Madeline Baer

Structure and transcription of a human gene for H1 RNA, the RNA component of human RNase P

The gene coding for H1 RNA, the RNA component of human RNase P, has been isolated and characterized from a human genomic DNA library. The sequence corresponding to the mature H1 RNA is almost identical to that previously identified using H1 RNA and a cDNA clone corresponding to it. The nucleotide sequence of the genomic clone contains an array of potential transcriptional control elements, some characteristic of transcription by RNA polymerase III and some characteristic of RNA polymerase II, as is also the case for U6 and certain other small stable RNAs. The transcription in vitro of the genomic clone shows that the gene is functional and is transcribed by RNA polymerase III. Southern hybridization analysis indicates that there is very likely only one copy of the gene for H1 RNA in the human genome.

Nucleotide sequence of the gene encoding the RNA subunit (M1 RNA) of ribonuclease P from Escherichia coli

The gene encoding the RNA subunit (M1 RNA) of RNAase P (EC 3.1.26.5) from Escherichia coli has been isolated, and its complete nucleotide sequence, including flanking regions, has been determined. The promoter region, similar to others near genes under stringent control, and the site of transcription termination have been identified. The transcript from the gene (M1 RNA) can be drawn in a secondary structure that has approximately 60% G-C base pairs. One hairpin loop of this hypothetical structure has five contiguous nucleotides complementary to invariant nucleotides in the TpsiCG loop of all E. coli tRNAs. The M1 gene, when subcloned in the plasmid pBR325, can be amplified. It directs production of functional M1 RNA. In an E. coli strain thermosensitive for RNAase P function, the size of the gene transcript is the same as in wild-type E. coli, but less mature M1 RNA is made in the mutant cells.

E. coli RNAase P has a required RNA component in vivo

RNAase P has been partially purified from three thermosensitive strains of E. coli and the thermal inactivation characteristics of each preparation have been determined. The RNAase P preparations from two of these mutant strains, ts241 and ts709, and the wild-type strain have been separated into RNA and protein components. Various mixtures of the reconstituted components have been checked in vitro for complementation of their thermal sensitivity properties. The protein component of RNAase P from ts241 and the RNA component of RNAase P from ts709, respectively, account for the thermal sensitivity of the rnaase P from the two strains. The amount of the RNA component of RNAase P is lower in ts709 than in ts241 or the wild-type parent, 4273. RNAase P partially purified from a revertant of the third mutant strain, A49, which maps at or near the ts241 mutation, has an altered charge when compared to the RNAase P from the parent strain, BF265. We conclude that mutations which affect either the protein or RNA component of RNAase P can confer thermal sensitivity on the enzyme both in vivo and in vitro.

Enzymatic cleavage of RNA by RNA

The discovery and characterization of the catalytic RNA subunit of the enzyme ribonuclease P ofEscherichia coli is described.

Differential effects of mutations in the protein and RNA moieties of RNase P on the efficiency of suppression by various tRNA suppressors

We have studied the efficiency of suppression by tRNA suppressors in vivo in strains of Escherichia coli that harbor a mutation in the rnpA gene, the gene for the protein component (C5) of RNase P, and in strains that carry several different alleles of the rnpB gene, the gene for the RNA component (M1) of RNase P. Depending on the genetic background, different efficiencies of suppression by the various tRNA suppressors were observed. Thus, mutations in rnpA have separable and distinct effects from mutations in rnpB on the processing of tRNA precursors by RNase P. In addition, the efficiency of suppression by several derivatives of E. coli tRNA(Tyr) Su3 changed as the genetic background was altered.

Preparation and characterization of RNase P from Escherichia coli

Publisher Summary This chapter describes a methodology for isolation of the RNase P holoenzyme from E. coli, preparation of the individual RNA and protein subunits, reconstitution of the holoenzyme from purified subunits, and detection of the enzymatic activity of RNase. The preparation of RNase P obtained by chromatography on Sepharose 4B is resuspended in buffer C and dialyzed against the same buffer. To improve the purification of C5 protein preparative polyacrylamide gel electrophoresis has been used with some success. This procedure involves electroelution of C5 protein from the gels followed by precipitation with acetone in order to remove traces of sodium dodecyl sulfate (SDS) from the preparation. The cell pellet is placed in a prechilled mortar, on ice, and mixed with twice its weight of cold alumina. The cells are ground by hand with a chilled pestle. When a homogeneous paste is obtained, 10 ml of buffer A is added and mixed with the paste. The supernatant is centrifuged again for 30 min at 15,500 rpm (30,000 g). About 90% of the overproduced C5 protein is in the supernatant (S30).

TRANSFER RNA PROCESSING AND GENE REGULATION

ABSTRACT Transcripts of tRNA genes in prokaryotes include sequences from adjacent genes coding for rRNA, protein or other tRNAs. RNA processing events do not merely tailor the gene transcripts to the final size of mature tRNAs but also must play a role in the regulation of expression of cotranscribed genes as is especially evident in mammalian mitochondria. A role in gene regulation is not so apparent for nuclear tRNA genes of eukaryotes. An important enzyme in the processing of the 5′ termini of tRNAs is RNase P, which is a ribonucleoprotein. The gene coding for the RNA subunit of this enzyme from E. coli has been cloned and the nucleotide sequence determined. The gene transcript has five contiguous nucleotides which are complementary to an invariant region in tRNA and which may be important in determining the substrate recognition properties of RNase P.