer for
tRNA and as a monomer for ACSLPhe(wt) (below; see Table
II)). In this regard, MiaA resembles the Tgt transglycosylases, whose
Vmaxapp/Kmapp
is 5-10-fold lower for an ACSLTyr microhelix compared with
an intact tRNATyr substrate (58). In contrast, the TrmA
methyltransferase uses a T-loop microhelix as a substrate almost as
well as intact tRNA molecules (reduction of
kcatapp/Kmapp
2.5-fold; (51)). At the other extreme, the TrmD methyltransferase depends strongly on intact tRNA tertiary structures and does not efficiently modify an ACSL microhelix (reduction of
Vmaxapp/Kmapp
300-fold (54)).
Recently, an attempt was made to classify tRNA modification enzymes into two families (61). The TrmA methyltransferase and other enzymes that modify the amino acid-accepting minihelix, which is composed of the acceptor and T-loop microhelices, generally do not depend on overall tRNA tertiary structure for their activities (61). In contrast, TrmD methyltransferase and other enzymes that modify the anticodon minihelix, which is composed of the anticodon and D-loop microhelices, have been found to strongly depend on intact tRNA three-dimensional structure (see Ref. 61). The exception to this classification scheme is bacterial Tgt transglycosylases, which modify ACSL microhelices moderately efficiently (see above) (58, 62) and interact strongly, but not exclusively, with the ACSL region of substrate tRNA molecules (57, 63). MiaA also presents an exception to the strictest application of this classification scheme. However, the significantly reduced substrate specificity of MiaA for the ACSLPhe(wt) microhelix compared with intact tRNAPhe(wt) (Table II) suggests that interactions of MiaA with regions other than the ACSLPhe are important for optimal activity. This conclusion is further supported by binding studies discussed below. Compilations of the tRNA molecules that contain ms2i6A37 or i6A37 modifications led to a consensus ACSL that includes possible secondary structure and sequence-specific elements required for MiaA recognition and modification (1, 8). Recent experiments by Y. Motorin and H. Grosjean3 using tRNASer isoaccepting species confirm that optimal MiaA activity may depend on primary, secondary, and tertiary interactions within tRNA substrates. The kinetic, binding, and footprinting properties of these and other mutant RNA substrates are currently being determined.
One noteworthy difference between MiaA and other purified tRNA
modification enzymes is its extremely low
Kmapp for native and synthetic
tRNA substrates (3 nM; Table II). By comparison, the
Kmapp of the Tgt
transglycosylases, TrmD methyltransferase, and TrmA methyltransferase
for synthetic tRNA substrates is 2.0, 3.3, and 2.8 µM,
respectively (51, 54, 58), which are about 3 orders of magnitude higher
than that of MiaA (Table II). Likewise, the kcatapp or
Vmaxapp of MiaA is about 10-fold
greater for synthetic tRNA substrates than that of the TrmA and Tgt
enzymes (51, 58). Together, these results show that MiaA is a
comparatively active enzyme with a high apparent affinity for its tRNA
substrates. Consistent with this interpretation, the kinetic properties
of MiaA for a synthetic, purified tRNAPhe(wt) substrate
were very similar to those for bulk tRNA isolated from a
miaA mutant (Table II). Thus, the presence of nonsubstrate, native tRNA molecules in the bulk tRNA preparations did not appreciably inhibit or affect MiaA substrate recognition or activity. This behavior
contrasts with the purified HisT pseudouridine synthase (56) and TrmD
methyltransferase,4 which are strongly
inhibited by nonsubstrate tRNA species. Moreover, the constancy of
kinetic properties of MiaA for synthetic, unmodified tRNAPhe(wt) and hypomodified bulk tRNA from a
miaA mutant implies that the other modifications present in
tRNA molecules do not significantly affect MiaA activity. Transparency
to other base modifications in tRNA was also documented for the Tgt
transglycosylase (62).
We used quantitative Western immunoblotting to determine that there are
about 660 MiaA monomers per cell in bacteria growing exponentially in
enriched minimal salts/glucose medium (Fig. 4). Previously, the
cellular amounts of tRNA modification enzymes have been measured only
indirectly by comparisons of specific activities (e.g. see
Ref. 64), and these studies have led to the generalization that tRNA
modification enzymes are present in few copies per cell (9). To the
contrary, our results show that MiaA is a relatively abundant cellular
protein compared with other biosynthetic enzymes (see Ref. 41). From
the kcatapp of MiaA for tRNA
(Table II) and the number of MiaA monomers per cell, we calculate that
the rate of i6A37 synthesis can approach 15,400 modifications per min per cell. The equation for the rate of MiaA
substrate tRNA synthesis in exponentially growing cells is (65):
d(tRNA)/dt = ((ln2)/(cell doubling time)) × (number of
MiaA substrate tRNA molecules per cell) = (ln2/54 min) × (198,000 total tRNA molecules × 12.9% MiaA substrates (7)) = 328 MiaA
substrate tRNA molecules synthesized per min per cell. Thus, there is
about a 47-fold excess (15,400/328) of MiaA catalytic capacity in
vivo. This calculation makes a number of necessary simplifying
assumptions, including that MiaA substrates are not limiting in
vivo, that MiaA activity is not subjected to additional regulation
(below), and that the in vitro kinetic properties of MiaA
can be extrapolated to the in vivo situation. We estimate
that the in vivo steady-state concentration of MiaA is about
1.0 µM in these exponentially growing bacteria (see
"Results"). By comparison, the steady-state in vivo
concentration of MiaA tRNA substrates can be calculated at about 42 µM, Kmapp(tRNA) of
MiaA 3 nM (Table II), and
Kdapp(tRNA) of MiaA
0.07
µM in the absence of DMAPP (below). Finally, we found
that the cellular amount of MiaA was regulated and decreased about
3-fold as bacterial cells entered stationary phase (Fig. 4), which
leads to a drop in the rate of tRNA synthesis (66).
Early work indicated that MiaA activity was strongly inhibited by unknown compounds in some substrate preparations (37). Overexpression of E. coli tRNAPhe(wt) in E. coli caused the accumulation of hypomodified tRNA species lacking the ms2i6A37 or i6A37 modifications (21), implying that MiaA activity can be saturated in vivo. We found that MiaA activity was strongly inhibited by ADP, ATP, and other NTPs (see "Results"; Fig. 7, A and B). This inhibition was classically competitive with the DMAPP substrate (Fig. 7) with a Kiapp of about 0.06 µM (Fig. 7, A and B).
The comparatively large cellular amount of MiaA and its high activity
and substrate affinities are likely needed to overcome inhibition by
NTPs and NDPs in vivo. We can estimate this inhibition by
first recalling that the number of MiaA substrate tRNA molecules synthesized per min is about 328 (see above) 0.54 µM
(see "Results"). The
Kmapp(tRNA) of MiaA
3
nM (Table II), so MiaA is saturated for its tRNA
substrates. The inhibition of MiaA catalytic capacity can be calculated
from the standard enzyme inhibition equation (v = Vmaxapp [S]/([S] + Kmapp (1 + ([I]/Kiapp))) and will depend
on the MiaA kinetic parameters for DMAPP (Table II) and the free, but
not total, intracellular concentrations of NTPs, NDPs, and DMAPP. To
our knowledge, these free concentrations are not known with certainty
for E. coli. Nevertheless, one estimate of
[NTP]free is 50 µM, based on the
Kmapp(ATP) of many kinases (67),
and the fact that ATP is by far the most abundant nucleotide compound
in enterobacterial cells (68). Assuming that [NTP]free + [NDP]free actually approaches 100 µM, then
[DMAPP]free would only have to be about 25 µM to allow MiaA to fully modify newly synthesized tRNA
substrate molecules. [DMAPP]free
25 µM
seems reasonable, because DMAPP and isopentenyl diphosphate (IPP) are
precursors to ubiquinone, which is abundant in E. coli (Fig.
1) (69). Moreover, 25 µM is near the
Kmapp of IPP·DMAPP isomerases
of yeast and other organisms (70). Thus, the amount and kinetic
properties of MiaA and its inhibition by NTPs and NDPs seem balanced to
just allow full tRNA substrate modification.
The competitive inhibition of MiaA by ATP or ADP likely indicates an important structure-function relationship for this class of prenyltransferases. MiaA homologs from several organisms lack significant amino acid similarities with other enzymes that use IPP and DMAPP as substrates, such as the Asp-Asp-Xaa-Xaa-Asp motif (71). MiaA homologs also lack conserved Cys and His residues positioned in possible metal binding motifs (e.g. Ref. 59). On the other hand, they do share an ATP/GTP P-loop binding motif, which is also present in Agrobacterium Ti-plasmid-encoded adenine isopentenyl-diphosphate transferases (ipt; tzs) that synthesize the free-base plant hormone i6A (cytokinin) (19, 34). The conservation of the P-loop motif and the competitive inhibition of DMAPP by NDPs and NTPs suggests that these families of tRNA and adenine prenyltransferases may use the P-loop motif to bind DMAPP instead of motifs used by other prenyltransferases (71, 72). This hypothesis will be tested in future studies. To our knowledge, MiaA is the first example of an RNA modification enzyme whose activity is regulated by compounds other than its substrates or products.
In the absence of the DMAPP substrate, MiaA bound to wild-type and
mutant synthetic tRNAPhe and ACSLPhe substrates
in a surprisingly complicated way (Figs. 8, 9, 10). The
Kdapp(tRNAPhe(wt)) of
MiaA 0.07 µM (see "Results"; Fig. 10), which was
about 11-fold lower than the
Kd(ACSLPhe(A11G)) (see "Results").
The stoichiometry of binding MiaA to intact tRNAPhe(wt) and
tRNAPhe(A37G) was 2:1 at saturation (Fig. 9), whereas it
was 1:1 for MiaA binding to ACSLPhe(wt) or
ACSLPhe(A11G) (Fig. 9). With the exception of the
nonphysiological mutant tRNAPhe(A37G), these molar binding
ratios were consistent with complex sizes detected by gel filtration
chromatography (see "Results"; Fig. 5). As expected
tRNAPhe(A37G) and ACLS(A11G) were not modified by MiaA, but
tRNAPhe(A37G) strongly and competitively inhibited MiaA for
tRNAPhe(wt) with a
Kiapp = 4.2 nM (Fig.
7C). Unexpectedly the complexes formed between MiaA and
tRNAPhe(wt) or tRNAPhe(A37G) at low tRNA to
protein ratios were larger (Complex 1; Fig. 8B)
than the MiaA2·tRNA dimers formed at saturation
(Complex 2; Fig. 8B and Fig. 9).
The results from gel filtration (Fig. 5), kinetic (Figs. 6 and 7), and binding (Figs. 8, 9, 10) experiments can be accounted for by a model in which MiaA binds to ACSLPhe structures as a monomer but binds to intact tRNA molecules as a multimer, either a dimer with half-site occupancy or a tetramer that dissociates into half-site occupied dimers. If intact tRNA molecules bind to the MiaA multimer preferentially and only bind to the monomer at higher tRNA concentrations, then apparent substrate inhibition of MiaA by tRNAPhe(wt) would result (Fig. 6A). One attractive feature of this model is that only the correct tRNA substrates may bind tightly and possibly in a positively cooperative way to MiaA multimers. Consequently, the association state of MiaA may contribute to the process of distinguishing between substrate and nonsubstrate tRNA molecules. Additional studies are needed to test features of this model directly, determine the kinetic order of the modification reaction, and learn whether DMAPP affects the MiaA association state.
After submission of this paper, J. A. Moore and C. D. Poulter published an independent purification and characterization of the E. coli MiaA tRNA prenyltransferase (Moore, J. A., and Poulter, C. D. (1997) Biochemistry 36, 604-614). Their paper, which largely complements the work reported here, shows that the order of substrate binding is tRNA then DMAPP. Apparent differences in the two studies in certain parameters, such as the Kmapp (tRNAPhe) of MiaA, will be resolved by futher experiments.