A novel endonucleolytic mechanism to generate the CCA 3' termini of tRNA molecules in Thermotoga maritima

The tRNA 3'-terminal CCA sequence is essential for aminoacylation of the tRNAs and for translation on the ribosome. The tRNAs are transcribed as larger precursor molecules containing 5' and 3' extra sequences. In the tRNAs that do not have the encoded CCA, the 3' extra sequence after the discriminator nucleotide is usually cleaved off by the tRNA 3' processing endoribonuclease (3' tRNase, or RNase Z), and the 3'-terminal CCA residues are added thereto. Here we analyzed Thermotoga maritima 3' tRNase for enzymatic properties using various pre-tRNAs from T. maritima, in which all 46 tRNA genes encode CCA with only one exception. We found that the enzyme has the unprecedented activity that cleaves CCA-containing pre-tRNAs precisely after the CCA sequence, not after the discriminator. The assays for pre-tRNA variants suggest that the CA residues at nucleotides 75 and 76 are required for the enzyme to cleave pre-tRNAs after A at nucleotide 76 and that the cleavage occurs after nucleotide 75 if the sequence is not CA. Intriguingly, the pre-tRNA(Met) that is the only T. maritima pre-tRNA without the encoded CCA was cleaved after the discriminator. The kinetics data imply the existence of a CCA binding domain in T. maritima 3' tRNase. We also identified two amino acid residues critical for the cleavage site selection and several residues essential for the catalysis. Analysis of cleavage sites by 3' tRNases from another eubacteria Escherichia coli and two archaea Thermoplasma acidophilum and Pyrobaculum aerophilum corroborates the importance of the two amino acid residues for the cleavage site selection.     

Long 5' leaders inhibit removal of a 3' trailer from a precursor tRNA by mammalian tRNA 3' processing endoribonuclease.

Mammalian tRNA 3' processing endoribonuclease (3' tRNase) can remove a 3' trailer from various pre-tRNAs without 5' leader nucleotides. To examine how 5[prime] leader sequences affect 3' processing efficiency, we performed in vitro 3' processing reactions with purified pig 3' tRNase and pre-tRNAArgs containing a 13-nt 3' trailer and a 5[prime] leader of various lengths. The 3' processing was slightly stimulated by 5[prime] leaders containing up to 7 nt, whereas leaders of 9 nt or longer severely inhibited the reaction. Structure probing indicated that the 5' leader sequences had little effect on pre-tRNA folding. Similar results were obtained using pre-tRNA(Val)s containing a 5' leader of various lengths. We also investigated whether 3'tRNase can remove 3' trailers that are stably base-paired with 5' leaders to form an extended acceptor stem. Even such small 5' leaders as 3 and 6 nt, when base-paired with a 3' trailer, severely hindered removal of the 3' trailer by 3' tRNase.     

Specific cleavage of target RNAs from HIV-1 with 5' half tRNA by mammalian tRNA 3' processing endoribonuclease.

Mammalian tRNA 3' processing endoribonuclease (3' tRNase) can be converted to an RNA cutter that recognizes four bases, with about a 65-nt 3'-truncated tRNA(Arg) or tRNA(Ala). The 3'-truncated tRNA recognizes the target RNA via four base pairings between the 5'terminal sequence and a sequence 1-nt upstream of the cleavage site, resulting in a pre-tRNA-like complex (Nashimoto M, 1995, Nucleic Acids Res 23:3642-3647). Here I developed a general method for more specific RNA cleavage using 3' tRNase. In the presence of a 36-nt 5' half tRNA(Arg) truncated after the anticodon, 3' tRNase cleaved the remaining 56-nt 3' half tRNA(Arg) with a 19-nt 3' trailer after the discriminator. This enzyme also cleaved its derivatives with a 5' extra sequence or nucleotide changes or deletions in the T stem-loop and extra loop regions, although the cleavage efficiency decreases as the degree of structural change increases. This suggests that any target RNA can be cleaved site-specifically by 3'tRNase in the presence of a 5' half tRNA modified to form a pre-tRNA-like complex with the target. Using this method, two partial HIV-1 RNA targets were cleaved site-specifically in vitro. These results also indicate that the sequence and structure of the T stem-loop domain are important, but not essential, for the recognition of pre-tRNAs by 3' tRNase.     

Minimum requirements for substrates of mammalian tRNA 3' processing endoribonuclease.

Mammalian tRNA 3' processing endoribonuclease (3' tRNase) removes a 3' trailer after the discriminator nucleotide from precursor tRNA (pre-tRNA). To elucidate the minimum requirements for 3' tRNase substrates, we tested small pre-tRNA(Arg) substrates lacking the D and anticodon stem-loop domain for cleavage by purified pig 3' tRNase. A small pre-tRNA (R-ATW) composed of an acceptor stem, an extra loop, a T stem-loop domain, a discriminator nucleotide, and a 3' trailer was cleaved more efficiently than the full-length wild type. The catalytic efficiencies of three R-ATW derivatives, which were constructed to destroy the original T stem base pairs, were also higher than that of the full-length wild type. Pig 3' tRNase efficiently processed a "minihelix" (R-ATM5) that consists of a T stem-loop domain, an acceptor stem, a discriminator nucleotide, and a 3' trailer, while the enzyme never cleaved a "microhelix" that is composed of a T loop, an acceptor stem, a discriminator nucleotide, and a 3' trailer. Five R-ATM5 derivatives that have one to seven base substitutions in the T loop were all cleaved slightly more efficiently than the full-length wild type and slightly less efficiently than R-ATM5. A helix ("minihelixDelta1") one base pair smaller than minihelices was a good substrate, while small helices containing a continuous 10-base pair stem were poor substrates. The cleavage of these three small substrates occurred after the discriminator and one to three nucleotides downstream of the discriminator. From these results, we conclude that minimum substrates for efficient cleavage by mammalian 3' tRNase are minihelices or minihelicesDelta1, in which there seem to be no essential bases.     

Selection of cleavage site by mammalian tRNA 3' processing endoribonuclease

Mammalian tRNA 3' processing endoribonuclease (3' tRNase) removes 3' trailers from pre-tRNAs by cleaving the RNA immediately downstream of the discriminator nucleotide. Although 3' tRNase can recognize and cleave any target RNA that forms a pre-tRNA-like complex with another RNA, in some cases cleavage occurs at multiple sites near the discriminator. We investigated what features of pre-tRNA determine the cleavage site using various pre-tRNAArg variants and purified pig enzyme. Because the T stem-loop and the acceptor stem plus a 3' trailer are sufficient for recognition by 3' tRNase, we constructed variants that had additions and/or deletions of base-pairs in the T stem and/or the acceptor stem. Pre-tRNAs lacking one and two acceptor stem base-pairs were cleaved one and two nucleotides and two and three nucleotides, respectively, downstream of the discriminator. On the other hand, pre-tRNA variants containing extra acceptor stem base-pairs were cleaved only after the discriminator. The cleavage site was shifted to one and two nucleotides downstream of the discriminator by deleting one base-pair from the T stem, but was not changed by additional base-pairs in the T stem. Pre-tRNA variants that contained an eight base-pair acceptor stem plus a six base-pair T stem, an eight base-pair acceptor stem plus a four base-pair T stem, or a six base-pair acceptor stem plus a six base-pair T stem were all cleaved after the original nucleotide. In general, pre-tRNA variants containing a total of more than 11 bp in the acceptor stem and the T stem were cleaved only after the discriminator, and pre-tRNA variants with a total of N bp (N is less than 12) were cleaved 12-N and 13-N nt downstream of the discriminator. Cleavage efficiency of the variants decreased depending on the degree of structural changes from the authentic pre-tRNA. This suggests that the numbers of base-pairs of both the acceptor stem and the T stem are important for recognition and cleavage by 3' tRNase.     

Functional importance of conserved nucleotides at the histone RNA 3' processing site.

Histone pre-mRNA 3' processing is controlled by a hairpin element preceding the processing site that interacts with a hairpin-binding protein (HBP) and a downstream spacer element that serves as anchoring site for the U7 snRNP. In addition, the nucleotides following the hairpin and surrounding the processing site (ACCCA'CA) are conserved among vertebrate histone genes. Single to triple nucleotide mutations of this sequence were tested for their ability to be processed in nuclear extract from animal cells. Changing the first four nucleotides had no qualitative and little if any quantitative effects on histone RNA 3' processing in mouse K21 cell extract, where processing of this gene is virtually independent of the HBP. A gel mobility shift assay revealing HBP interactions and a processing assay in HeLa cell extract (where the contribution of HBP to efficient processing is more important) showed that only one of these mutations, predicted to extend the hairpin by one base pair, affected the interaction with HBP. Mutations in the next three nucleotides affected both the cleavage efficiency and the choice of processing sites. Analysis of these novel sites indicated a preference for the nucleotide 5' of the cleavage site in the order A > C > U > G. Moreover, a guanosine in the 3' position inhibited cleavage. The preference for an A is shared with the cleavage/polyadenylation reaction, but the preference order for the other nucleotides is different [Chen F, MacDonald CC, Wilusz J, 1995, Nucleic Acids Res 23:2614-2620].     

Sequence-dependent cleavage site selection by RNase Z from the cyanobacterium Synechocystis sp. PCC 6803

Biosynthesis of transfer RNA requires processing from longer precursors at the 5'- and 3'-ends. In eukaryotes, in archaea, and in those bacteria where the 3'-terminal CCA sequence is not encoded, 3' processing is carried out by the endonuclease RNase Z, which cleaves after the discriminator nucleotide to generate a mature 3'-end ready for the addition of the CCA sequence. We have identified and cloned the gene coding for RNase Z in the cyanobacterium Synechocystis sp. PCC 6803. The gene has been expressed in Escherichia coli, and the recombinant protein was purified. The enzymatic activity of RNase Z from Synechocystis has been studied in vitro with a variety of substrates. The presence of C or CC after the discriminator nucleotide modifies the cleavage site of RNase Z so that it is displaced by one and two nucleotides to the 3'-side, respectively. The presence of the complete 3'-terminal CCA sequence in the precursor of the tRNA completely inhibits RNase Z activity. The inactive CCA-containing precursor binds to Synechocystis RNase Z with similar affinity than the mature tRNA. The properties of the enzyme described here could be related with the mechanism by which CCA is added in this organism, with the participation of two separate nucleotidyl transferases, one specific for the addition of C and another for the addition of A. This work is the first characterization of RNase Z from a cyanobacterium, and the first from an organism with two separate nucleotidyl transferases.     

Cleavage site determinants in the mammalian polyadenylation signal.

Using a series of position and nucleotide variants of the SV40 late polyadenylation signal we have demonstrated that three sequence elements determine the precise site of 3-end cleavage in mammalian pre-mRNAs: an upstream AAUAAA element, a down-stream U-rich element consisting of five nucleotides, at least four of which are uridine, and a nucleotide preference at the site of cleavage in the order A > U > C >> G. Cleavage occurs no closer than 11 bases, but no further than 23 bases from the AAUAAA element. The downstream U-rich element is usually located 10-30 bases from the cleavage site. The relative position of the AAUAAA and the U-rich elements define the approximate region within a 13 base domain in which cleavage will occur. The exact position of cleavage is then determined by the local nucleotide sequence in the order of preference noted above. This model accounts for nearly three quarters of polyadenylation signals surveyed and is consistent with previous experimental observations.     

The C nucleotide at the mature 5′ end of the Escherichia coli proline tRNAs is required for the RNase E cleavage specificity at the 3′ terminus as well as functionality

Proline tRNA 3′-maturation in Escherichia coli occurs through a one-step RNase E endonucleolytic cleavage immediately after the CCA determinant. This processing pathway is distinct from the 3′-end maturation of the other tRNAs by avoiding the widespread use of 3′ → 5′ exonucleolytic processing, 3′-polyadenylation and subsequent degradation. Here, we show that the cytosine (C) at the mature 5′-terminus of the proK and proL tRNAs is required for both the RNase E cleavage immediately after the CCA determinant and their functionality. Thus, changing the C nucleotide at the mature 5′-terminus of the proL and proK tRNAs to the more common G nucleotide led to RNase E cleavages 1–4 nucleotides downstream of the CCA determinant. Furthermore, the 5′-modified mutant tRNAs required RNase T and RNase PH for their 3′-maturation and became substrates for polyadenylation and degradation. Strikingly, the aminoacylation of the 5′-modified proline tRNAs was blocked due to the change in the recognition element for prolyl-tRNA-synthetase. An analogous modification of the pheV 5′-mature terminus from G to C nucleotide did not support cell viability. This result provides additional support for the importance of first nucleotide of the mature tRNAs in their processing and functionality.     

Crystal structure of the tRNA 3' processing endoribonuclease tRNase Z from Thermotoga maritima

The maturation of the tRNA 3' end is catalyzed by a tRNA 3' processing endoribonuclease named tRNase Z (RNase Z or 3'-tRNase) in eukaryotes, Archaea, and some bacteria. The tRNase Z generally cuts the 3' extra sequence from the precursor tRNA after the discriminator nucleotide. In contrast, Thermotoga maritima tRNase Z cleaves the precursor tRNA precisely after the CCA sequence. In this study, we determined the crystal structure of T. maritima tRNase Z at 2.6-A resolution. The tRNase Z has a four-layer alphabeta/betaalpha sandwich fold, which is classified as a metallo-beta-lactamase fold, and forms a dimer. The active site is located at one edge of the beta-sandwich and is composed of conserved motifs. Based on the structure, we constructed a docking model with the tRNAs that suggests how tRNase Z may recognize the substrate tRNAs.     

Identity elements and aminoacylation of plant tRNATrp.

Mutation of the Arabidopsis thaliana tRNA (Trp)(CCA) anticodon or of the A73 discriminator base greatly diminishes in vitro aminoacylation with tryptophan, indicating the importance of these nucleotides for recognition by the plant tryptophanyl-tRNA synthetase. Mutation of the tRNA (Trp)(CCA) anticodon to CUA so as to translate amber nonsense codons permits tRNA (Trp)(CCA) to be aminoacylated by A.thaliana lysyl-tRNA synthetase. Thus, translational suppression by tRNA (TRP)(CCA) observed in plant cells includes significant incorporation of lysine into protein.     

A model for C74 addition by CCA-adding enzymes: C74 addition, like C75 and A76 addition, does not involve tRNA translocation

The CCA-adding enzyme adds CCA to the 3'-end of tRNA one nucleotide at a time, using CTP and ATP as substrates. We found previously that tRNA does not rotate or translocate on the enzyme during the addition of C75 and A76. We therefore predicted that the growing 3'-end of tRNA must, upon addition of each nucleotide, refold to reposition the new 3'-hydroxyl equivalently relative to the solitary nucleotidyltransferase motif. Cocrystal structures of the class I archaeal Archaeoglobus fulgidus enzyme, poised for addition of C75 and A76, confirmed this prediction. We have also demonstrated that an evolutionarily flexible beta-turn facilitates progressive refolding of the 3'-terminal C74 and C75 residues during C75 and A76 addition. Although useful cocrystals corresponding to C74 addition have not yet been obtained, we now show experimentally that tRNA does not rotate or translocate during C74 addition. We therefore propose, based on the existing A. fulgidus cocrystal structures, that the same flexible beta-turn functions as a wedge between the discriminator base (N73) and the terminal base pair of the acceptor stem, unstacking and repositioning N73 to attack the incoming CTP. Thus a single flexible beta-turn would orchestrate consecutive addition of all three nucleotides without significant movement of the tRNA on the enzyme surface.     

Purification of potential 3'' processing nucleases using synthetic tRNA precursors.

The synthetic tRNA precursors, tRNA-C-114C]U and tRNA-C-C-A-[14C]C-C, as well as poly (a) and diesterase-treated tRNA, have been used to identify and purify potential 3'processing nucleases. Four activities have been separated by this analysis; and three of them have been characterized. Two of the enzymes, which are well-separated on hydroxylapatite columns, act on poly(A), require K+ and Mg2+ for activity, and have molecular weights of about 90,000. These activities have properties previously ascribed to RNase II. The third enzyme does not act on poly(A), requires Mg2+ for activity, and has a molecular weight of about 60,000. It is identical to RNase D, previously characterized as an exonuclease acting on tRNAs with altered structure. Each of the enzymes can remove nucleotides from the tRNA precursor containing extra nucleotides beyond the 3'terminus, whereas they are relatively inactive with intact tRNA or tRNA-C-U. The greatest specificity was displayed by RNase D. The possibility that RNase D is a 3'processing nuclease is discussed.     

Endonucleolytic processing of CCA-less tRNA precursors by RNase Z in Bacillus subtilis

In contrast to Escherichia coli, where the 3' ends of tRNAs are primarily generated by exoribonucleases, maturation of the 3' end of tRNAs is catalysed by an endoribonuclease, known as RNase Z (or 3' tRNase), in many eukaryotic and archaeal systems. RNase Z cleaves tRNA precursors 3' to the discriminator base. Here we show that this activity, previously unsuspected in bacteria, is encoded by the yqjK gene of Bacillus subtilis. Decreased yqjK expression leads to an accumulation of a population of B.subtilis tRNAs in vivo, none of which have a CCA motif encoded in their genes, and YqjK cleaves tRNA precursors with the same specificity as plant RNase Z in vitro. We have thus renamed the gene rnz. A CCA motif downstream of the discriminator base inhibits RNase Z activity in vitro, with most of the inhibition due to the first C residue. Lastly, tRNAs with long 5' extensions are poor substrates for cleavage, suggesting that for some tRNAs, processing of the 5' end by RNase P may have to precede RNase Z cleavage.     

A story with a good ending: tRNA 3'-end maturation by CCA-adding enzymes

CCA-adding enzymes (tRNA nucleotidyltransferases) are responsible for the maturation or repair of the functional 3' end of tRNAs. These enzymes are remarkable because they polymerize the essential nucleotides CCA onto the 3' terminus of tRNA precursors without using a nucleic acid template. Recent crystal structures, plus three decades of enzymology, have revealed the elegant mechanisms by which CCA-adding enzymes achieve their substrate specificity in a nucleic acid template independent fashion. The class I CCA-adding enzyme employs both an arginine sidechain and backbone phosphates of the bound tRNA to recognize incoming nucleotides. It switches from C to A addition through changes in the size and shape of the nucleotide-binding pocket, which is progressively altered by the elongating 3' terminus of the tRNA. By contrast, the class II CCA-adding enzyme uses only amino acid sidechains, which form a protein template for incoming nucleotide selection.