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.     

A site in a tRNA precursor that can be processed by the whole RNase P enzyme but not by the RNA alone

A precursor molecule for 10 Sb RNA, the RNA moiety of the RNA processing enzyme RNase P, was purified, characterized for enzymatic activity, and compared to 10 Sb RNA and to RNase P. In these studies the K RNA, a dimeric precursor of tRNAGln-tRNALeu, coded by bacteriophage T4, was used as a substrate. This precursor contains two RNase P cleavage sites, one at each 5' end of the two tRNAs. The precursor 10 Sb and 10 Sb RNAs have the capacity to cleave the precursor tRNA molecule but only at the 5' end of tRNALeu, not at the 5' end of tRNAGln. Even when a substrate was prepared that contained only one site for RNase P (the one next to tRNAGln), this substrate was not cleaved by the RNA alone while the whole enzyme was effective in processing this substrate. The possible function of the protein of RNase P in the enzymatic reaction is discussed.     

Structural requirements for processing of synthetic tRNAHis precursors by the catalytic RNA component of RNase P

Experiments were conducted to investigate structural features of the aminoacyl stem region of precursor histidine tRNA critical for the proper cleavage by the catalytic RNA component of RNase P that is responsible for 5' maturation. Histidine tRNA was chosen for study because tRNAHis has an 8 base pair instead of the typical 7-base pair aminoacyl stem. The importance of the 3' proximal CCA sequence in the 5'-processing reaction was also investigated. Our results show that the tRNAHis precursor patterned after the natural Bacillus subtilis gene is cleaved by catalytic RNAs from B. subtilis or Escherichia coli, leaving an extra G residue at the 5'-end of the aminoacyl stem. Replacing the 3' proximal CCA sequence in the substrate still allowed the catalytic RNA to cleave at the proper position, but it increased the Km of the reaction. Changing the sequence of the 3' leader region to increase the length of the aminoacyl stem did not alter the cleavage site but reduced the reaction rate. However, replacing the G residue at the expected 5' mature end by an A changed the processing site, resulting in the creation of a 7-base pair aminoacyl stem. The Km of this reaction was not substantially altered. These experiments indicate that the extra 5' G residue in B. subtilis tRNAHis is left on by RNase P processing because of the precursor's structure at the aminoacyl stem and that the cleavage site can be altered by a single base change. We have also shown that the catalytic RNA alone from either B. subtilis or E. coli is capable of cleaving a precursor tRNA in which the 3' proximal CCA sequence is replaced by other nucleotides.     

Cleavage of T4 species I ribonucleic acid by Escherichia coli ribonuclease III.

T4 Species I RNA, a molecule 140 nucleotides in length with some structural features very much like a tRNA, is specifically cleaved by an enzymatic activity in Escherichia coli extracts to give three segments with 19, 48 and 73 nucleotides. We report the purification and characterization of the E. coli RNase which cleaves two 3' phosphodiester bonds of T4 Species I RNA. This reaction has many properties in common with those catalyzed by E. coli RNase III, although the optimal salt conditions for T4 Species I RNA cleavage differ significantly from those for other RNase III-catalyzed reactions. The reaction is not catalyzed by extracts from an E. coli strain lacking RNase III activity. Furthermore, T4 Species I RNA is cleaved by highly purified E. coli RNase III to yield the same three specific fragments. We conclude that this specific cleavage is due to the action of RNase III, and that the requirement for lower ionic strength may reveal further important properties about this RNA processing enzyme.     

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.     

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.     

Recognition of the 5' leader of pre-tRNA substrates by the active site of ribonuclease P

The bacterial tRNA processing enzyme ribonuclease P (RNase P) is a ribonucleoprotein composed of a approximately 400 nucleotide RNA and a smaller protein subunit. It has been established that RNase P RNA contacts the mature tRNA portion of pre-tRNA substrates, whereas RNase P protein interacts with the 5' leader sequence. However, specific interactions with substrate nucleotides flanking the cleavage site have not previously been defined. Here we provide evidence for an interaction between a conserved adenosine, A248 in the Escherichia coli ribozyme, and N(-1), the substrate nucleotide immediately 5' of the cleavage site. Specifically, mutations at A248 result in miscleavage of substrates containing a 2' deoxy modification at N(-1). Compensatory mutations at N(-1) restore correct cleavage in both the RNA-alone and holoenzyme reactions, and also rescue defects in binding thermodynamics caused by A248 mutation. Analysis of pre-tRNA leader sequences in Bacteria and Archaea reveals a conserved preference for U at N(-1), suggesting that an interaction between A248 and N(-1) is common among RNase P enzymes. These results provide the first direct evidence for RNase P RNA interactions with the substrate cleavage site, and show that RNA and protein cooperate in leader sequence recognition.     

Eukaryotic RNase P: role of RNA and protein subunits of a primordial catalytic ribonucleoprotein in RNA-based catalysis

Ribonuclease P (RNase P) is an essential enzyme that processes the 5' leader sequence of precursor tRNA. Eubacterial RNase P is an RNA enzyme, while its eukaryotic counterpart acts as catalytic ribonucleoprotein, consisting of RNA and numerous protein subunits. To study the latter form, we reconstitute human RNase P activity, demonstrating that the subunits H1 RNA, Rpp21, and Rpp29 are sufficient for 5' cleavage of precursor tRNA. The reconstituted RNase P precisely delineates its cleavage sites in various substrates and hydrolyzes the phosphodiester bond. Rpp21 and Rpp29 facilitate catalysis by H1 RNA, which seems to require a phylogenetically conserved pseudoknot structure for function. Unexpectedly, Rpp29 forms a catalytic complex with M1 RNA of E. coli RNase P. The results uncover the core components of eukaryotic RNase P, reveal its evolutionary origin in translation, and provide a paradigm for studying RNA-based catalysis by other nuclear and nucleolar ribonucleoprotein enzymes.     

The 5' leader of precursor tRNAAsp bound to the Bacillus subtilis RNase P holoenzyme has an extended conformation

RNase P catalyzes the 5' maturation of transfer RNA (tRNA). RNase P from Bacillus subtilis comprises a large RNA component (130 kDa, P RNA) and a small protein subunit (14 kDa, P protein). Although P RNA alone can efficiently catalyze the maturation reaction in vitro, P protein is strictly required under physiological conditions. We have used time-resolved fluorescence resonance energy transfer on a series of donor-labeled substrates and two acceptor-labeled P proteins to determine the conformation of the pre-tRNA 5' leader relative to the protein in the holoenzyme-pre-tRNA complex. The resulting distance distribution measurements indicate that the leader binds to the holoenzyme in an extended conformation between nucleotides 3 and 7. The conformational mobility of nucleotides 5-8 in the leader is reduced, providing further evidence that these nucleotides interact with the holoenzyme. The increased fluorescence intensity and lifetime of the 5'-fluorescein label of these leaders indicate a more hydrophobic environment, consistent with the notion that such interactions occur with the central cleft of the P protein. Taken together, our data support a model where the P protein binds to the 5' leader between the fourth and seventh nucleotides upstream of the cleavage site, extending the leader and decreasing its structural dynamics. Thus, P protein acts as a wedge to separate the 5' from the 3' terminus of the pre-tRNA and to position the cleavage site in the catalytic core. These results reveal a structural basis for the P protein dependent discrimination between precursor and mature tRNAs.     

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.     

Non-ribosomal nucleotide sequences in 7-S RNA, the immediate precursor of 5.8-S ribosomal RNA in yeast.

The topography and the length of the non-ribosomal sequences present in 7-S RNA, the immediate precursor of 5.8-S ribosomal RNA, from the yeast Saccharomyces carlsbergensis were determined by analyzing the nucleotide sequences of the products obtained after complete digestion of 7-S RNA with RNase T1. The results show that 7-S RNA contains approximately 150 non-ribosomal nucleotides. The majority (90%) of the 7-S RNA molecules was found to have the same 5'-terminal pentadecanucleotide sequence as mature 5.8-S rRNA. The remaining 10% exhibited 5'-terminal sequences identical to those of 5.9-S RNA, which has the same primary structure as 5.8-S rRNA except for a slight extension at the 5' end [Rubin, G.M. (1974) Eur. J. Biochem. 41, 197--202]. These data show that the non-ribosomal nucleotides present in 7-S RNA are all located 3'-distal to the mature 5.8-S rRNA sequence. Moreover, it can be concluded that 5.9-S RNA is a stable rRNA rather than a precursor of 5.8-S rRNA. The 3'-terminal sequence of 5.8-S rRNA (U-C-A-U-U-UOH) is recovered in a much longer oligonucleotide in the T1 RNase digest of 7-S RNA having the sequence U-C-A-U-U-U-(C-C-U-U-C-U-C)-A-A-A-C-A-(U-U-C-U)-Gp. The sequences enclosed in brackets are likely to be correct but could not be established with absolute certainty. The arrow indicates the bond cleaved during processing. The octanucleotide sequence -A-A-A-C-A-U-U-C- located near the cleavage site shows a remarkable similarity to the 5'-terminal octanucleotide sequence of 7-S RNA (-A-A-A-C-U-U-U-C-). We suggest that these sequences may be involved in determining the specificity of the cleavages resulting in the formation of the two termini of 5.8-S rRNA.