Validity of hypothesis that
[>>t] is compatible withA-tRNAGlx but notN-tRNAGlx
Since our primary focus here is to understand the role played by the
H8-L -H9 motif in recognizing/discriminating
tRNAGlx D-helix (augmented versus non-augmented), the
genomic tRNAGln and tRNAGlusequences of all 13 bacterial species were examined (Figure S5) for the
presence/absence of augmented/non-augmented D-helix in
tRNAGlx. The results are summarized in Table S2. The
genomic tRNAGlx is annotated with A (augmented)
or N (non-augmented) corresponding to each bacterial species.
We first test our hypothesis that the rigid proteobacterial
α-[>>t ]-H8-L -H9
conformational motif favorably interacts withA -tRNAGlx and unfavourably withN -tRNAGlx on three proteobacterial species
whose GluRSs display the α-[>>t ]
conformation — ECO(D-), SML(D-) and XOP(D-) — all associated withA -tRNAGlu andN -tRNAGln. In absence of gatCAB (all are D-),
these three GluRSs must strictly discriminate againstN -tRNAGln and favorably interact withA -tRNAGlu. Our hypothesis matches with the
strict requirement of the GluRSs to beN -tRNAGln-discriminatory. The hypothesis was
then tested for other GluRSs (see below).
Two proteobacterial species BTE(D+) and PAE(D+) contain gatCAB in their
genomes suggesting that there is no strict requirement forBte (D+)-GluRS and Pae (D+)-GluRS to discriminate against
tRNAGln. Both genomes are associated withA -tRNAGlu andN -tRNAGln and both display the
[>>t ] conformation
(α- and β-). In absence of
experimental data, our hypothesis suggests that both must be
tRNAGln-discriminatory. The other proteobacterial
species HPY (A -tRNAGlu;N -tRNAGlu) contain twin GluRSs. Among the two,Hpy (T1)-GluRS displayed the dynamic
α-[>>t ] conformation, compatible
with A - but not N -tRNAGlx.Hpy (T1)-GluRS has been experimentally shown to recognizeA -tRNAGlu and notN -tRNAGln, which is compatible with our
hypothesis. Hpy (T2)-GluRS has been experimentally shown to be
non-functional and therefore is not included in this discussion.
Two non-proteobacterial ND-GluRSs (Mtu -GluRS andBbu -GluRS; being ND they must recognize both
tRNAGlu and tRNAGln) displayed the
[>>t] motif. Both
tRNAGlu and tRNAGln areA -type in the MTU genome, compatible with the presence ofA -tRNAGlx-recognizing
[>>t ]-H8-L -H9 motif. In
contrast, the BBU genome containsA -tRNAGlu/N -tRNAGln.
The [>>t ]-H8-L -H9 motif inBbu -GluRS is thus compatible withA -tRNAGlu but not withN -tRNAGln, although as ND-GluRS it must
recognize both. It is possible that with a large addendum to its
C-terminal domain and the presence of K in place of D at the tip of the
turn that protrudes into tRNA, the geometry of Bbu -GluRS with
tRNA may be non-canonical and not the same as the structural models
considered here.
All other (four) GluRSs originating from six bacterial species (Table
S2) display the
[t>> ]-H8-L -H9 motif,
compatible with both A - and N -type of RNA. Of these, TEL
(A -tRNAGlu; N -tRNAGlu)
contain ND-GluRS which must recognize both tRNAGlu and
tRNAGlu, compatible with our hypothesis. TMA
(A -tRNAGlu;N/A -tRNAGlu) genome contains twin GluRSs.Tma (T1)-GluRS has been experimentally shown to recognize both
tRNAGlu and tRNAGln, also compatible
with our hypothesis. TTH (A -tRNAGlu;A -tRNAGlu) and EMG
(A -tRNAGlu; A -tRNAGlu)
genomes contain GlnRS and are devoid of gatCAB, making the corresponding
GluRSs discriminatory towards A -tRNAGln. Since
the [t>> ]-H8-L -H9 motif,
displayed by these two proteins, is favorably disposed to interact withA -tRNAGln, the origin ofA -tRNAGln-discrimination for these two
non-proteobacterial GluRSs must arise from interactions between the
GluRSs and some non-D-helix element in tRNA. For Tth -GluRS, it
has been experimentally shown that
tRNAGln-discrimination is mediated by R358 at the anti
codon-binding C-terminal domain of Tth -GluRS (26).
Correlation between
H8-L-H9 sequence and genomic A/N-tRNAGlnin proteobacteria
The above discussion, where experimentally determined bacterial GluRS
crystal structures were used to probe their role, especially the
H8-L -H9 motif, in recognizing or discriminating againstA /N -tRNAGlx, identified certain
sequence/structural features of the H8-L -H9 motif to be
responsible for A -tRNAGlx recognition andN -tRNAGlx discrimination. This was especially
found to be true for the proteobacterial class whose genomes are known
to mostly contain A -tRNAGlu andN -tRNAGln. Experimental evidence forA -tRNAGlu recognitionN -tRNAGln discrimination by proteobacterial
GluRSs also point towards the importance of the A/N -feature of
tRNAGlx in tRNAGlx-specificity of
proteobacterial GluRSs, for example the role of augmented versus
non-augmented D-helix of tRNAGlx in E. coli(18), H. pylori (10) and A. ferroxidans (11). Here we
probed the importance of the H8-L -H9 motif in proteobacterial
GluRSs, extending the analysis to cases for which GluRS structures are
not available.
The analysis is restricted to a small subset of T1/T2 or D/ND
probacterial GluRS from three classes (γ-,ε- and α-). Specifically, we
focus on three class-specific groups of proteobacteria: i)
ε-proteobacterial GluRS(T1/T2) pairs from 11 species (CJR, HPY, WSU,
ABU, NIS, TDN, NAM, SKU, SDL, SUN, NSA), ii) γ-proteobacterial
GluRS(T1/T2) pairs from 7 species (AFE, MCA, HHA, AEH, NOC, CBU, TGR),
iii) α-proteobacterial GluRS(D+) from four species (BJA, OCA, NHA, RPD)
and α-proteobacterial GluRS(ND) from 9 species (SME, ATU, RET, LAS, AEX,
HCI, CCR, PZU, PUB). Sequence variations of the H8-L-H9 motif were
analyzed in parallel with the corresponding D-helix features (either
‘augmented’ or ‘non-augmented’) of tRNAGln and
tRNAGlu isoacceptors.
All ε-proteobacterial species considered were associated with aA -tRNAGlu, aN -tRNAGln1 isoacceptor and the complete absence
of the tRNAGln2 isoacceptor
(34CUG36) and (Figure 6A). For all
GluRS(T1), the length of the loop between H8 and H9 helices is
12-residues long while in all GluRS(T2) it is 13-residue long. The
12-residue long loop exhibited the ‘H(N)-G-D-Q(D)-E’ signature motif
(except HPY, which exhibits a ‘Y-Q-D-K-E’ motif) with a conserved Arg at
position 266 (Fig. 6A and Figure S6). We had already shown that the
YQDKE sequence stretch of HYP forms a dynamic
α-[>>t ] conformation, incompatible
with N -tRNAGln, where YQDK formed a Type II’
-turn. The central QD stretch in the -turn in HPY is replaced by an
equally turn-compatible GD stretch in all other ε-proteobacterial
GluRS(T1) proteins, and therefore we predict that, likeHpy -GluRS, all other ε-proteobacterial GluRS(T1) proteins
considered here will beN -tRNAGln-discriminatory. On the other hand
since structural comparison showed that a 13-residue long loop, without
the HGD(Q/A) motif at the center, gave rise to the
[<<t ]-H8-L -H9 motif, compatible
with A -tRNAGlx, we also predict that if
H8-L -H9 motif is the sole basis for
tRNAGlx-specificity, then GluRS(T2) will charge bothN -tRNAGln1 andA -tRNAGlu.
Unlike ε-proteobacterial species, the seven γ-proteobacterial genomes
considered here possess two tRNAGln isoacceptors
(Figure 6B and Figure S7): N -tRNAGln1(34UUG36) andA -tRNAGln2(34CUG36). All H8-L -H9 motifs
of GluRS(T1) displayed a 12-residue long loop with a central sequence
signature HGDQE (and Arg266), implying an
α-[>>t ] conformation compatible
with A - but not N -tRNAGlx. The only
exception is TGR (Thioalkalivibrio sulfidiphilus ) that was an
outlier and appeared in cluster B in Figure 3B; it displays the YEVDG
sequence, compatible with a [<<t ]
conformation (compatible with both A - andN -tRNAGlx). Canonical
[>>t ] conformation-competent
sequence motifs were also absent in all GluRS(T2), indicating that these
also adopt the [<<t ] conformation.
Therefore, our hypothesis predicts all GluRS(T2) andTgr -GluRS(T1) to be tRNAGln-non-discriminatory
while all other GluRS(T1) to beN -tRNAGlx-discriminatory (X = Glu/Gln). This is
consistent with experimental results (11) on AFE (A.
ferrooxidans ) where it was shown that GluRS(T1) discriminates againstN -tRNAGln1(34UUG36) while GluRS(T2) is
non-discriminatory.
The genomes of all four α-proteobacterial GluRSs(D+) considered here
contain two tRNAGln isoacceptors (Figure 6C and Figure
S8): N -tRNAGln1 andA -tRNAGln2. All GluRSs also display the
α-[>>t ] conformation promoting
sequence motif HGDQE (and Arg266), implying that all are expected to
discriminate N -tRNAGln1 overA -tRNAGln2 orA -tRNAGlu.
Among the genomes of six (SME, ATU, RET, LAS, AEX, HCI)
α-proteobacterial GluRSs(ND) considered here, two (AEX and HCI) contain
two one and the rest contain two tRNAGln isoacceptors
(Figure 6D and Figure S9): N -tRNAGln1 andA -tRNAGln2, and,A -tRNAGlu. The GluRSs do not contain sequence
motifs compatible with the [>>t ]
conformation implying that all are expected to be non-discriminating
type. This is consistent with their functional requirement of compulsory
glutamylation of both tRNAGln and
tRNAGlu, in absence of GlnRS in their genomes.
The genomes of other three (CCR, PZU, PUB) α-proteobacterial GluRSs(ND)
considered here (Figure 6E and Figure S9) contain only one
tRNAGln isoacceptor
(A -tRNAGln1) andA -tRNAGlu. The GluRSs show the
α-[>>t ] conformation promoting
sequence motif HGDDE (CCR, PZU) or YQDKE (PUB) along with Arg266; all
three appear in “tRNAGln-discriminatory” clusters in
Figure 3B (CCR/PZU in cluster F and PUB in cluster E). This implies that
all are expected to discriminate N -tRNAGlx overA -tRNAGlx. AlthoughN -tRNAGlx discriminatory, since their genomes
do not contain N -tRNAGlx, essentially the
GluRSs are non-discriminatory, consistent with their functional
requirement.