Suppressor and novel mutants of bacteriophage T4 tRNA(Gly)

We have isolated a weak UGA suppressor of phage T4 tRNA(Gly) in which the anticodon is changed from UCC to UCA. Two secondary mutants lacking suppressor activity are atypical in accumulating tRNA(Gly). Both mutations change the T stem of the cloverleaf model. One involved a G to A change at the 5' base position of the middle base-pair; the second involves a C to U change at a constant base position next to the T loop. The precursor RNAs of the mutants were cleaved in vitro with the catalytic RNA subunit of RNase P. Relative to normal precursor RNA, the precursor mutated at the middle base-pair position of the T stem was cleaved more rapidly, whereas the precursor mutated at the base-pair position next to the T loop was cleaved more slowly.     

RNase E cleavage in the 5' leader of a tRNA precursor

In this study, we have used various tRNA(Tyr)Su3 precursor (pSu3) derivatives that are processed less efficiently by RNase P to investigate if the 5' leader is a target for RNase E. We present data that suggest that RNase E cleaves the 5' leader of pSu3 both in vivo and in vitro. The site of cleavage in the 5' leader corresponds to the cleavage site for a previously identified endonuclease activity referred to as RNase P2/O. Thus, our findings suggest that RNase P2/O and RNase E activities are of the same origin. These data are in keeping with the suggestion that the structure of the 5' leader influences tRNA expression by affecting tRNA processing and indicate the involvement of RNase E in the regulation of cellular tRNA levels.     

The kinetics and specificity of cleavage by RNase P is mainly dependent on the structure of the amino acid acceptor stem.

Cleavage by RNase P of the tRNA(His precursor yields a mature tRNA with an 8 base pair amino acid acceptor stem instead of the usual 7 base pair stem. Here we show, both in vivo and in vitro, that this is mainly dependent on the primary structure and length of the acceptor stem in the precursor. Furthermore, the tRNA(His) precursor used in this study was processed with a change in both kinetic constants, Km and kcat, in comparison to the kinetics of cleavage of the precursor to tRNA(Tyr)Su3. Cleavage of a chimeric tRNA precursor showed that these altered kinetics were due to a difference in the primary structure and in the length of the acceptor stems of these two tRNA precursors. We also studied the cleavage reaction as a function of base substitutions at positions -1 and/or +73 in the precursor to tRNA(His). Our results suggest that the nucleotide at position +73 in tRNA(His) plays a significant role in the kinetics of cleavage of its precursor, possibly in product release. In addition, it appears that the C5 protein of RNase P is involved in the interaction between the enzyme and its substrate in a substrate-dependent manner, as previously suggested.     

Interaction between Escherichia coli RNase P RNA and the discriminator base results in slow product release.

We suggested previously that a purine at the discriminator base position in a tRNA precursor interacts with the well-conserved U294 in M1 RNA, the catalytic subunit of Escherichia coli RNase P. Here we investigated this interaction and its influence on the kinetics of cleavage as well as on cleavage site selection. The discriminator base in precursors to tRNA(Tyr)Su3 and tRNA(Phe) was changed from A to C and cleavage kinetics were studied by wild-type M1 RNA and a mutant M1 RNA carrying the compensatory substitution of a U to a G at position 294 in M1 RNA. Our data suggest that the discriminator base interacts with the residue at position 294 in M1 RNA. Although product release is a rate-limiting step both in the absence and in the presence of this interaction, its presence results in a significant reduction in the rate of product release. In addition, we studied cleavage site selection on various tRNA(His) precursor derivatives. These precursors carry a C at the discriminator base position. The results showed that the mutant M1 RNA harboring a G at position 294 miscleaved a wild-type tRNA(His) precursor and a tRNA(His) precursor carrying an inosine at the cleavage site. The combined data suggest a functional interaction between the discriminator base and the well-conserved U294 in M1 RNA. This interaction is suggested to play an important role in determining the rate of product release during multiple turnover cleavage of tRNA precursors by M1 RNA as well as in cleavage site selection.     

Polarity effects in the lactose operon of Escherichia coli

An intergenic RNA segment between lacY and lacA of the lactose operon in Escherichia coli is cleaved by RNase P, an endoribonuclease. The cleavage of the intergenic RNA was ten times less efficient than cleavage of a tRNA precursor in vitro. Fragments of the RNase P cleavage product are detectable in vivo in the wild-type strain but not in a mutant strain at the restrictive temperature. The cleavage product that contains lacA in the wild-type strain was quickly degraded. When this intergenic segment was cloned upstream of a reporter gene, the expression of the reporter gene was also inhibited substantially in wild-type E.coli, but not in a temperature sensitive mutant strain in RNase P at the restrictive temperature. These results support data regarding the natural polarity between lacZ versus lacA, the downstream gene.     

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.     

The nucleotide sequence of a precursor to the glycine- and threonine-specific transfer ribonucleic acids of Escherichia coli.

The primary nucleotide sequence of an Escherichia coli tRNA precursor molecule has been determined. This precursor RNA, specified by the transducing phage lambdah80dglyTsuA36 thrT tyrT, accumulates in a mutant strain temperature-sensitive for RNase P activity. The 170-nucleotide precursor RNA is processed by E. coli extracts to form mature tRNA Gly 2 suA36 and tRNA Thr ACU/C. The sequence of the precursor is pG-U-U-C-C-A-G-G-A-U-G-C-G-G-G-C-A-U-C-G-U-A-U-A-A-U-G-G-C-U-A-U-U-A-C-C-U-C-A-G-C-C-U-N-C-U-A-A-G-C-U-G-A-U-G-A-U-G-C-G-G-G-T-psi-C-G-A-U-U-C-C-C-G-C-U-G-C-C-C-G-C-U-C-C-A-A-G-A-U-G-U-G-C-U-G-A-U-A-U-A-G-C-U-C-A-G-D-D-G-G-D-A-G-A-G-C-G-C-A-C-C-C-U-U-G-G-U-mt6A-A-G-G-G-U-G-A-G-m7G-U-C-G-G-C-A-G-T-psi-C-G-A-A-U-C-U-G-C-C-U-A-U-C-A-G-C-A-C-C-A-C-U-UOH(tRNA sequences are italicized). It contains the entire primary nucleotide sequences of tRNA Gly2 suA36 and tRNA Thr ACU/C, including the common 3'-terminal sequence, CCA. Nineteen additional nucleotides are present, with 10 at the 5' end, 3 at the 3' end, and the remaining 6 in the inter-tRNA spacer region. RNase P cleaves the precursor specifically at the 5' ends of the mature tRNA sequences.     

The precursor tRNA 3'-CCA interaction with Escherichia coli RNase P RNA is essential for catalysis by RNase P in vivo

The L15 region of Escherichia coli RNase P RNA forms two Watson-Crick base pairs with precursor tRNA 3'-CCA termini (G292-C75 and G293-C74). Here, we analyzed the phenotypes associated with disruption of the G292-C75 or G293-C74 pair in vivo. Mutant RNase P RNA alleles (rnpBC292 and rnpBC293) caused severe growth defects in the E. coli rnpB mutant strain DW2 and abolished growth in the newly constructed mutant strain BW, in which chromosomal rnpB expression strictly depended on the presence of arabinose. An isosteric C293-G74 base pair, but not a C292-G75 pair, fully restored catalytic performance in vivo, as shown for processing of precursor 4.5S RNA. This demonstrates that the base identity of G292, but not G293, contributes to the catalytic process in vivo. Activity assays with mutant RNase P holoenzymes assembled in vivo or in vitro revealed that the C292/293 mutations cause a severe functional defect at low Mg2+ concentrations (2 mM), which we infer to be on the level of catalytically important Mg2+ recruitment. At 4.5 mM Mg2+, activity of mutant relative to the wild-type holoenzyme, was decreased only about twofold, but 13- to 24-fold at 2 mM Mg2+. Moreover, our findings make it unlikely that the C292/293 phenotypes include significant contributions from defects in protein binding, substrate affinity, or RNA degradation. However, native PAGE experiments revealed nonidentical RNA folding equilibria for the wild-type versus mutant RNase P RNAs, in a buffer- and preincubation-dependent manner. Thus, we cannot exclude that altered folding of the mutant RNAs may have also contributed to their in vivo defect.     

Interplay among processing and degradative enzymes and a precursor ribonucleic acid in the selective maturation and maintenance of ribonucleic acid molecules

In order to understand why the first tRNA (tRNAGln) in the T4 tRNA gene cluster is not produced when T4 infects an RNase III- mutant of Escherichia coli, RNA metabolism was analyzed in RNase III- RNase P- (rnc, rnp) cells infected with bacteriophage T4. After such an infection a new dimeric precursor RNA molecule of tRNAGln and tRNALeu has been identified and analyzed. This molecule is structurally very similar to K band RNA that accumulates in rnc+ rnp strains. It is four nucleotides shorter than K RNA at the 5' end. This molecule like K RNA contains two RNase P processing sites at the 5' ends of each tRNA. Both sites are accessible to RNase P. However, while in the K RNA the site at the 5' end of tRNALeu (the site in the middle of the substrate) is more efficiently cleaved than the other site, this differential is even increased in the Ks (K like) molecule. This difference is sufficiently large that in vivo in the RNase III- strain the smaller precursor of tRNAGln is degraded rather than being matured to tRNAGln by RNase P. This information contributes to the elucidation of the key role of RNase III in the processing of T4 tRNA. It shows the dependence of RNase P activity at the 5' end of tRNAGln on a correct and specific cleavage by RNase III at a position six nucleotides proximal to the RNase P site, and it explains why in the absence of RNase III the first tRNA in the T4 tRNA cluster, tRNAGln, does not accumulate.     

Kinetics of the processing of the precursor to 4.5 S RNA, a naturally occurring substrate for RNase P from Escherichia coli.

A study was made of the cleavage by M1 RNA and RNase P of a non-tRNA precursor that can serve as a substrate for RNase P from Escherichia coli, namely, the precursor to 4.5 S RNA (p4.5S). The overall efficiency of cleavage of p4.5S by RNase P is similar to that of wild-type tRNA precursors. However, unlike the reaction with wild-type tRNA precursors, the reaction catalyzed by the holoenzyme with p4.5S as substrate has a much lower Km value than that catalyzed by M1 RNA with the same substrate, indicating that the protein subunit plays a crucial role in the recognition of p4.5S. A model hairpin substrate, based on the sequence of p4.5S, is cleaved with greater efficiency than the parent molecule. The 3'-terminal CCC sequence of p4.5 S may be as important for cleavage of this substrate as the 3'-terminal CCA sequence is for cleavage of tRNA precursors.     

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.     

Determinants of Escherichia coli RNase P cleavage site selection: a detailed in vitro and in vivo analysis.

The location of the Escherichia coli RNase P cleavage site was studied both in vitro and in vivo. We show that selection of the cleavage site is dependent on the nucleotide at the cleavage site and the length of the acceptor-stem. Within the acceptor-stem the number of nucleotides on the 5'-half of the acceptor-stem appears to be the important determinant, rather than the number of base pairs in the acceptor-stem. We also demonstrate that the length of the T-stem and a G to C substitution at position 57 in the tRNA(Tyr)Su3 precursor influence the location of the cleavage site under certain conditions. With respect to the function of the subunits of RNase P our data suggest that the nucleotide at position 333 in M1 RNA, and the C5 protein, are important for the identification of the cleavage site.     

Metal ion and substrate structure dependence of the processing of tRNA precursors by RNase P and M1 RNA

A synthetic tRNA precursor analog containing the structural elements of Escherichia coli tRNA(Phe) was characterized as a substrate for E. coli ribonuclease P and for M1 RNA, the catalytic RNA subunit. Processing of the synthetic precursor exhibited a Mg2+ dependence quite similar to that of natural tRNA precursors such as E. coli tRNA(Tyr) precursor. It was found that Sr2+, Ca2+, and Ba2+ ions promoted processing of the dimeric precursor at Mg2+ concentrations otherwise insufficient to support processing; very similar behavior was noted for E. coli tRNA(Tyr). As noted previously for natural tRNA precursors, the absence of the 3'-terminal CA sequence in the synthetic precursor diminished the facility of processing of this substrate by RNase P and M1 RNA. A study of the Mg2+ dependence of processing of the synthetic tRNA dimeric substrate radiolabeled between C75 and A76 provided unequivocal evidence for an alteration in the actual site of processing by E. coli RNase P as a function of Mg2+ concentration. This property was subsequently demonstrated to obtain (Carter, B. J., Vold, B.S., and Hecht, S. M. (1990) J. Biol. Chem. 265, 7100-7103) for a mutant Bacillus subtilis tRNAHis precursor containing a potential A-C base pair at the end of the acceptor stem.     

Phylogenetic comparative mutational analysis of the base-pairing between RNase P RNA and its substrate.

We have studied the base-pairing between the 3'-terminal CCA motif of a tRNA precursor and RNase P RNA by a phylogenetic mutational comparative approach. Thus, various derivatives of the Escherichia coli tRNA(Ser)Su1 precursor harboring all possible substitutions at either the first or the second C of the 3'-terminal CCA motif were generated. Cleavage site selection on these precursors was studied using mutant variants of M1 RNA, the catalytic subunit of E. coli RNase P, carrying changes at positions 292 or 293, which are involved in the interaction with the 3'-terminal CCA motif. From our data we conclude that these two C's in the substrate interact with the well-conserved G292 and G293 through canonical Watson-Crick base-pairing. Cleavage performed using reconstituted holoenzyme complexes suggests that this interaction also occurs in the presence of the C5 protein. Furthermore, we studied the interaction using various derivatives of RNase P RNAs from Mycoplasma hyopneumoniae and Mycobacterium tuberculosis. Our results suggest that the base-pairing between the 3'-terminal CCA motif and RNase P is present also in other bacterial RNase P-substrate complexes and is not limited to a particular bacterial species.     

Transfer RNA precursors are accumulated in Escherichia coli in the absence of RNase E

A temperature-sensitive Escherichia coli mutant, which contains a heat-labile RNase E, fails to produce 5-S rRNA at a non-permissive temperature. It accumulates a number of RNA molecules in the 4-12-S range. One of these molecules, a 9-S RNA, is a precursor to 5-S rRNA [Ghora, B. K. and Apirion, D. (1978) Cell, 15, 1055-1056]. These molecules were purified and processed in a cell-free system. Some of these RNA molecules, after processing, give rise to products the size of transfer RNA, but not to 5-S-rRNA. Further characterization of the processed products of one such precursor molecule shows that it contains tRNA1Leu and tRNA1His. RNase E is necessary but not sufficient for the processing of this molecule to mature tRNAs in vitro. The accumulation of such tRNA precursors in an RNase E mutant cell and the obligatory participation of RNase E in its processing indicate that RNase E functions in the maturation of transfer RNAs as well as of 5-S rRNA.     

RPM2, independently of its mitochondrial RNase P function, suppresses an ISP42 mutant defective in mitochondrial import and is essential for normal growth.

RPM2 is identified here as a high-copy suppressor of isp42-3, a temperature-sensitive mutant allele of the mitochondrial protein import channel component, Isp42p. RPM2 already has an established role as a protein component of yeast mitochondrial RNase P, a ribonucleoprotein enzyme required for the 5' processing of mitochondrial precursor tRNAs. A relationship between mitochondrial tRNA processing and protein import is not readily apparent, and, indeed, the two functions can be separated. Truncation mutants lacking detectable RNase P activity still suppress the isp42-3 growth defect. Moreover, RPM2 is required for normal fermentative yeast growth, even though mitochondrial RNase P activity is not. The portion of RPM2 required for normal growth and suppression of isp42-3 is the same. We conclude that RPM2 is a multifunctional gene. We find Rpm2p to be a soluble protein of the mitochondrial matrix and discuss models to explain its suppression of isp42-3.     

Processing of histidine transfer RNA precursors. Abnormal cleavage site for RNase P

The 5'-terminal guanylate residue (G-1) of mature Escherichia coli tRNA(His) is generated as a result of an unusual cleavage by RNase P (Orellana, O., Cooley, L., and S?ll, D. (1986) Mol. Cell. Biol. 6, 525-529). We have examined the importance of the unique acceptor stem structure of E. coli tRNA(His) in determining the specificity of RNase P cleavage. Mutant tRNA(His) precursors bearing substitutions of the normal base G-1 or the opposing, potentially paired base, C73, can be cleaved at the +1 position, in contrast to wild-type precursors which are cut exclusively at the -1 position. These data indicate that the nature of the base at position -1 is of greater importance in determining the site of RNase P cleavage than potential base pairing between nucleotides -1 and 73. In addition, processing of the mutant precursors by M1-RNA or P RNA under conditions of ribozyme catalysis yields a higher proportion of +1-cleaved products in comparison to the reaction catalyzed by the RNase P holoenzyme. This lower sensitivity of the holoenzyme to alterations in acceptor stem structure suggests that the protein moiety of RNase P may play a role in determining the specificity of the reaction and implies that recognition of the substrate involves additional regions of the tRNA. We have also shown that the RNase P holoenzyme and tRNA(His) precursor of Saccharomyces cerevisiae, unlike their prokaryotic counterparts, do not possess these abilities to carry out this unusual reaction.