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Kinetics of the Interaction of Translation Factor SelB fromEscherichia coli with Guanosine Nucleotides and Selenocysteine Insertion Sequence RNA*

Open AccessPublished:July 07, 2000DOI:https://doi.org/10.1074/jbc.M002496200
      The kinetics of the interaction of GTP and GDP with SelB, the specific translation factor for the incorporation of selenocysteine into proteins, have been investigated using the stopped-flow method. Useful signals were obtained using intrinsic (i.e. tryptophan) fluorescence, the fluorescence of methylanthraniloyl derivatives of nucleotides, or fluorescence resonance energy transfer from tryptophan to the methylanthraniloyl group. The affinities of SelB for GTP (K d = 0.74 μm) and GDP (K d = 13.4 μm) were considerably lower than those of other translation factors. Of functional significance is the fact that the rate constant for GDP release from its complex with SelB (15 s1) is many orders of magnitude larger than for elongation factor Tu, explaining why a GDP/GTP exchange factor is not required for the action of SelB. In contrast, the rate of release of GTP is 2 orders of magnitude slower and not significantly faster than for elongation factor Tu. Using a fluorescently labeled 17-nucleotide RNA minihelix that represents a binding site for the protein and that is part of the fdhF selenocysteine insertion sequence element positioned immediately downstream of the UGA triplet coding for selenocysteine incorporation, the kinetics of the interaction were studied. The high affinity of the interaction (K d ∼ 1 nm) appeared to be increased even further when selenocysteyl-tRNASec was bound to SelB, but to be independent of the presence or nature of the guanosine nucleotide at the active site. These results suggest that the affinity of SelB for its RNA binding site is maximized when charged tRNA is bound and decreases to allow dissociation and reading of codons downstream of the selenocysteine codon after selenocysteine peptide bond formation.
      Sec
      selenocysteine
      DTT
      dithiothreitol
      Pipes
      piperazine-N,N′-bis(2-ethanesulfonic acid)
      mantdGT(D)P
      3′-methylanthraniloyl-2′-deoxy-GT(D)P
      mantGT(D)P
      2′(3′)-methylanthraniloyl-GT(D)P
      EF-Tu
      elongation factor Tu
      SECIS
      selenocysteine insertion sequence
      The specialized translation factor SelB is the key molecule for the specific incorporation of the amino acid selenocysteine into polypeptides. SelB binds guanine nucleotides; selenocysteyl-tRNASec 1; and a secondary structure of the mRNA, the SECIS element. In bacterial selenoprotein mRNAs, this SECIS element is located immediately downstream of the UGA codon directing selenocysteine insertion, whereas in Archaea and Eukarya, it is situated in the 3′-nontranslated region (for a review, see Ref.
      • Hüttenhofer A.
      • Böck A.
      ). Binding of SelB to the SECIS structure is mediated by an ∼17-kDa C-terminal domain of the protein (domain IVb) that maintains its binding capacity when separated from the rest of the molecule. Minimization of the SECIS element showed that a 17-nucleotide long minihelix (see Fig. 1) was still able to bind to SelB or its C-terminal domain and to promote selenocysteine incorporation in vivo. On the other hand, the N-terminal two-thirds of SelB, which display sequence similarity to elongation factor Tu, are still capable of binding selenocysteyl-tRNASec when separated from the mRNA binding domain (
      • Kromayer M.
      • Wilting R.
      • Tormay P.
      • Böck A.
      ,
      • Liu Z.
      • Reches M.
      • Groisman I.
      • Engelberg-Kulka H.
      ).
      Figure thumbnail gr1
      Figure 1Structures of fdhF andfdnG SECIS elements. Bases that are protected by SelB against chemical modification (
      • Hüttenhofer A.
      • Westhof E.
      • Böck A.
      ) are marked bytriangles. The shaded regions were synthesizedin vitro and used for the experiments reported here.
      The formation of the quaternary complex is essential for the decoding of the UGA codon with selenocysteine at the ribosome. It is assumed that within this complex, SelB attains a conformation rendering it compatible for interaction with the ribosome, which is required for triggering GTPase activity. It is further assumed that GTP hydrolysis changes the conformation such that the charged tRNA is released. Therefore, in this model, SelB has two functions: the first one is to discriminate between an UGA codon programmed for selenocysteine insertion by the SECIS element and an ordinary UGA stop codon that lacks the SECIS element (
      • Hüttenhofer A.
      • Böck A.
      ). Furthermore, in bacterial mRNAs coding for selenocysteine, SelB also tethers the tRNA to the ribosomal A site (
      • Hüttenhofer A.
      • Böck A.
      ,
      • Heider J.
      • Baron C.
      • Böck A.
      ).
      Characterization of the kinetics of the SelB interaction with its substrates and the conformational changes involved is therefore of paramount importance for understanding the decoding process. Equilibrium dialysis measurements of the interaction of the protein with guanine nucleotides had indicated that GDP binding is approximately an order of magnitude weaker that GTP binding (
      • Forchhammer K.
      • Leinfelder W.
      • Böck A.
      ). This could obviate the necessity for a guanine nucleotide release factor. It has also been observed that SelB forms a tighter complex with the SECIS element in the presence of selenocysteyl-tRNASec than in its absence (
      • Baron C.
      • Heider J.
      • Böck A.
      ). This study deals with a detailed analysis of the association and dissociation kinetics of guanine nucleotides with SelB alone and in the presence of the mRNA. Additionally, the kinetics of the interaction of SelB with SECIS elements and the influence of guanine nucleotides and selenocysteyl-tRNASec thereon were investigated.

      DISCUSSION

      The results presented show that SelB displays a relatively low affinity for GTP and GDP in binary complexes between the protein and the nucleotides. In contrast to EF-Tu, the GTP affinity is greater (by an order of magnitude) than the GDP affinity. This difference is mainly due to a dramatic difference in the GDP affinities in the two systems, with the GTP affinities being more similar. Although GDP is bound more weakly than GTP, its kinetics of interaction with SelB are considerably faster. Thus, the association rate constant is almost an order of magnitude faster for GDP than for GTP (cf. a factor of 3 for EF-Tu) (
      • Wagner A.
      • Simon I.
      • Sprinzl M.
      • Goody R.S.
      ). In terms of affinity, this effect is more than compensated for by the fact that GDP is released ∼2 orders of magnitude faster than GTP so that GTP binds more strongly than GDP.
      The weaker binding of GDP compared with GTP and, in particular, the high dissociation rate constant are consistent with the idea that an exchange factor for SelB is not required since the intrinsic GDP dissociation rate is of the same order of magnitude as the exchange factor-catalyzed dissociation rate with EF-Tu. Somewhat similar properties have been seen for the E. coli signal recognition particle receptor FtsY (
      • Moser C.
      • Mol O.
      • Goody R.S.
      • Sinning I.
      ), i.e. weak affinities for GTP and GDP and high exchange rates. However, in this case, GDP is bound more strongly than GTP and exchanges rapidly in comparison with EF-Tu or the Ras family of proteins, but more slowly than GTP.
      When the kinetic results obtained in this work are compared with those for other GTPases, the most striking difference observed is the rapid rate of spontaneous GDP dissociation. With the exception of FtsY, all other GTPases involved in signal transduction and regulation that have been characterized in detail with respect to their kinetic properties have slow (∼103s1 for EF-Tu) (
      • Wagner A.
      • Simon I.
      • Sprinzl M.
      • Goody R.S.
      ) or very slow (106 to 104s1 for Ras and Ras-related proteins) (
      • John J.
      • Sohmen R.
      • Feuerstein J.
      • Linke R.
      • Wittinghofer A.
      • Goody R.S.
      ,
      • Simon I.
      • Zerial M.
      • Goody R.S.
      ) GDP dissociation rates, which are accelerated dramatically by exchange factors. The same is also true for the heterotrimeric G-proteins; and in the case of GTPases involved in signal transduction, it is easy to understand why GDP release must occur in a manner that is dependent on activation or recruitment of an exchange factor, this process being dependent, in general, on primary activation events in the signal transduction pathway. For ribosomal elongation factors, the requirement for an exchange factor is more difficult to rationalize; and in the case described here, SelB appears to function without one, proving that the kinetic properties of the GTPase can be designed so that the principle already known from other elongation factors can work even if the GDP produced concomitantly with peptide bond formation is bound weakly to the factor and dissociates spontaneously, allowing rebinding of GTP and the start of another cycle.
      The experiments reported on the kinetics of the interaction of stem-loop constructs with SelB show that this is a high affinity interaction, with a K d value of ∼1.3 nm. The association rate constant approaches the diffusion-controlled limit, and dissociation in the absence of tRNA occurs with a half-life of a few seconds. Neither the association nor the dissociation kinetics are affected by the presence or the nature of the guanosine nucleotide at the active site of the GTPase domain. Conversely, as is to be expected based on thermodynamic considerations, the presence of a stem-loop construct at its binding site does not affect the kinetics of the nucleotide interaction with SelB. Thus, there is no coupling between the nucleotide binding site and the RNA binding site, meaning that recruitment of SelB to its binding site on mRNA is not dependent on the state of the nucleotide binding site.
      Although we were not able to identify a usable spectroscopic signal for the interaction of selenocysteyl-tRNASec with SelB, there was a clear indication that the rate of dissociation from the stem-loop structure was slower when the charged tRNA was bound, suggesting an interaction between the two binding sites. The fact that a similar slowing down of the dissociation rate from mRNA was seen with the isolated domain IVb suggests that an interaction between the tRNA binding domain and the mRNA binding domain occurs in the absence of tRNA that is weakened or removed when tRNA is bound. Further studies with additional signals will be needed to clarify the nature of the interactions between the two different types of RNA binding sites. The possible functional significance of this effect is that the full affinity of SelB for its binding site on RNA is only realized when selenocysteyl-tRNASec is bound. Binding of the charged tRNA thus increases the stability of the SelB·GTP·selenocysteyl-tRNASec·mRNA quaternary complex and thereby favors the conformation of SelB able to interact with the ribosome. This interaction triggers GTP hydrolysis and most probably the release of the tRNA in the vicinity of the ribosomal A site (
      • Hüttenhofer A.
      • Böck A.
      ). As a consequence, there will be an increase in the rate of dissociation of SelB from its mRNA binding site. This is required for the translation of downstream codons since SelB complexed to its SECIS site exerts a considerable kinetic barrier to the processivity of translation (
      • Suppmann S.
      • Persson B.C.
      • Böck A.
      ).

      Acknowledgments

      We thank Andrea Beste for the synthesis of mant nucleotides.

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