Absence Of RNase Research Articles

Reverse transcriptase. The use of cloned Moloney murine leukemia virus reverse transcriptase to synthesize DNA from RNA.

Reverse transcriptase (RT) is the key enzyme required for conversion of RNA to DNA. Cloning of Moloney murine leukemia virus (MMLV) RT has enable engineering an RT that lacks endogenous RNase H activity. RT catalyzes cDNA synthesis more efficiently in the absence of RNase H. We describe here a number of properties of MMLV RT and RNase H-minus MMLV RT not summarized in a single location elsewhere, providing a basis for best use of these enzymes in cDNA synthesis. In addition, general guidelines and detailed protocols are provided for use of MMLV RTs in one tube double-stranded cDNA synthesis, in [32P]cDNA synthesis, and in RT-PCR and long RT-PCR.

Differential Sensitivities of Portions of the mRNA for Ribosomal Protein S20 to 3′-Exonucleases Dependent on Oligoadenylation and RNA Secondary Structure

The 3'-exonucleolytic decay of the mRNA for ribosomal protein S20 has been reconstituted in vitro using purified RNase II and crude extracts enriched for polynucleotide phosphorylase (PNPase) activity. We show that RNase II can catalyze the degradation of the 5' two-thirds of the S20 mRNA and that prior oligoadenylation of the 3' termini of truncated S20 mRNA substrates can significantly stimulate the initiation of degradation by RNase II. The intact S20 mRNA is, however, insensitive to attack by RNase II and polyadenylation of its 3'-end cannot overcome the natural resistance of the S20 mRNA to RNase II. Complete degradation of either the entire S20 mRNA without prior endonucleolytic cleavage or the 3'-terminal 147-residue fragment is dependent on both oligoadenylation and PNPase activity. Moreover, this process can take place in the absence of RNase E activity. Our data point to the importance of oligoadenylation in facilitating 3'-exonucleolytic activity and indicate that there are alternative degradative pathways. The implications for mRNA decay are discussed.

Site and mechanism of antisense inhibition by C-5 propyne oligonucleotides.

Antisense gene inhibition occurs when an oligonucleotide (ON) has sufficient binding affinity such that it hybridizes its reverse complementary target RNA and prevents translation either by causing inactivation of the RNA (possibly by RNase H) or by interfering with a cellular process such as stalling a ribosome. The mechanisms underlying these processes were explored. Cellular antisense inhibition was evaluated in a microinjection assay using ON modifications which precluded or allowed in vitro RNase H cleavage of ON/RNA hybrids. RNase H-independent inhibition of protein synthesis could be achieved by targeting either the 5'-untranslated region or the 5'-splice junction of SV40 large T antigen using 2'-O-allyl phosphodiester ONs which contained C-5 propynylpyrimidines (C-5 propyne). Inhibition at both sites was 20-fold less active than inhibition using RNase H-competent C-5 propyne 2'-deoxy phosphorothioate ONs. In vitro analysis of association and dissociation of the two classes of ONs with complementary RNA showed that the C-5 propyne 2'-O-allyl phosphodiester ON bound to RNA as well as the C-5 propyne 2'-deoxy phosphorothioate ON. In vitro translation assays suggested that the two classes of ONs should yield equivalent antisense effects in the absence of RNase H. Next, ON/T antigen RNA hybrids were injected into the nuclei and cytoplasm of cells. Injection of C-5 propyne 2'-O-allyl phosphodiester ON/RNA hybrids resulted in expression of T antigen, implying that the ONs dissociated from the RNA in cells which likely accounted for their low potency. In contrast, when C-5 propyne 2'-deoxy phosphorothioate ON/T antigen RNA complexes were injected into the nucleus, the duplexes were stable enough to completely block T antigen translation, presumably by RNA inactivation. Thus, a dramatic finding is that C-5 propyne 2'-deoxy phosphorothioate ONs, once hybridized to RNA, are completely effective at preventing mRNA translation. The implication is that further increases in complex stability coupled with effective RNase H cleavage will not result in enhanced potency. We predict that the development of more effective ONs will only come from modifications which increase the rate of ON/RNA complex formation within the nucleus.

Specific chromosomal sites enhancing homologous recombination in Escherichia coli mutants defective in RNase H

To clone new replication origin(s) activated under RNase H-defective (rnh-) conditions in Escherichia coli cells, whole chromosomal DNA digested with EcoRI was to with a Kmr DNA fragment and transformed into an rnh- derivative host. From the Kmr transformants, we obtained eight kinds of plasmid-like DNA, each of which contained a specific DNA fragment, termed "Hot", derived from the E. coli genome. Seven of the Hot DNAs (HotA-G) mapped to various sites within a narrow DNA replication termination region (about 280 kb), without any particular selection. Because Hot DNA could not be transformed into a mutant strain in which the corresponding Hot region had been deleted from the chromosome, the Hot DNA, though obtained as covalently closed circular (ccc) DNA, must have arisen by excision from the host chromosome into which it had initially integrated, rather than by autonomous replication of the transformed species. While Hot DNA does not have a weak replication origin it does have a strong recombinational hotspot active in the absence of RNase H. This notion is supported by the finding that Chi activity was present on all Hot DNAs tested and no Hot-positive clone without Chi activity was obtained, with the exception of a DNA clone carrying the dif site.

Mechanism of DNA A protein-dependent pBR322 DNA replication. DNA A protein-mediated trans-strand loading of the DNA B protein at the origin of pBR322 DNA.

pBR322 DNA can be replicated via a DNA A-dependent pathway mediated by its binding to the two DNA A-binding sites (dnaA boxes) present near the plasmid origin. DNA synthesis requires the transcription of RNA II (the leading-strand primer precursor) to generate a specific unwound structure in the region containing the dnaA boxes. In this structure, the DNA containing the dnaA boxes can take the form of either a RNA II-parental H strand pBR322 DNA hybrid opposed by the displaced parental L strand (in the absence of RNase H and DNA polymerase I), or a nascent leading strand-parental H strand DNA duplex opposed by the displaced parental L strand (in the presence of RNase H and DNA polymerase I). These findings defined three types of potential sites for productive DNA A binding: (i) the displaced parental L single strand, (ii) a hairpin formed by the inverted repeat of the two dnaA boxes, and (iii) either the RNA-DNA duplex or the nascent leading strand-parental DNA duplex. By using a combination of: (i) inhibition of the replication of a plasmid carrying oriC by oligonucleotides of various dnaA box sequences and conformation, (ii) a gel mobility shift assay to measure DNA A binding to the same oligonucleotide substrates, (iii) replication of pBR322 DNA templates with either one or no dnaA box, and (iv) photocross-linking to demonstrate DNA A binding to an RNA-DNA hybrid, evidence is presented here that DNA A-mediated pBR322 DNA replication proceeds by a mechanism in which DNA A binds to the duplex side of the unwound origin structures and loads the DNA B protein in trans to the displaced parental L strand DNA.

Association of poly(ADP-ribose) polymerase with the nuclear matrix: The role intermolecular disulfide bond formation, RNA retention, and cell type

The recovery of the enzyme poly(ADP-ribose) polymerase (pADPRp) in the nuclease- and 1.6 M NaCl-resistant nuclear subfraction prepared from a number of different sources was assessed by Western blotting. When rat liver nuclei were treated with DNase I and RNase A followed by 1.6 M NaCl, ~ 10% of the nuclear pADPRp was recovered in the sedimentable fraction. The proportion of pADPRp recovered with the residual fraction decreased to < 5% of the total nuclear polymerase when nuclei were prepared in the presence of the sulfhydryl blocking reagent iodoacetamide and increased to ~ 50% of the total nuclear pADPRp when nuclei were treated with the sulfhydryl cross-linking reagent sodium tetrathionate (NaTT) prior to fractionation. To determine whether this effect of disulfide bond formation was unique to rat liver nuclei, nuclear matrix/cytoskeleton structures were prepared in situ by sequentially treating monolayers of tissue culture cells with Nonidet-P40, DNase I and RNase A, and 1.6 M NaCl ( S. H. Kaufmann and J. H. Shaper (1991) Exp. Cell Res. 192, 511–523). When nuclear monolayers were prepared from HTC rat hepatoma cells, CaLu-1 human lung carcinoma cells, and CHO hamster ovary cells in the absence of NaTT, pADPRp was undetectable in the nuclease- and 1.6 M NaCl-resistant fraction. In contrast, when nuclear monolayers were isolated in the presence of NaTT, from 5% (CaLu-1) to 26% (HTC cells) of the total nuclear pADPRp was recovered with the nuclease- and salt-resistant fraction. Examination of these residual structures by SDS-polyacrylamide gel electrophoresis under nonreducing conditions suggested that pADPRp was present as a component of disulfide cross-linked complexes. Further analysis by immunofluorescence revealed that the pADPRp was diffusely distributed throughout the CaLu-1 or CHO nuclear matrix. In addition, when matrices were prepared in the absence of RNase A, pADPRp was also observed in the residual nucleoli. These observations reveal that the recovery of pADPRp with a nuclease- and salt-resistant nuclear subfraction is dependent on the source of the nuclei and on the conditions used to fractionate those nuclei. In addition, these observations raise the possibility that there might be different functional classes of pADPRp molecules within the nucleus.

RNase T affects Escherichia coli growth and recovery from metabolic stress

To determine the essentiality and role of RNase T in RNA metabolism, we constructed an Escherichia coli chromosomal rnt::kan mutation by using gene replacement with a disrupted, plasmid-borne copy of the rnt gene. Cell extracts of a strain with mutations in RNases BN, D, II, and I and an interuppted rnt gene were devoid of RNase T activity, although they retained a low level (less than 10%) of exonucleolytic activity on tRNA-C-C-[14C]A due to two other unidentified RNases. A mutant lacking tRNA nucleotidyltransferase in addition to the aforementioned RNases accumulated only about 5% as much defective tRNA as did RNase T-positive cells, indicating that this RNase is responsible for essentially all tRNA end turnover in E. coli. tRNA from rnt::kan strains displayed a slightly reduced capacity to be aminoacylated, raising the possibility that RNase T may also participate in tRNA processing. Strains devoid of RNase T displayed slower growth rates than did the wild type, and this phenotype was accentuated by the absence of the other exoribonucleases. A strain lacking RNase T and other RNases displayed a normal response to UV irradiation and to the growth of bacteriophages but was severely affected in its ability to recover from a starvation regimen. The data demonstrate that the absence of RNase T affects the normal functioning of E. coli, but it can be compensated for to some degree by the presence of other RNases. Possible roles of RNase T in RNA metabolism are discussed.

Template switching by reverse transcriptase during DNA synthesis

The ability of reverse transcriptase to make template switches during DNA synthesis is implicit in models of retrovirus genome replication, as well as in recombination and oncogene transduction. In order to understand such switching, we used in vitro reactions with purified nucleic acids and enzymes. The assay system involved the use of an end-labeled DNA primer so as to allow the quantitation of elongation on a donor template relative to the amount of elongation achieved by template switching (by means of sequence homology) when an acceptor template RNA was added. We examined several variables that affected the efficiency of the reaction: (i) the reaction time, (ii) the relative amounts of acceptor and donor template, (iii) the extent of sequence overlap between the donor and acceptor templates, and (iv) the presence or absence of RNase H activity associated with the reverse transcriptase. The basic reaction, with RNA templates and normal reverse transcriptase, yielded as much as 83% template switching. In the absence of RNase H, switching still occurred but the efficiency was lowered. Also, when the donor template was changed from RNA to DNA, there was still switching; not surprisingly, this was largely unaffected by the presence or absence of RNase H. Finally, we examined the action of the RNase H on RNA templates after primary transcription but prior to template switching. We found that in most cases, both ends of the original RNA template were able to maintain an association with the DNA product. This result was consistent with the work of others who have shown that RNase H acts as an endonuclease.

Isolation and characterization of herC, a mutation of Escherichia coli affecting maintenance of ColE1

Two modes of ColE1 DNA replication are known, one dependent on RNase H, and the other RNase H independent. The cer114 mutant of the ColE1 replicon is defective in both modes and carries a single base pair alteration 95 bp upstream of the replication origin. An Escherichia coli mutant which restored maintenance of the cer114 replicon was isolated. This host suppressor mutant is defective in RNase H and carries a herC mutation located at 62 min of the E. coli chromosome. The herC mutation is recessive to its wild-type allele and supports maintenance of the mutant replicon in the absence of RNase H. The herC mutation alone conferred cold-sensitive growth, suggesting that the herC gene product is essential for cell growth. The 1832 bp E. coli DNA fragment, containing the wild-type allele of the herC mutation, was cloned and an open reading frame for the HerC protein was determined.

A distinct form of ribonuclease H from calf thymus stimulates its homologous DNA‐polymerase‐α–primase complex

A ribonuclease H which degrades RNA specifically in RNA-DNA hybrids and, moreover, stimulates its homologous DNA-polymerase-primase complex was purified from calf thymus. The enzyme consists of a single polypeptide of molecular mass 78 kDa. It requires divalent cations for activity, and prefers Mg2+ over Mn2+. Ribonuclease H is optimally active at neutral pH and in 75 mM potassium acetate and is strongly sensitive to N-ethylmaleimide. [3H]Poly(rA).poly(dT), [3H]poly(rC).poly(dI), and [3H]RNA.M13-DNA are degraded to 3-9-mer oligoribonucleotides with similar kinetics, whereas double- or single-stranded DNA, and double- and single-stranded RNA remain unaffected. The enzyme stimulates in vitro DNA synthesis by the immunoaffinity-purified calf-thymus DNA-polymerase-alpha-primase complex threefold. When ribonuclease H is present in a three-fold molar excess to the polymerase-primase complex, twice as much primer is formed as in the absence of ribonuclease H. Ribonuclease H also stimulates the elongation rate of DNA polymerase alpha by a factor of 2-3, independent of whether primase-primed DNA templates or templates primed with oligonucleotides are used. Our results suggest that this form of ribonuclease H is a likely candidate for a genuine primer-removing enzyme in mammalian cells.

Transcriptional Activation of pBR322 DNA Can Lead to Duplex DNA Unwinding Catalyzed by the Escherichia coli Preprimosome

Mechanisms that could operate to initiate pBR322 DNA replication in the absence of RNase H and DNA polymerase I are described. Two different pathways leading to extensive unwinding of pBR322 DNA have been observed under DNA replication reaction conditions in vitro. In the presence of RNA polymerase and DNA gyrase, specifically initiated RNA II (the leading-strand primer precursor) can form an RNA-DNA hybrid with the template that starts just upstream of the origin of DNA replication and continues for about 3 kilobases. Subsequent digestion of the RNA in this RNA-pBR322 DNA hybrid results in the formation of a highly unwound DNA termed form I. If DNA gyrase is absent during the RNA polymerase-catalyzed elongation of RNA II, a stable RNA-pBR322 DNA hybrid can still form that is localized to the origin region of the genome. Formation of this hybrid activates the primosome assembly site present on the lagging-strand DNA template, by displacing it to a single-stranded conformation, thereby allowing preprimosome assembly. Once assembled, the DNA helicase activity of the preprimosome, in the presence of the single-stranded DNA binding protein and DNA gyrase but in the absence of any further transcription, can also result in extensive unwinding of pBR322 DNA. The product of this reaction, form I DNA, is more unwound than form I DNA. The formation of both form I and form I DNA is inhibited by the presence of excess RNA I, as well as by RNase H at concentrations sufficient to catalyze the normal processing of RNA II required for initiation of leading-strand DNA synthesis. These results suggest that RNA II-pBR322 DNA hybrid formation is essential to permit preprimosome assembly during pBR322 DNA replication under conditions where both RNase H and DNA polymerase I are absent.

Multiple mechanisms for initiation of ColE1 DNA replication: DNA synthesis in the presence and absence of ribonuclease H

A transcript (RNA II) of plasmid ColE1 that hybridizes with the template DNA is cleaved by RNAase H and used as a primer by DNA polymerase I. However, the plasmid can replicate in bacteria lacking both enzymes, apparently using a different mechanism of initiation of replication. Here we report in vivo and in vitro studies on initiation of DNA replication in the presence or absence of either or both enzymes. Hybridization of RNA II with the template DNA is always required for initiation. Hybridized RNA II is cleaved by RNAase H to form a primer or used as a primer without cleavage by RNAase H. Hybridization also creates a single-stranded region on the nontranscribed strand that can serve as a template for synthesis of the lagging strand in a reaction that does not require DNA polymerase I. Lagging strand synthesis terminates 17 nucleotides upstream of the normal replication origin, forcing unidirectional replication.

Genetic analysis of constitutive stable DNA replication in rnh mutants of Escherichia coli K12.

Escherichia coli rnh mutants deficient in ribonuclease H (RNase H) are capable of DNA replication in the absence of protein synthesis. This constitutive stable DNA replication (SDR) is dependent upon the recA+ gene product. The requirement of SDR for recA+ can be suppressed by rin mutations (for recA+-independent), or by lexA(Def) mutations which inactivate the LexA repressor. Thus, there are at least three genetically distinct types of SDR in rnh mutants: recA+-dependent SDR seen in rnh- rin+ lexA+ strains, recA+-independent in rnh- rin- lexA+, and recA+-independent in rnh- rin+ lexA(Def). The expression of SDR in rin- and lexA(Def) mutants demonstrated a requirement for RNA synthesis and for the absence of RNase H. The suppression of the recA+ requirement by rin mutations was shown to depend on some new function of the recF+ gene product. In contrast, the suppression by lexA-(Def) mutations was not dependent on recF+. The lexA3 mutation inhibited recA+-dependent SDR via reducing the amount of recA+ activity available, and was suppressed by the recAo254 mutation. The SDR in rnh- rin- cells was also inhibited by the lexA3 mutation, but the inhibition was not reversed by the recAo254 mutation, indicating a requirement for some other lexA+-regulated gene product in the recA+-independent SDR process. A model is presented for the regulation of the expression of these three types of SDR by the products of the lexA+, rin+ and recF+ genes.

An alternative protein factor which binds the internal promoter of Xenopus 5S ribosomal RNA genes.

In small oocytes of Xenopus species, two sets of 5S RNA genes, oocyte-type and somatic-type, are fully activated. The 5S RNA transcripts are temporarily stored, half in association with TFIIIA to form a 7S particle, the other half in association with tRNA and two proteins (p48 and p43) to form a 42S particle. It has been established previously that TFIIIA binds to the internal control region of 5S RNA genes and promotes their transcription. Here we show that protein can be translocated from the 42S particles to 5S RNA genes, but only after treatment of the particles with ribonuclease. Nevertheless, once transferred, stable protein-DNA complexes are formed and DNase-protection experiments show that binding is specific to the gene promoter, covering exactly the same sequence as TFIIIA. The DNA-binding protein is identified as p48 which, after isolation by ion-exchange chromatography, will bind to 5S RNA genes in the absence of ribonuclease.

RNase H and replication of ColE1 DNA in Escherichia coli.

Amber mutations within the rnh (RNase H) gene of Escherichia coli K-12 were isolated by selecting for bacteria capable of replicating in a sup+ background replication-defective cer-6 mutant of the ColE1 replicon. The cer-6 mutation is an alteration of one base pair located 160 nucleotides upstream of the unique replication origin of this plasmid. Subsequently, we determined the DNA alterations present within these mutants. ColE1 DNA replicated in rnh(Am) recA cells, indicating that (i) RNase H, which has been shown to be absolutely required for in vitro initiation of ColE1 DNA replication, is dispensable in vivo, and (ii) ColE1 replication in the absence of RNase H is not dependent on "stable DNA replication," which has been reported to be an alternative mode of chromosomal DNA replication. Another class of bacterial mutations was also isolated. These mutations, named herB, suppressed cer-6 replication in rnh+ bacteria. herB mutations mapped close to the polA gene on the E. coli chromosome and increased the activity of DNA polymerase I. These findings suggest that when the DNA polymerase I has an opportunity to initiate DNA synthesis before RNase H acts, the replication-defective cer-6 mutant or the wild-type ColE1 replicates in E. coli.

Escherichia coli mutants suppressing replication-defective mutations of the ColE1 plasmid.

Mutants of Escherichia coli K-12 have been isolated that suppress cer mutants, ColE1 mutants that are unable to replicate as the plasmid. These host suppressors were designated her, for host factor affecting ColE1 replication. Each her suppressor showed a characteristic pattern of suppression depending on the cer mutation used for selecting the mutant bacteria. One of the suppressors, named herA, that suppressed cer6, a single-base-pair alteration 160 base pairs upstream of the ColE1 replication origin, was genetically identified as an alteration of the rnh gene (RNase H). HerA was recessive to its wild-type allele. RNase H activity of herA cell extracts was defective. Conversely, rnh mutants that were isolated independently of ColE1 replication supported replication of cer6 DNA. Some rnh mutants manifested the HerA phenotype only above a certain transition temperature, and their RNase H activity was found to be temperature sensitive. Therefore, replication of cer6 DNA in vivo is sensitive to RNase H activity. Under the conditions that suppressed cer6, the wild-type colE1 replicon replicated normally. Then, ColE1 replication in vivo proceeds in the absence of RNase H activity, which has been shown to be required for in vitro replication of the DNA.

Interaction of Substrate Analogs with Bovine Pancreatic Ribonuclease A as Studied by &lt;sup&gt;1&lt;/sup&gt;H Nuclear Magnetic Resonance

The 270 MHz 1H NMR spectra of 3'-UMP and 3'-CMP were observed in the presence of a two-fold molar excess of bovine pancreatic RNase A [EC 3.1.27.5] at various pHs. For the C(5), C(6), and C(1') protons of these nucleotides, the pH profiles of chemical shifts induced by binding to RNase A were obtained by plotting the differences between chemical shifts in the presence and the absence of RNase A against pH. Such profiles were bell-shaped for the C(5) and C(6) protons of both 3'-UMP and 3'-CMP. However the profiles of C(1') protons were not bell-shaped but appeared to consist of two bell-shaped curves and reflect the dissociations of at least three ionizable groups. The observations for the C(1') protons suggest that there are at least two forms of complexes different from each other in the interaction reflecting the chemical shift of the C(1') proton. In order to clarify the interacting sites of ribonucleotides affecting the induced shift profile of the C(1') proton, the pH titration curves were observed for 3'-dCMP in the presence of RNase A. The induced shift profile was bell-shaped for the C(1') proton as well as for the C(5) proton of the base. This indicates that the interaction at the O(2')H [or O(2')] sites of ribonucleotides causes the two forms of complexes of 3'-UMP and 3'-CMP with RNase A. The interacting sites and modes were discussed with these and the pH titration curves of His-12, His-119, and Phe-120 of RNase A in the presence of a three-fold molar excess of ribonucleotides.

Determination of the chemical shift of the 1′ proton of cytidine 2′-phosphate in the presence and absence of RNase via phosphorus-31 detection of proton-proton decoupling

The investigation of the conformations of cellular phosphates in solution typically relies on the interpretation of the chemical shifts and scalar couplings of protons. In many cases of potential interest, such as the binding of cellular phosphates to enzymes, the proton signals are obscured by those of the enzyme. A partial solution to this problem is given by heteronuclear two-dimensional magnetic resonance which allows the determination of proton spectral information via phosphorus-31 detection. The two-dimensional approach bypasses the inability to determine proton spectral information that is obscured in a conventional experiment but is typically limited to reporting only on those protons which are scalar coupled to the phosphorus-31 nucleus. Chemical shift information about an additional group of protons, those coupled to the protons coupled to the phosphorus-31 nucleus, can be determined by phosphorus-31 detection of proton-proton decoupling. This indirect approach observes the effect of proton-proton decoupling on heteronuclear population transfer. The method is used to determine the chemical shift of the 1′ proton of cytidine 2′-phosphate in the presence and absence of RNase.

Processing of bacteriophage T4 transfer RNAs structural analysis and In Vitro processing of precursors that accumulate in RNase E − strains

Infection of the Escherichia coli RNA-processing mutant rne with the bacteriophage strain T4Δ27 (a deletion in the transfer RNA region that leaves intact only three of the ten final RNA molecules) results in a reduced accumulation of the three mature RNAs: species 1 (a small RNA of unknown function), tRNALeu and tRNAGln, as well as an accumulation of two new RNAs: 10·1 S and p2Sp1 (a precursor of species 1). Structural analyses of these molecules showed that 10·1 S RNA contains tRNAGln, tRNALeu and species 1 RNA (in this order), while p2Sp1 is similar to the distal part of the 10·1 S RNA and contains only species 1 RNA. The 10·1 S and p2Sp1 molecules share a common 3′ terminus, which is apparently formed by termination of transcription. Using cell extracts, in which RNase E was inactivated, as the source of processing enzymes and 10·1 S RNA as the substrate, production of the final three RNA molecules took place, albeit at lower rates. Heating extracts even at relatively high temperatures did not prevent the RNase E− extracts from processing p2Sp1 to species 1. These observations can be explained in at least two ways: RNase E is directly involved in the production of the three final RNA molecules, but there is at least one other enzyme, which either normally or only in the absence of RNase E carries out similar cleavages; alternatively RNase E is involved indirectly in the processing of bacteriophage T4 10·1 S RNA by affecting the activity of at least one other RNA processing enzyme. We prefer the second possibility. Thus, these studies demonstrate the existence of new T4 tRNA precursors and host RNA-processing enzyme(s), and lead to a new concept in RNA processing, whereby one enzyme can affect very drastically the function of another enzyme.

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.