Aminoglycoside interactions with RNAs and nucleases.

One of the major challenges in medicine today is the development of new antibiotics as well as effective antiviral agents. The well-known aminoglycosides interact and interfere with the function of several noncoding RNAs, among which ribosomal RNAs (rRNAs) are the best studied. Aminoglycosides are also known to interact with proteins such as ribonucleases. Here we review our current understanding of the interaction between aminoglycosides and RNA. Moreover, we discuss briefly mechanisms behind the inactivation of aminoglycosides, a major concern due to the increasing appearance of multiresistant bacterial strains. Taken together, the general knowledge about aminoglycoside and RNA interaction is of utmost importance in the process of identifying/developing the next generation or new classes of antibiotics. In this perspective, previously unrecognized as well as known noncoding RNAs, apart from rRNA, are promising targets to explore.

Monitoring the structure of Escherichia coli RNase P RNA in the presence of various divalent metal ions.

Lead(II)-induced cleavage can be used as a tool to probe conformational changes in RNA. In this report, we have investigated the conformation of M1 RNA, the catalytic subunit of Escherichia coli RNase P, by studying the lead(II)-induced cleavage pattern in the presence of various divalent metal ions. Our data suggest that the overall conformation of M1 RNA is very similar in the presence of Mg(2+), Mn(2+), Ca(2+), Sr(2+) and Ba(2+), while it is changed compared to the Mg(2+)-induced conformation in the presence of other divalent metal ions, Cd(2+) for example. We also observed that correct folding of some M1 RNA domains is promoted by Pb(2+), while folding of other domain(s) requires the additional presence of other divalent metal ions, cobalt(III) hexamine or spermidine. Based on the suppression of Pb(2+) cleavage at increasing concentrations of various divalent metal ions, our findings suggest that different divalent metal ions bind with different affinities to M1 RNA as well as to an RNase P hairpin-loop substrate and yeast tRNA(Phe). We suggest that this approach can be used to obtain information about the relative binding strength for different divalent metal ions to RNA in general, as well as to specific RNA divalent metal ion binding sites. Of those studied in this report, Mn(2+) is generally among the strongest RNA binders.

Several regions of a tRNA precursor determine the Escherichia coli RNase P cleavage site.

The RNase P cleavage reaction was studied as a function of the number of base-pairs in the acceptor-stem and/or T-stem of a natural tRNA precursor, the tRNA(Tyr)Su3 precursor. Our data suggest that the location of the Escherichia coli RNase P cleavage site does not depend merely on the lengths of the acceptor-stem and T-stem as previously suggested. Surprisingly, we find that precursors with only four base-pairs in the acceptor-stem are cleaved by M1 RNA and by holoenzyme. Furthermore, we show that both disruption of base-pairing, and alteration of the nucleotide sequence (without disruption of base-pairing) proximal to the cleavage site result in aberrant cleavage. Thus, the identity of the nucleotides near the cleavage site is important for recognition of the cleavage site rather than base-pairing. The important nucleotides are those at positions -2, -1, +1, +72, +73 and +74. We propose that the nucleotide at position +1 functions as a guiding nucleotide. These results raise the possibility that Mg2+ binding near the cleavage site is dependent on the identity of the nucleotides at these positions. In addition, we show that disruption of base-pairing in the acceptor-stem affects both Michaelis-Menten constants, Km and kcat.

Reaction in vitro of some mutants of RNase P with wild-type and temperature-sensitive substrates.

The reaction of wild-type and two mutant derivatives of RNase P have been examined with wild-type and mutant substrates. We show that a mutant derivative of tRNA(Tyr)Su3, tRNA(Tyr)Su3A15, in which the G15.C48(57) base-pair essential for folding of the tRNA moiety is altered, is a temperature-sensitive suppressor in vivo. The precursor to tRNA(Tyr)Su3A15 is cleaved in a temperature-sensitive manner in vitro by RNase P and with a higher Km compared to the precursor to tRNA(Tyr)Su3. The precursor to tRNA(Tyr)Su3A2, another temperature-sensitive suppressor in vivo in which the G2.C71(80) base-pair in the acceptor stem is changed to A2.C71(80), behaves like the precursor to tRNA(Tyr)Su3 in vitro; that is, it is not cleaved in a temperature-sensitive manner. Therefore, there are at least two ways in which a suppressor tRNA can acquire a temperature-sensitive phenotype in vivo. One of the mutant derivatives of RNase P we have tested, rnpA49, which affects the protein cofactor of the enzyme, has a decreased kcat compared to wild-type, which can explain its phenotype in vivo.

Cleavage site selection by M1 RNA the catalytic subunit of Escherichia coli RNase P, is influenced by pH.

We have studied cleavage site selection by M1 RNA, the catalytic subunit of Escherichia coli RNase P, under various reaction conditions using tRNA precursors which are cleaved at two positions. Our results showed that the preference of cleavage site changed with variations in pH or Mg2+ concentration. By contrast, no difference in cleavage site selection was observed with increasing pH in the presence of Ca2+ as the only divalent metal ion. Depending on the identity of the nucleotide at position "+ 72", replacement of Mg2+ with Ca2+ resulted in a change of the main cleavage site irrespective of pH. We conclude that cleavage in the presence of Ca2+ compared to cleavage in the presence of Mg2+ has different structural requirements at and near the cleavage site. UV cross-linking revealed that close points between M1 RNA and its substrate were the same irrespective of pH or the identity of the divalent cation. Our results also showed that the observed pH effects are due to changes in the catalytic cleavage rates rather than to global, structural rearrangements. These data are discussed in terms of metal ion binding near the cleavage sites in the enzyme-substrate complex.

Manganese ions induce miscleavage in the Escherichia coli RNase P RNA-catalyzed reaction.

Cleavage by the endoribonuclease RNase P requires the presence of divalent metal ions, of which Mg2+ promotes most efficient cleavage. Here we have studied the importance of there being Mg2+ in RNase P RNA catalysis. It is demonstrated that addition of Mn2+ resulted in a shift of the cleavage site and that this shift was associated with a change in the kinetic constants, in particular kcat. Our data further suggest that the influence of Mn2+ on cleavage site recognition depends on the -1/+73 base-pair in the substrate and the +73/294 base-pair in the RNase P RNA-substrate (RS)-complex. Based on our data we suggest that cleavage in the presence of Mg2+ as the only divalent metal ion proceeds through an intermediate which involves the establishment of the +73/294 base-pair in the RS-complex. By contrast, addition of Mn2+ favours an alternative pathway which results in a shift of the cleavage site. We also studied the influence of Mn2+ on cleavage site recognition and the kinetics of cleavage using various RNase P RNA derivatives carrying substitutions in the region of RNase P RNA that base-pair with the 3' terminal end of the substrate. From these results we conclude that a change in the structure of this RNase P RNA domain influences the involvement of a divalent metal ion(s) in the chemistry of cleavage.

Identification of a region within M1 RNA of Escherichia coli RNase P important for the location of the cleavage site on a wild-type tRNA precursor.

To investigate the function of the catalytic subunit of Escherichia coli RNase P, M1 RNA, we studied cleavage by different M1 RNA mutants of wild-type precursors to tRNA(TyrSu3), tRNA(His) and tRNA(SerSu1). We showed that deletion or substitution of the conserved nucleotides G291, G292, U294 and A295 in M1 RNA resulted in a shift of the cleavage site for the tRNA(SerSu1) precursor, whereas the other two tRNA precursors were cleaved at the normal position. By using chimeric tRNA precursors in which the acceptor-stem of the tRNA(TyrSu3) precursor was replaced by the acceptor-stem derived from the tRNA(SerSu1) precursor, we showed that the aberrant cleavage by M1 RNA mutants could be reversed by substituting the nucleotide at position -2 in one of the chimeric precursors. These results suggest, in support of our previous findings, that different tRNA precursors are processed differently and that the primary structure of the amino acid acceptor-stem of a tRNA precursor plays a significant role in the RNase P cleavage reaction. Furthermore, in agreement with a previous report, a truncated tRNA(TyrSu3) precursor was cleaved aberrantly by a mutant M1 RNA in which the nucleotide at position 92 had been deleted. In contrast, a corresponding truncated tRNA(SerSu1) precursor was cleaved at the same position both by the wild-type and by this mutant M1 RNA. We conclude that not only the primary structures of the acceptor-stems of tRNA precursors, but also the primary structures in different regions of M1 RNA determine the location of the cleavage site on various tRNA precursors. Here we have identified the region G291 to A295 to be important for the selection of the cleavage site on the tRNA(SerSu1) precursor. We discuss the possibility that the conformation of M1 RNA in the enzyme-substrate complex is dependent on the identity of the substrate.

Residues in Escherichia coli RNase P RNA important for cleavage site selection and divalent metal ion binding.

We have used genetics as a tool to study the importance of an internal loop (P7) of Escherichia coli RNase P RNA (M1 RNA) in cleavage site selection and the binding of a divalent metal ion(s). The preferred cleavage site on a model tRNA precursor substrate shifted as a result of base-substitutions and deletions within this loop, in particular when changes were introduced at positions directly involved in base-pairing with the 3'-terminal RCCA motif of the substrate. Additionally, these changes in M1 RNA resulted in alterations in the binding of a divalent metal ion(s) in the vicinity of this internal loop as revealed by lead(II)-induced cleavage. From these data we conclude that the structural integrity of the P7 loop is important for both cleavage site selection and divalent metal ion binding. Cross-linking experiments using precursors carrying a 4-thioU immediately 5' of two independent cleavage sites suggest that close contact points between M1 RNA and nucleotides at these cleavage sites depend on the interaction between M1 RNA and the 3'-terminal RCCA motif of the substrate. Our findings further support the view that there are at least two different ways for the tRNA domain of a tRNA precursor to interact with M1 RNA. These results support a model in which base-pairing between M1 RNA and its substrate results in a re-coordination of a divalent metal ion(s) such that cleavage at the correct position is accomplished.

Differential effects of mutations in the protein and RNA moieties of RNase P on the efficiency of suppression by various tRNA suppressors.

We have studied the efficiency of suppression by tRNA suppressors in vivo in strains of Escherichia coli that harbor a mutation in the rnpA gene, the gene for the protein component (C5) of RNase P, and in strains that carry several different alleles of the rnpB gene, the gene for the RNA component (M1) of RNase P. Depending on the genetic background, different efficiencies of suppression by the various tRNA suppressors were observed. Thus, mutations in rnpA have separable and distinct effects from mutations in rnpB on the processing of tRNA precursors by RNase P. In addition, the efficiency of suppression by several derivatives of E. coli tRNA(Tyr) Su3 changed as the genetic background was altered.

Growth rate regulation of 4.5 S RNA and M1 RNA the catalytic subunit of Escherichia coli RNase P.

We have studied the expression of 4.5 S RNA and M1 RNA, the catalytic subunit of Escherchia coli RNase P, under various growth conditions. Both RNA species increase in abundance as a function of growth rate. There are roughly 450 molecules of 4.5 S RNA and 80 molecules of M1 RNA per cell at 0.4 doubling per hour, and this is increased to 5300 and 1060 molecules per cell, respectively, at 2.7 doublings per hour. Deletion of both relA and spoT, the two genes that are responsible for synthesis of ppGpp, does not affect the rate of synthesis of either RNA species. However, deletion of fis renders the expression of 4.5 S RNA independent of growth rate, but has little effect on the expression of M1 RNA. These data suggest that the expression of both 4.5 S RNA and M1 RNA genes are growth-rate regulated, but not through the same mechanism. The growth-rate dependent accumulation of 4.5 S RNA depends on FIS-mediated trans-activation, whereas that of M1 RNA is not governed by ppGpp or by FIS.

RNase P RNA structure and cleavage reflect the primary structure of tRNA genes.

The function of RNase P RNA depends on its folding in space. A majority of RNase P RNAs from various bacteria show a similar secondary structure to that of Escherichia coli (M1 RNA). However, there are exceptions as exemplified by the RNase P RNA derived from the low GC-content Gram-positive bacteria Bacillus subtilis and Mycoplasma hyopneumoniae (Hyo P RNA). Previous studies using M1 RNA and Hyo P RNA suggest differences both with respect to the kinetics of cleavage as well as to cleavage site recognition. Here we have studied cleavage by these two structurally different RNase P RNAs as a function of changes in the 5' leader and the 3'-terminal CCA motif in the substrate. Our data suggest that the nucleotide at the -2 position in the 5' leader plays a role both for cleavage site recognition and for the rate of cleavage. However, depending on the identity of the -2 residue differences in the cleavage pattern comparing these two types of RNase P RNAs were observed. The results also suggest that the identity of the -1/+73 base-pair in the substrate influences the cleavage site recognition process. These findings will be related to differences in structure comparing these types of RNase P RNAs and the "RCCA-RNase P RNA" interaction. In addition, our findings will be discussed with respect to the primary structure of the tRNA genes in different bacteria.

Genetic implication for an interaction between release factor one and ribosomal protein L7/L12 in vivo.

Strains with mutant alleles of the genes encoding release factor one (RF1), prfA, and ribosomal protein L7/L12, rplL, have been analyzed. The prfA1 allele has previously been shown to be associated with temperature sensitivity for growth at 43 degrees C and increased misreading of the nonsense codons UAG and UAA. Here we show that the prfA1 mutation is a nucleotide substitution that results in an arginine to proline change at amino acid position 137 in RF1. This alters the factor in a domain which may be involved in ribosome-binding. The rplL564 allele codes for a mutant form of protein L7/L12 that has a change in its conserved, translation-factor binding, domain. The rplL564 mutation suppresses the temperature sensitive phenotype and the increased translational misreading associated with the prfA1 allele, while other studied rplL mutant alleles do not. These data are consistent with an interaction between L7/L12 and RF1 in vivo.

Characterization of the Borrelia burgdorferi RNase P RNA gene reveals a novel tertiary interaction.

Characterization of the RNase P RNA gene derived from Borrelia burgdorferi reveals covariation of the conserved nucleotides at positions corresponding to nucleotides 128 and 230 in Escherichia coli RNase P RNA (M1 RNA). Single base substitutions at either of these positions in M1 RNA resulted in a lack of complementation of the temperature-sensitive phenotype associated with rnpA49 in vivo whereas complementation was observed for the double mutant M1 RNA or wild-type M1 RNA. Our in vitro data showed that M1 RNA harbouring a substitution at 128 or 230 cleaved a tRNA precursor both in the absence and presence of C5 with reduced efficiency compared to the wild-type and the double mutant M1 RNA. We conclude that the nucleotides at positions 128 and 230 establish a long-range tertiary interaction in RNase P RNA. Our data also suggest that this interaction together with the identity of the nucleotide at position 230 is important for Pb2+ induced cleavage at specific positions in M1 RNA.

Differential role of the intermolecular base-pairs G292-C(75) and G293-C(74) in the reaction catalyzed by Escherichia coli RNase P RNA.

We present a systematic investigation of the thermodynamic and kinetic role of the intermolecular G292-C(75 )and G293-C(74 )Watson-Crick base-pairs in the reaction catalyzed by Escherichia coli RNase P RNA. Single turnover kinetics were analyzed for wild-type RNase P RNA and two variants with a single G to C exchange (C292 or C293), either acting on wild-type precursor tRNA (ptRNA) or derivatives carrying a complementary change at the tRNA 3'-end (G(74)CA or CG(75)A). Ground state binding of tRNA was studied using three different methods, including a novel fluorescence-based assay measuring equilibrium binding. We conclude that: (1) the role of the G293-C(74 )interaction is essentially confined to Watson-Crick base-pairing, with no indication for crucial tertiary contacts involving this base-pair; (2) the G293-C(74 )pair, although being as important for ptRNA ground state binding as G292-C(75), is much less crucial to catalytic performance than the G292-C(75) pair; (3) disruption of the G292-C(75 )base-pair results in preferential destabilization of enzyme transition-state complexes; and (4) the identity of the G292-C(75) pair, as part of the higher-order structural context consisting of coplanar G292-C(75)-A258 and G291-G259-A(76 )triples, contributes to high affinity binding of ptRNA and catalytic efficiency.

Catalysis by the RNA subunit of RNase P--a minireview.

RNase P, an enzyme that contains both RNA and protein components, cleaves tRNA precursors to generate mature 5' termini. The catalytic activity of RNase P resides in the RNA component, with the protein cofactor affecting the rate of the cleavage reaction. The reaction is also influenced by the nature of the tRNA substrate.

Mutations in ribosomal proteins L7/L12 perturb EF-G and EF-Tu functions.

In vitro cycling rates of E. coli ribosomes and of elongation factors EF-Tu and EF-G have been obtained and these are compatible with translation rates in vivo. We show that the rate of translocation is faster than 50 s-1 and therefore that the EF-G function is not a rate limiting step in protein synthesis. The in vivo phenotype of some L7/L12 mutants could be accounted for by perturbed EF-Tu as well as EF-G functions. The S12 mutants that we studied were, in contrast, only perturbed in their EF-Tu function, while their EF-G interaction was not impaired in relation to wild type ribosomes.

RNase P RNA-mediated catalysis.

The endoribonuclease RNase P is involved in the processing of tRNA precursors to generate mature 5' termini. The catalytic activity of RNase P is associated with an RNA, RNase P RNA. A specific interaction between the 3' end of the substrate and RNase P RNA, to form an RNase P RNA-substrate complex, is referred to as the '73-294-interaction'. This interaction has an important role for efficient and correct cleavage to occur. Here our understanding of the contribution of the 73-294-interaction and metal ions, with respect to efficient and correct cleavage in RNase P RNA-mediated catalysis, will be discussed.

RNase P--a 'Scarlet Pimpernel'.

RNase P is responsible for the maturation of the 5'-termini of tRNA molecules in all cells studied to date. This ribonucleoprotein has to recognize and identify its cleavage site on a large number of different precursors. This review covers what is currently known about the function of the catalytic subunit of Escherichia coli RNase P, M1 RNA, and the protein subunit, C5, in particular with respect to cleavage-site selection. Recent genetic and biochemical data show that the two C residues in the 3'-terminal CCA sequence of a precursor interact with the enzyme through Watson-Crick base-pairing. This is suggested to result in unfolding of the amino acid acceptor-stem and exposure of the cleavage site. Furthermore, other close contact points between M1 RNA and its substrate have recently been identified. These data, together with the two existing three-dimensional structure models of M1 RNA in complex with its substrate, establish a platform that will enable us to seek an understanding of the underlying mechanism of cleavage by this elusive enzyme.

Involvement of ribosomal protein L7/L12 in control of translational accuracy.

The effects of two mutations, which map at the rplL locus and both give a changed 50S ribosomal protein L7/L12, were studied. Both mutations are associated with an increased misreading of all three nonsense codons in vivo and ribosomes from the mutants give an increased misreading of the phenylalanine codon UUU by tRNALeu in vitro. The rplL-associated misreading in vitro is not limited to a particular type of mRNA or tRNA. Results from a translational proofreading assay, using mutant ribosomes, suggest that protein L7/L12 is involved in the control of translational accuracy by contributing to the efficiency of a translational proofreading step(s).