Polycistronic Precursor Research Articles

Insertional editing of mitochondrial tRNAs of Physarum polycephalum and Didymium nigripes.

tRNAs encoded on the mitochondrial DNA of Physarum polycephalum and Didymium nigripes require insertional editing for their maturation. Editing consists of the specific insertion of a single cytidine or uridine relative to the mitochondrial DNA sequence encoding the tRNA. Editing sites are at 14 different locations in nine tRNAs. Cytidine insertion sites can be located in any of the four stems of the tRNA cloverleaf and usually create a G. C base pair. Uridine insertions have been identified in the T loop of tRNALys from Didymium and tRNAGlu from Physarum. In both tRNAs, the insertion creates the GUUC sequence, which is converted to GTPsiC (Psi = pseudouridine) in most tRNAs. This type of tRNA editing is different from other, previously described types of tRNA editing and resembles the mRNA and rRNA editing in Physarum and Didymium. Analogous tRNAs in Physarum and Didymium have editing sites at different locations, indicating that editing sites have been lost, gained, or both since the divergence of Physarum and Didymium. Although cDNAs derived from single tRNAs are generally fully edited, cDNAs derived from unprocessed polycistronic tRNA precursors often lack some of the editing site insertions. This enrichment of partially edited sequences in unprocessed tRNAs may indicate that editing is required for tRNA processing or at least that RNA editing occurs as an early event in tRNA synthesis.

Localization and processing from a polycistronic precursor of novel snoRNAs in maize.

We have shown previously that groups of U14 snoRNA genes are clustered with other, novel snoRNAs in maize. These genes are transcribed polycistronically from an upstream promoter to give a precursor snoRNA, which is processed by a splicing-independent mechanism. The clusters contain both box C/D snoRNAs, thought to guide rRNA O-ribose methylations, and the first plant box H/ACA snoRNA so far identified, thought to guide an rRNA pseudo-uridylation. Here we show that four novel snoRNAs identified as members of U14-containing gene clusters each show distinct sub-nucleolar localizations. Two of the snoRNAs (snoR2, a box H/ACA snoRNA, and snoR3, a box C/D snoRNA) colocalise closely with nucleolar rDNA transcription sites. A third box C/D snoRNA, U49, is localised to a more extended region which includes the transcription sites. On the other hand snoR1, another box C/D snoRNA, is located in a quite different region of the nucleolus, and shows a similar distribution to that of 7-2/MRP, a snoRNA involved in the later pre-rRNA cleavage reactions. This may indicate that this snoRNA is involved at later stages of processing, whereas the other snoRNAs are involved early or cotranscriptionally. Probes to intergenic spacer regions of the precursor snoRNA have been used to determine the location of the precursor. This shows a clear labelling of both the dense fibrillar component of the nucleolus, and of coiled bodies. This distribution implies that the polycistronic precursor is imported into the nucleolus for processing to the mature snoRNAs, and that the import or processing pathway involves coiled bodies.

Trans mRNA splicing in trypanosomes: cloning and analysis of a PRP8-homologous gene from Trypanosoma brucei provides evidence for a U5-analogous RNP.

In trypanosomes all mRNAs are generated through trans mRNA splicing, requiring the functions of the small nuclear RNAs U2, U4 and U6. In the absence of conventional cis mRNA splicing, the structure and function of a U5-analogous snRNP in trypanosomes has remained an open question. In cis splicing, a U5 snRNP-specific protein component called PRP8 in yeast and p220 in man is a highly conserved, essential splicing factor involved in splice-site recognition and selection. We have cloned and sequenced a genomic region from Trypanosoma brucei, that contains a PRP8/p220-homologous gene (p277) coding for a 277 kDa protein. Using an antibody against a C-terminal region of the trypanosomal p277 protein, a small RNA of approximately 65 nucleotides could be specifically co-immunoprecipitated that appears to be identical with a U5 RNA (SLA2 RNA) recently identified by Dungan et al. (1996). Based on sedimentation, immunoprecipitation and Western blot analyses we conclude that this RNA is part of a stable ribonucleoprotein (RNP) complex and associated not only with the p277 protein, but also with the common proteins present in the other trans-spliceosomal snRNPs. Together these results demonstrate that a U5-analogous RNP exists in trypanosomes and suggest that basic functions of the U5 snRNP are conserved between cis and trans splicing.

In Vitro Analysis of Mutations Causing Myoclonus Epilepsy with Ragged-Red Fibers in the Mitochondrial tRNALys Gene: Two Genotypes Produce Similar Phenotypes

Cytoplasts from patients with myoclonus epilepsy with ragged-red fibers harboring a pathogenic point mutation at either nucleotide 8344 or 8356 in the human mitochondrial tRNA(Lys) gene were fused with human cells lacking endogenous mitochondrial DNA (mtDNA). For each mutation, cytoplasmic hybrid (cybrid) cell lines containing 0 or 100% mutated mtDNAs were isolated and their genetic, biochemical, and morphological characteristics were examined. Both mutations resulted in the same biochemical and molecular genetic phenotypes. Specifically, cybrids containing 100% mutated mtDNAs, but not those containing the corresponding wild-type mtDNAs, exhibited severe defects in respiratory chain activity, in the rates of protein synthesis, and in the steady-state levels of mitochondrial translation products. In addition, aberrant mitochondrial translation products were detected with both mutations. No significant alterations were observed in the processing of polycistronic RNA precursor transcripts derived from the region containing the tRNA(Lys) gene. These results demonstrate that two different mtDNA mutations in tRNA(Lys), both associated with the same mitochondrial disorder, result in fundamentally identical defects at the cellular level and strongly suggest that specific protein synthesis abnormalities contribute to the pathogenesis of myoclonus epilepsy with ragged-red fibers.

Hierarchies of RNA-processing signals in a trypanosome surface antigen mRNA precursor.

Nearly all trypanosome mRNAs are synthesized as polycistronic precursors, from which mature mRNAs are excised by trans splicing and polyadenylation. Polyadenylation of a procyclic acidic repetitive protein (PARP, or procyclin) transcript was studied by transient transfection of constructs bearing a chloramphenicol acetyltransferase gene linked to the PARP intergenic region. Polyadenylation usually occurred at A residues, about 100 bases upstream of a trans-splicing acceptor signal. The wild-type polyadenylation site has a cryptic trans-splicing signal about 100 bp downstream: deletion or inversion of this signal results in polyadenylation at multiple sites, upstream of other cryptic trans-splicing signals. The PARP mRNA precursor appears to contain a hierarchy of possible processing signals, the function of cryptic ones being revealed only when the dominant ones are deleted or moved. Correct polyadenylation can be restored by addition of trans-splicing signals from other loci. The results indicate that polyadenylation is coupled to downstream trans splicing but that the products of the trans-splicing reaction are not necessarily functional mRNAs.

Hierarchies of RNA-processing signals in a trypanosome surface antigen mRNA precursor.

Nearly all trypanosome mRNAs are synthesized as polycistronic precursors, from which mature mRNAs are excised by trans splicing and polyadenylation. Polyadenylation of a procyclic acidic repetitive protein (PARP, or procyclin) transcript was studied by transient transfection of constructs bearing a chloramphenicol acetyltransferase gene linked to the PARP intergenic region. Polyadenylation usually occurred at A residues, about 100 bases upstream of a trans-splicing acceptor signal. The wild-type polyadenylation site has a cryptic trans-splicing signal about 100 bp downstream: deletion or inversion of this signal results in polyadenylation at multiple sites, upstream of other cryptic trans-splicing signals. The PARP mRNA precursor appears to contain a hierarchy of possible processing signals, the function of cryptic ones being revealed only when the dominant ones are deleted or moved. Correct polyadenylation can be restored by addition of trans-splicing signals from other loci. The results indicate that polyadenylation is coupled to downstream trans splicing but that the products of the trans-splicing reaction are not necessarily functional mRNAs.

A nuclear mutation in maize blocks the processing and translation of several chloroplast mRNAs and provides evidence for the differential translation of alternative mRNA forms.

A mutant designated crp1 (chloroplast RNA processing 1) was identified in a screen for transposon-induced maize mutants with defects in chloroplast gene expression. crp1 is a recessive, nuclear mutation that causes the loss of the cytochrome f/b6 complex and a reduction in photosystem I. The molecular basis for these protein losses is unique relative to previously described mutants with defects in organelle gene expression; it involves defects in the metabolism of two organellar mRNAs and in the translation of two organellar proteins. Mutants lack the monocistronic forms of the petB and petD mRNAs (encoding cytochrome f/b6 subunits), but contain normal levels of their polycistronic precursors. Pulse-labeling experiments revealed normal synthesis of the petB gene product, but a large decrease in synthesis of the petD gene product. These results suggest that petD sequences are more efficiently translated in a monocistronic than in a polycistronic context, thereby providing evidence that the elaborate RNA processing typical of chloroplast transcripts can play a role in controlling gene expression. Structural predictions suggest that the petD start codon lies in a stable hairpin in the polycistronic RNA, but remains unpaired in the monocistronic transcript. Thus, processing to a monocistronic form may increase translational efficiency by releasing the translation initiation region from inhibitory interactions with upstream RNA sequences. Synthesis of a third cytochrome f/b6 subunit, encoded by the petA gene, was undetectable in crp1, although its mRNA appeared unaltered. Two mechanisms are consistent with the simultaneous loss of both petA and petD protein synthesis: the translation of the petA and petD mRNAs might be coupled via a mechanism independent of crp1, or the crp1 gene may function to coordinate the expression of the two genes, which encode subunits of the same complex.

Accurate polyadenylation of procyclin mRNAs in Trypanosoma brucei is determined by pyrimidine-rich elements in the intergenic regions.

Polycistronic precursor RNAs from trypanosomes are processed into monocistronic mRNAs by the excision of intergenic sequences and the addition of a 39-nucleotide spliced leader by trans splicing. These mRNAs are also polyadenylated, yet they do not contain the hexamer AAUAAA within their 3' untranslated regions (UTRs). To identify the signals required for the accurate polyadenylation of mRNAs, we tested the effects of deletions in either the procyclin 3' UTR or the downstream intergenic region on the polyadenylation of transcripts from a reporter gene. Deletion of the entire 3' UTR does not affect polyadenylation, but a crucial element is located in the intergenic region and includes a pyrimidine-rich sequence from positions 79 to 112 followed by an AG dinucleotide. Related motifs are also found a similar distance downstream of other genes in both the procyclin and the variant surface glycoprotein expression sites. These sequences bear a strong resemblance to splice acceptor sites, but they are generally several hundred base pairs upstream of the major splice acceptor site of the next gene in the transcription unit. There is evidence, however, that some of them can give rise to alternatively spliced transcripts with unusually long 5' UTRs.

A common pyrimidine-rich motif governs trans-splicing and polyadenylation of tubulin polycistronic pre-mRNA in trypanosomes.

In trypanosomes, the generation of monocistronic mRNAs from polycistronic precursors is achieved via RNA processing, namely trans-splicing of the spliced leader sequence at the 5' end and cleavage/polyadenylation at the 3' end of the mRNA coding region. Recent evidence raised the intriguing possibility that these two reactions are coupled. To begin a dissection of the signals required for mRNA 5'-end and 3'-end formation and to uncover potential interactions between trans-splicing and polyadenylation, we mutagenized the intergenic region between the beta- and alpha-tubulin genes of Trypanosoma brucei. Block substitutions identified the pyrimidine-rich sequences at the alpha-tubulin 3'-splice-acceptor site as a major determinant for accurate trans-splicing downstream and 3'-end formation upstream. In addition to the utilization of cryptic 3'-splice sites, obliteration of the polypyrimidine tracts led to aberrant poly(A)+ site choice, even in the presence of the wild-type poly(A)+ site and neighboring sequences. Taken together, these results indicate that the polypyrimidine-rich sequences act as a bifunctional element that affects RNA processing both upstream and downstream from itself. This is consistent with the possibility that the polypyrimidine tract is recognized by both the trans-splicing and polyadenylation machineries, either sequentially or simultaneously.

Abnormal RNA processing associated with a novel tRNA mutation in mitochondrial DNA. A potential disease mechanism.

A patient with a mitochondrial myopathy and biochemically proven profound complex I deficiency has a new mutation in mtDNA. This A-to-G transition at position 3302, involving the aminoacyl stem of tRNA(Leu(UUR)), is associated with abnormal mitochondrial RNA processing. Northern analysis demonstrates marked accumulation of a polycistronic RNA precursor containing sequence for 16 S rRNA, tRNA(Leu(UUR)), and ND1. Comparison of skeletal muscle and skin fibroblasts suggests that the processing error may be quantitatively less severe in this tissue, and biochemical analysis shows that fibroblasts do not express a biochemical defect despite containing the mutation. Important qualitative differences in the processing of this RNA precursor were found when comparing muscle and skin fibroblasts. In muscle, processing appears to occur first at the 5'-end of the tRNA, generating 16 S rRNA plus a tRNA + ND1 intermediate. In fibroblasts, processing occurs at the 3'-end of the tRNA, generating a 16 S rRNA + tRNA intermediate. We suggest that the mutation at position 3302 induces abnormal mitochondrial RNA processing that is linked to the biochemical defect (profound loss of complex I activity), either by qualitative or quantitative abnormalities in the ND1 message. The restriction to skeletal muscle of both the processing error and the biochemical defect suggests that the observed tissue differences in RNA processing play a protective role in skin fibroblasts.

Genes encoding the major surface glycoprotein in Leishmania are tandemly linked at a single chromosomal locus and are constitutively transcribed

The major surface glycoprotein of Leishmania, gp63, is encoded by a small multi-gene family of tandemly linked genes which map to a single chromosome. For Leishmania major, there are five 3.1 kilobasepair (kb) direct repeat units which include a 1.8-kb open reading frame and a 1.3-kb intergenic or spacer region. In addition, there is a single gene copy linked as a direct repeat but separated from the tandem array of gp63 genes by about 8 kb. The restriction enzyme map of the repeat unit is highly conserved among the gene copies. The regions which flank the tandemly repeated genes diverge outside of the 3.1-kb repeat unit. Transcription of the gp63 gene locus is constitutive as the 3-kb transcript was present in promastigotes as well as in amastigotes. A minor 6-kb gp63 gene transcript was also detected in Northern blot analysis which could signify the transcription of the genes as a polycistronic or multigene precursor RNA.

Accuratein vitrocleavage by RNase in of phosphorothioate-substituted RNA processing signals in bacteriophage T7 early mRNA

To test the ability of an RNA processing enzyme to cleave chemically-modified RNA substrates, RNA transcripts containing RNase III cleavage sites were enzymatically synthesized in vitro to contain specific phosphorothioate diester internucleotide linkages. One transcript (R1.1 RNA) was generated using phage T7 RNA polymerase and a cloned segment of phage T7 DNA containing the R1.1 RNase III processing site. The second transcript was the phage T7 polycistronic early mRNA precursor, which was synthesized using E. coli RNA polymerase and T7 genomic DNA. The RNA transcripts contained phosphorothioate diester groups at positions including the scissile bonds. The modified RNAs were stable to incubation in Mg2+-containing buffer, and were specifically cleaved by RNase III. RNA oligonucleotide sequence analysis showed that the modified R1.1 RNA processing site was the same as the canonical site and contained a phosphorothioate bond. Furthermore, RNase III cleaved the phosphorothioate internucleotide bond with 5' polarity. RNase III cleavage of phosphorothioate substituted T7 polycistronic early mRNA precursor produced the same gel electrophoretic pattern as that obtained with the control transcript. Thus, RNase III cleavage specificity is not altered by phosphorothioate internucleotide linkages.

Transcription initiation and RNA processing of a yeast mitochondrial tRNA gene cluster.

Expression of 5 yeast mitochondrial tRNA genes (Ala, Ile, Tyr, Asn and Metm), localized upstream from the oxil gene has been analyzed by in vitro capping using guanylyltransferase, northern hybridization and S1 nuclease mapping in the wild type and a rho-strain. The 5 tRNA sequences belong to the same transcriptional unit which is initiated 133 bp upstream from the tRNA(Ala) gene at a promoter sequence TTATAAGTA. Furthermore, a truncated tRNA(Tyr) transcript, 2 nucleotides shorter than mature tRNA(Tyr) has been found, only in the rho-strain. This minor transcript may result from secondary transcription initiation at a variant nonanucleotide sequence, ATATAAGGA, which overlaps the tRNA(Tyr) coding sequence by 3 nucleotides. The polycistronic precursor has proven to be useful in investigation of the mechanisms of tRNA processing. Maturation of this primary transcript proceeds exclusively by precise endonucleolytic cleavages at the 5' and 3'-ends of tRNA sequences.

Evidence that the methylation inhibitor cycloleucine causes accumulation of a discrete ribosomal RNA precursor in hamster mitochondria.

A novel RNA fraction, 'Cy RNA,' that accumulates in mitochondria when hamster cells are treated with the methylation inhibitor cycloleucine, has been characterized by high resolution acrylamide gel electrophoresis and DNA-RNA hybridization. Cy RNA ran in gels as a discrete band, with an apparent chain length of 2 600. It hybridized specifically to restriction fragments containing genes for the mitochondrial ribosomal RNAs. We infer that Cy RNA is a discrete polycistronic ribosomal RNA precursor transcript, whose processing is dependent on normal methylation.

Origins of transcripts of the yeast mitochondrial var 1 gene.

Transcription of the yeast mitochondrial var 1 gene encoding the mitochondrial small ribosomal subunit protein, var 1, has been examined in wild type and petite strains by Northern hybridization, S 1 nuclease protection experiments, and analysis of primary transcripts by in vitro labeling of mitochondrial RNAs with [alpha-32P]GTP and guanylyl transferase. We have identified a transcription unit in which a polycistronic precursor gives rise to the mRNAs for ATPase subunit 9 (the product of the oli 1 gene), var 1, and tRNASerUCN. Potential cleavage sites have been identified within this precursor that separates oli 1, tRNASerUCN, and var 1 transcripts. Transcriptional initiation of the polycistronic precursor occurs from one of two nearby copies of the sequence, 5'-ATATAAGTA-3', located approximately 550 and 630 base pairs 5' to the oli 1 gene. A closely related sequence, 5'-TTATAAGTA-3', is found within the var 1 gene and may serve as a transcriptional initiation site for one of a pair of primary transcripts detected in a petite retaining the var 1[40.0] allele. The other primary transcript in this strain initiates upstream in a region of the var 1 gene containing no recognizable initiation sites.

Accurate processing and pseudouridylation of chloroplast transfer RNA in a chloroplast transcription system

The trancription of a cloned trnV1-trnN1-trnR1 cluster from Euglena gracilis chloroplast (ct) DNA and the processing of a tRNA(Val)-tRNA(Asn)-tRNA(Arg) polycistronic precursor were studied in a spinach ct transcription extract. A soluble ct RNA polymerase selectively transcribes the trnV1-trnN1-trnR1-trnL1 locus in the EcoG fragment from the Euglena ct genome. Restriction enzyme modified templates and RNA fingerprint analysis were used to confirm that the tRNA genes were correctly transcribed. The tRNA(Val)-tRNA(Asn)-tRNA(Arg) polycistronic precursor transcribed by RNA polymerase III in a HeLa cell extract was used as a substrate to demonstrate that a ct tRNA precursor molecule is correctly processed by the ct tRNA processing enzymes. The oligonucleotide pattern of tRNAs processed in vitro from the tRNA(Val)-tRNA(Asn)-RNA(Arg) polycistronic precursor is indistinguishable from tRNA(Val), tRNA(Asn) and tRNA(Arg) transcribed by the ct RNA polymerase and processed in the ct transcription extract. The 3'-CCAOH is added to the tRNAs by a 3' nucleotidyltransferase after correct processing of the 3' terminus. Correct pseudouridylation was demonstrated for uridine residues in a tRNA(Met) m molecule transcribed from a spinach ct trnM1 locus. Thus, the enzymatic activities involved in tRNA biosynthesis in vitro include DNA-dependent (tDNA) RNA polymerase, a 5'-processing activity (RNase P-like), a 3'-exonuclease, an endoribonuclease involved in 3'-tRNA maturation, a tRNA nucleotidyltransferase, and pseudouridylate synthetase.

Aberrant glycoprotein mRNA synthesized by the internal deletion mutant of vesicular stomatitis virus.

The internal deletion mutant (DI-LT) derived from the heat-resistant strain of vesicular stomatitis virus synthesized an aberrant polyadenylated mRNA in vivo and in vitro. No normal glycoprotein message could be detected among the in vivo transcription products. The abnormal RNA contained a transcript of the partially deleted polymerase gene covalently linked to the 3' end of the glycoprotein message. The polyadenylate is located at the 3' end of the molecule and is most probably encoded by the remnant polymerase gene polyadenylation signal. This aberrant RNA may be synthesized because of either a failure to terminate transcription at the end of the glycoprotein gene or an inability to process an abnormal polycistronic precursor.

Mapping of transcription units in the bacteriophage T4 tRNA gene cluster.

Abstract The transfer RNA gene cluster of bacteriophage T4 (about 1600 base-pairs long) is comprised of ten genes that code for eight tRNA and two other stable RNA species. It is arranged in two tight subclusters separated by a spacer of 600 base-pairs. We studied the transcription of these genes in vitro by using DNA templates derived from a series of T4 mutants partially deleted within the tRNA genetic region. The mixtures of primary products made by RNA polymerase on the different T4 DNA templates were fractionated by gel electrophoresis into discrete RNA species. Wildtype and deletion-specific transcripts of the tRNA gene cluster were isolated and their sizes and stable RNA sequence composition were determined. Three primary transcripts of the intact tRNA operon, one major and two minor species, were identified. The major RNA species measured about 3000 nucleotides and harboured the sequences of both subclusters of stable RNA genes. The two minor RNA species (2300 and 1700 nucleotides, respectively) lacked the sequences of the promoterdistal gene subcluster. The existence of a fourth minor primary transcript (about 3500 nucleotides) that contains the sequences of both gene subclusters is inferred from the results. On the basis of these data and the location of a promoter for the T4 tRNA gene cluster by Fukada et al. (1980b), we suggest two alternative schemes for transcriptional organization of the T4 tRNA genes. Both schemes envisage a major transcription unit defined by a promoter 1000 base-pairs upstream, and a terminator 300 base-pairs downstream from the tRNA genes. The existence of weak terminator(s) in the spacer region is also proposed. Primary transcripts of the tRNA operon were processed with S-100 extracts of Escherichia coli and nine T4-specific stable RNAs were identified among the products. This indicates that E. coli contains all the enzymes necessary for the processing of polycistronic T4 tRNA precursors. The in vitro synthesized primary transcripts provide useful substrates for the study of processing enzymes involved in the early steps of tRNA maturation.

Precise localization and nucleotide sequence of the two mouse mitochondrial rRNA genes and three immediately adjacent novel tRNA genes

The complete DNA sequence of the ribosomal RNA region of mouse L cell mitochondrial DNA has been determined. Genes for the small (12S) and large (16S) rRNAs have been precisely located by direct sequencing of the termini of the two mature rRNAs. A comparison of the lengths (956 and 1582 nucleotides) and terminal sequences of the mature rRNAs with the DNA coding sequences indicates that mouse mt rRNAs are not spliced. Computer analysis of the complete DNA sequence has identified three potential transfer RNA genes. A gene for phenylalanine tRNA is located immediately adjacent to the 5′ end of the 12S rRNA gene, a valine tRNA gene occupies the entire region between the 12S and 16S rRNA genes and a leucine tRNA gene is located immediately adjacent to the 3′ end of the 16S gene. Hybridization of 32P-labeled, tRNA-sized mtRNA to selected DNA restriction endonuclease fragments from the rRNA region confirms the existence of small, abundant mtRNAs transcribed from these DNA sequences. All three tRNA genes and both rRNA genes are transcribed from the heavy strand of mtDNA. The mt rRNA sequences exhibit notable homologies to other rRNAs and, in particular, to those of E. coli. Within the 3′ terminal 50 nucleotides, the mouse mt 12S rRNA contains a potential 10 bp hairpin structure and a sequence of 15 consecutive nucleotides common to the RNA of the small ribosomal subunit in all systems, but does not contain the mRNA binding site (ACCUCC) found in E. coli and corn chloroplast rRNAs. The mt tRNA genes do not have the 3′ terminal CCA sequence encoded in the DNA, nor do they contain any intervening sequences. Two of the three tRNAs would lack many features which are known to be strictly conserved in all other nonorganelle tRNAs which have been sequenced. The fact that all the genes in this region are directly contiguous with at most one intervening nucleotide suggests that the entire region is transcribed into a polycistronic precursor RNA which is processed by endonucleolytic cleavages. The organization of the genes of the rRNA operon of mouse mtDNA, when compared to the organization of rRNA and tRNA genes in bacterial or eucaryotic nuclear genomes, provides evidence for the endosymbiotic hypothesis of the biogenesis of mammalian mitochondria.

An Escherichia coli endonuclease responsible for primary cleavage of in vitro transcripts of bacteriophage T4 tRNA gene cluster.

An endonuclease activity was isolated from 100,000 g supernatant fraction of Escherichia coli using in vitro primary transcripts of T4 tRNA gene cluster as assay substrates. The endonuclease cleaves the polycistronic RNA precursors into fragments containing monomeric and dimeric stable RNA sequences. The result strongly suggest that this enzyme participates in the early steps of T4 tRNA maturation pathway preceding the action of RNase P.