Recognition of local nucleotide conformation in contrast to sequence by a rRNA processing endonuclease.

RNase M5 of Bacillus subtilis cleaves twice in a double-helical region of a 179-nucleotide precursor of 5S rRNA to yield mature 5S rRNA (116 nucleotides) plus fragments (21 and 42 nucleotides) derived from both termini. Previous experiments had shown that the major recognition elements for the highly specific RNase M5 are in the mature domain of the precursor. However, one precursor residue, a G adjacent to the 5' cleavage site, significantly enhances the rate of its own cleavage as well as that of the 3' precursor fragment, so it must be an important component of the features recognized by the enzyme. This G residue is opposed in the helical substrate region to a C residue, which is at the 3' terminus of the mature domain, presenting the question of whether RNase M5 specifically contacts the cleavage site on the basis of nucleotide sequence (the G residue per se) or on the basis of more general aspects of helical conformation. We tested these alternatives by fabricating partially synthetic test substrates for RNase M5. Experiments were performed on 5' and 3' half-molecules derived from mature 5S rRNA. The 3'-terminal C was removed by periodate oxidation and beta elimination and replaced in a T4 RNA ligase condensation with each of the four mononucleoside bisphosphates. Artificial "precursor" segments containing each of the four nucleotides adjacent to the 5' cleavage site were added to the 5' terminus of the 5S rRNA half-molecule. We then annealed the modified half-molecules to yield test substrates containing all permutations of complementary in contrast to noncomplementary nucleotides at the cleavage site. The susceptibilities of these test substrates show that conformation, not sequence, is the important feature in the locale of the cleaved bonds.     

Synthesis of a large precursor to ribosomal RNA in a mutant of Escherichia coli

A mutant of E. coli, isolated by Kindler and Hofschneider as a strain defective in RNase III activity, forms a 30S precursor of ribosomal RNA ("30S pre-rRNA"). The half-life of the 30S pre-rRNA in growing cells at 30 degrees , estimated by the rate of specific (3)[H]uridine incorporation, is about 1 min. In rifampicin-treated cells, the RNA is metabolized to mature rRNA with a half-life of about 2 min.The 30S pre-rRNA has been highly purified. DNA-RNA hybridization tests demonstrate that it contains both 16S and 23S rRNA sequences. Also, in cultures treated with rifampicin, the cleavage products of radioactive 30S pre-rRNA include 25S and 17.5S RNA species, destined to becomes 23S and 16S rRNA. Thus, each 30S chain probably contains one 16S and one 23S RNA sequence, as well as additional sequences. Two independent techniques indicate that the additional portions account for about 27% of the total lenght: (1) By comparison to the sedimentation rate and electrophoretic mobility of marker RNAs, the 30S pre-RNA has an apparent molecular weight of 2.3 x 10(6) +/- 5%, or 28% more than the sum of 16S and 23S rRNA; (2) 27% of the 30S pre-rRNA is not competed away from hybridization by mature 16S and 23S rRNA.Thus, bacteria appear to make a pre-rRNA similar in some respects to that observed in eukaryotes; though in normal E. coli cells, the pre-rRNA is ordinarily cleaved endonucleolytically during its formation.     

Yeast RNase P: catalytic activity and substrate binding are separate functions.

During tRNA biosynthesis the 5'-leader sequences in precursor tRNAs are removed by the ribonucleoprotein RNase P, an enzyme whose RNA moiety is required for activity. To clarify some aspects of the enzyme mechanism, we examined substrate binding and product formation with mutant precursor tRNAs. Mutations G-1----A or U-2----C in the Schizosaccharomyces pombe sup3-e tRNASer, which cause mispairing at or near the top of the acceptor stem, prevent the removal of the 5'-leader sequences by Saccharomyces cerevisiae RNase P. Equilibrium binding studies involving specific gel retardation of RNase P-precursor tRNA complexes showed that complexes with wild-type and A-1 and C-2 mutant precursor tRNAs had very similar dissociation constants (average Kd for sup3 = 1.5 +/- 0.2 nM). Thus, the 5'-terminal nucleotides of mature tRNA, on the 3' proximal side of the RNase P cleavage site, affect the enzyme's catalytic function but not substrate binding. The catalytic integrity of the RNA component of RNase P is not essential for binding of tRNA precursors, as demonstrated by gel retardation of micrococcal nuclease-inactivated enzyme. This suggests a possible role for the protein component of the enzyme in substrate binding. Upon restoration of base pairing to the acceptor stem in the A-1 or C-2 mutants, we found that, in addition to a requirement for pairing at these positions, conservation of the wild-type first and second nucleotides of the tRNA was necessary to obtain maximal cleavage by RNase P. This indicates a distinct sequence preference of this enzyme.     

Ribonuclease P substrate specificity: cleavage of a bacteriophage phi80-induced RNA.

RNase P can cleave in vitro a bacteriophage phi80-induced RNA which is 62 nucleotides long [M3 RNA, G. Pieczenik et al. (1972) Arch. Biochem. Biophys. 152, 152-165] to yield two specific fragments 25 and 37 nucleotides long. As is the case for another substrate of RNase P; the precursor to Escherichia coli 4.5S RNA, the cleavage site in M3 RNA is at the end of a long double-stranded region immediately adjacent to a single-stranded segment. Similar nucleotide sequences span the cleavage site in both substrates. These and other features of the reaction of RNase P with M3 and 4.5S precursor RNA are different from some aspects of the reaction of this enzyme with tRNA precursor molecules. A qualitative scheme is presented that is directed towards the understanding of the differences in RNase P cleavage site specificity for these substrates.     

Use of T4 RNA ligase to construct model substrates for a ribosomal RNA maturation endonuclease

RNase M5 of Bacillus subtilis specifically cleaves a 179-nucleotide precursor 5S rRNA to yield mature 5S rRNA (116 nucleotides) and two fragments derived from the termini. Possible recognition elements for RNase M5 within the precursor structure include nucleotide sequences arranged with 2-fold rotational and translational symmetry about the substrate bonds. We have used bacteriophage T4 RNA ligase to construct, from synthetic oligonucleotides and mature or precursor 5S rRNA fragments, test substrates lacking these symmetry elements. The susceptibilities of the artificial substrates to RNase M5 demonstrate that the symmetrically arranged sequences are not used in the RNase M5 interaction with the precursor. Additionally, the synthetic protocols permitted the invention of an acid-soluble assay for RNase M5 and, potentially, other specific endoribonucleases.     

Complete nucleotide sequence of a 16S ribosomal RNA gene from Escherichia coli.

The complete nucleotide sequence of the 16S RNA gene from the rrnB cistron of Escherichia coli has been determined by using three rapid DNA sequencing methods. Nearly all of the structure has been confirmed by two to six independent sequence determinations on both DNA strands. The length of the 16S rRNA chain inferred from the DNA sequence is 1541 nucleotides, in close agreement with previous estimates. We note discrepancies between this sequence and the most recent version of it reported from direct RNA sequencing [Ehresmann, C., Stiegler, P., Carbon, P. & Ebel, J.P. (1977) FEBS Lett. 84, 337-341]. A few of these may be explained by heterogeneity among 16S rRNA sequences from different cistrons. No nucleotide sequences were found in the 16S rRNA gene that cannot be reconciled with RNase digestion products of mature 16S rRNA.     

Processing of the 5'' end of Escherichia coli 16S ribosomal RNA.

We have isolated and partially characterized an endonuclease involved in processing the 5' end of 16S rRNA of Escherichia coli. A mutant strain that is deficient in this enzyme accumulates a new precursor of 16S rRNA, named 16.3S rRNA. This rRNA has the 3' end of mature 16S rRNA but is about 60 nucleotides longer at the 5' end. In vitro, the enzyme preparation cleaves an RNA fragment of about 60 nucleotides from the 5' end of 16.3S rRNA in 30S ribosomal subunits, yielding the mature 5' end of 16S rRNA. In the mutant strain the 16.3S rRNA is associated with a full complement of 21 ribosomal proteins in 30S subunits. These particles, which comprise 50% of the total 30S subunits, are present on polyribosomes.     

RNase E is required for the maturation of ssrA RNA and normal ssrA RNA peptide-tagging activity

During recent studies of ribonucleolytic "degradosome" complexes of Escherichia coli, we found that degradosomes contain certain RNAs as well as RNase E and other protein components. One of these RNAs is ssrA (for small stable RNA) RNA (also known as tm RNA or 10Sa RNA), which functions as both a tRNA and mRNA to tag the C-terminal ends of truncated proteins with a short peptide and target them for degradation. Here, we show that mature 363-nt ssrA RNA is generated by RNase E cleavage at the CCA-3' terminus of a 457-nt ssrA RNA precursor and that interference with this cleavage in vivo leads to accumulation of the precursor and blockage of SsrA-mediated proteolysis. These results demonstrate that RNase E is required to produce mature ssrA RNA and for normal ssrA RNA peptide-tagging activity. Our findings indicate that RNase E, an enzyme already known to have a central role in RNA processing and decay in E. coli, also has the previously unsuspected ability to affect protein degradation through its role in maturation of the 3' end of ssrA RNA.     

T7 early RNAs and Escherichia coli ribosomal RNAs are cut from large precursor RNAs in vivo by ribonuclease 3

The early region of T7 DNA is transcribed as a single unit in a Ribonuclease III-deficient E. coli strain to produce large molecules essentially identical to those produced in vitro by E. coli RNA polymerase. As with the in vitro RNAs, these molecules are cut by purified RNase III in vitro to produce the messenger RNAs normally observed in vivo. Thus, the normal pathway for producing the T7 early messenger RNAs in vivo appears to involve endonucleolytic cleavage by RNase III. The uninfected RNase III-deficient strain contains several RNAs not observed in the parent strain. Patterns of labeling in vivo suggest that the largest of these RNAs, about 1.8 x 10(6) daltons, may be a precursor to the 16S and 23S ribosomal RNAs. When this large molecule is treated in vitro with purified RNase III, molecules the size of precursor 16S and 23S ribosomal RNAs are released; hybridization competition experiments also indicate that the 1.8 x 10(6) dalton RNA does indeed represent ribosomal RNA. Thus, RNase III cleavage seems to be part of the normal pathway for producing at least the 16S and 23S ribosomal RNAs in vivo. Several smaller molecules are also released from the 1.8 x 10(6) dalton RNA by RNase III, but it is not yet established whether any of these contain 5S RNA sequences.     

A protein subunit of human RNase P, Rpp14, and its interacting partner, OIP2, have 3'-->5' exoribonuclease activity

The processing of precursor tRNAs at their 5' and 3' termini is a fundamental event in the biosynthesis of tRNA. RNase P is generally responsible for endonucleolytic removal of a leader sequence of precursor tRNA to generate the mature 5' terminus. However, much less is known about the RNase P counterparts or other proteins that are active at the tRNA 3' terminus. Here we show that one of the human RNase P subunits, Rpp14, together with one of its interacting protein partners, OIP2, is a 3'-->5' exoribonuclease with a phosphorolytic activity that processes the 3' terminus of precursor tRNA. Immunoprecipitates of a crude human RNase P complex can process both ends of precursor tRNA by hydrolysis, but purified RNase P has no exonuclease activity. Rpp14 and OIP2 may be part of an exosome activity.     

Identification of a precursor molecular for the RNA moiety of the processing enzyme RNase P.

A precursor molecule for 10Sb (M1) RNA, the RNA moiety of the RNA processing enzyme ribonuclease P (EC 3.1.26.5), is accumulated transiently in an Escherichia coli strain containing a plasmid that carries the 10Sb RNA gene. The same RNA precursor molecule is accumulated, in relatively large quantities, in a temperature-sensitive RNase E- mutant at the nonpermissive temperature. The RNA precursor includes 10Sb RNA and an extra 3' fragment that contains a termination stem and loop. It can be processed in vitro to a molecule the size of 10Sb RNA. None of the four endoribonucleases of E. coli--RNase III, RNase E, RNase F, or RNase P--takes part in this cleavage reaction. Therefore, we suggest that the processing of the precursor-10Sb RNA to 10Sb RNA is carried out by a thus-far unidentified endoribonuclease. The accumulation of a RNA molecule in a RNase E- mutant that does not contain a cleavage site for RNase E has been encountered previously and can be explained by assuming the existence of a RNA processing complex in the E. coli cell.     

Covalent crosslinking of tRNA1Val to 16S RNA at the ribosomal P site: identification of crosslinked residues.

N-Acetylvalyl-tRNA1Val (AcVal-tRNA1Val) was bound to the P site of uniformly 32P-labeled 70S ribosomes from Escherichia coli and crosslinked to 16S RNA in the 30S ribosomal subunit by irradiation with light of 300-400 nm. To identify the crosslinked nucleotide in 16S RNA. AcVal-tRNA1Val-16S [32P]RNA was digested completely with RNase T1 and the band containing the covalently attached oligonucleotides from tRNA and rRNA was isolated by polyacrylamide gel electrophoresis. The crosslinked oligonucleotide, and the 32P-labeled rRNA moiety released from it by photoreversal of the crosslink at 254 nm, were then analyzed by secondary hydrolysis with pancreatic RNase A and RNase U2.The oligonucleotide derived from 16S RNA was found to be the evolutionarily conserved sequence, U-A-C-A-C-A-C-C-G1401, and the nucleotide crosslinked to tRNA1Val, C1400. The identity of the covalently attached residue in the tRNA was established by using AcVal-tRNA1Val-16S RNA prepared from unlabeled ribosomes. This complex was digested to completion with RNase T1 and the resulting RNA fragments were labeled at the 3' end with [5'-32P]pCp. The crosslinked T1 oligonucleotide isolated from the mixture yielded one major end-labeled component upon photoreversal. Chemical sequence analysis demonstrated that this product was derived from the anticodon-containing pentadecanucleotide of tRNA1Val, C-A-C-C-U-C-C-C-U-cmo5U-A-C-m6A-A-G39(cmo5U, 5-carboxymethoxyuridine). A similar study of the crosslinked oligonucleotide revealed that the residue covalently bound to 16S was cmo5U34, the 5' or wobble base of the anticodon. The adduct is believed to result from formation of a cyclobutane dimer between cmo5U34 of tRNA1Val and C1400 of the 16S RNA.     

Gene for lysine tRNA1 may be a progenitor of the highly repetitive and transcribable sequences present in the salmon genome.

When salmon total DNA was transcribed in a HeLa cell extract, a discrete 6S RNA was found to be synthesized by RNA polymerase III. We isolated several phage clones containing the 6S RNA gene from a salmon genomic library and determined the sequences of two representative clones. The 5' part of the gene showed remarkable sequence homology with the lysine tRNA1 molecule. This homology extended to secondary structures, and the numbers of nucleotides in the stem and loop structures in the 6S RNA were the same as those in lysine tRNA1. Further, the pseudouridylic acid residues synthesized by HeLa pseudouridylate synthase(s) were determined to be at uridine-27 and uridine-55, which are the positions of these modified nucleosides in lysine tRNA1. These results strongly suggest that the lysine tRNA1 gene is a progenitor of the highly repetitive and transcribable sequences in the salmon genome.