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F J Schmidt

Publications and source records attributed to F J Schmidt.

47 records · Page 3Linked to original sources
PubMed1978

An Escherichia coli ribonuclease which removes an extra nucleotide from a biosynthetic intermediate of bacteriophage T4 proline transfer RNA.

The biosynthesis of bacteriophage T4 tRNAPro, tRNASer, and tRNAIle requires enzymatic removal of extra nucleotides from the 3' terminus of the respective precursor RNAs. A ribonuclease activity capable of catalyzing such reactions has been partially purified from uninfected Escherichia coli using an artificial precursor RNA as substrate. A number of ribonuclease activities were resolved during purification. Use of E. coli strain BN, a mutant known to be deficient in the relevant ribonuclease activity, permitted us to identify it in wild-type cells. This activity was designated the BN ribonuclease. BN ribonuclease had an apparent molecular weight of 35,000 as measured by Sephadex gel filtration. Mg2+ was required for activity, which was optimal at [Mg2+] of 2mM. Activity did not require monovalent cations K+ or Na+. BN ribonuclease was less efficient at removing extra residues in the biosynthesis of tRNASer and tRNAIle than in the biosynthesis of tRNAPro.

Base Sequence↗
PubMed1977

Preparation of Escherichia coli tRNAs terminating of modified nucleosides by the use of CTP(ATP):tRNA nucleotidyltransferase and polynucleotide phosphorylase.

Two procedures were investigated for the modification of tRNAs at the 3'-terminal nucleoside. The first involved the incubation of an enzymatically abreviated tRNA (tRNA-C-COH) with appropriate nucleoside triphosphates in the presence of CTP(ATP):tRNA nucleotidyltransferase from Escherichia coli and yeast. The E. coli enzyme did not utilize 2'- or 3'-deoxyadenosine 5'-triphosphate as substrates, but affected incorporation of the 2'- and 3'-O-methyladenosine triphosphates onto tRNA-C-Cou to the extent of 30 and 37%, respectively. Although incorporation of the deoxynucleotides could not be effected using the E. coli enzyme, yeast CTP(ATP:tRNA nucleotidyltransferase produced the desired tRNAs in yields of 45-65%. The second modification procedure involved incubation of tRNA-C-COH with (appropriately blocked) nucleoside diphosphates in the presence of polynucleotide phosphorylase. This procedure afforded the tRNAs terminating in 2'- and 3'-deoxyadenosine in yields of 4% (and the yield of the former was increased to 36% when the incubation was carried out in the presence of 20% methanol). The yields of tRNAs terminating in 2'- and 3'-O-methyladenosing produced by this procedure were 55 and 17%, respectively. Because only single isomers of most of the tRNAs terminating in 2'- and 3'-deoxy- and O-methyladenosine are aminoacylated, attempts were made to obtain the other isomericaminoacyl-tRNA by enzymatic introduction of chemically preaminoacylated nucleotides onto tRNA-C-COH. Although incubation of tRNA-C-COH with three aminoacylated nucleoside 5'-triphosphates and E. coli CTP(ATP):tRNA nucleotidyltransferase did not result in production of the desired tRNAs to a detectable extent, incubation with 2'-deoxy-3'-O-L-phenylalanyladenosine 5'-diphosphate and polynucleotide phosphorylase afforded E. coli tRNA terminating with the corresponding aminoacylated deoxynucleoside.

Adenosine Diphosphate↗
PubMed1976

Transfer ribonucleic acid biosynthesis. Substrate specificity of ribonuclease P.

Bacteriophage T4 synthesizes proline and serine tRNA species which are derived from a common precursor RNA. The processing of this precursor RNA involves the replacement of a U-A-A terminus in serine tRNA by C-C-A prior to precursor cleavage. In the present work we have examined in detail the cleavage of T4 proline-serine precursor RNA by the previously identified ribonuclease P. Ribonuclease P accurately cleaves precursor RNA terminating in either C-C-A or U-A-A to generate the 5' termini characteristic of both mature tRNA species. These cleavages do not depend solely on the nucleotide sequence of the precursor RNA since isolated oligonucleotides spanning the cleavage sites are not substrates for the enzyme. Two types of experiments show that RNase P kinetically favors precursor RNA ending C-C-A over that ending U-A-A. Isolated preparations of precursor RNA containing the C-C-A sequence were cleaved more rapidly by RNase P than precursor RNA ending U-A-A. In addition, the serine tRNA generated by limited cleavage of a mixed population of precursor RNA ending C-C-A or U-A-A was enriched 3-fold in the C-A-A sequence relative to the starting material. Bacteriophage T4 proline-serine precursor RNA, in contrast to other tRNA precursors, accumulates in measurable amounts in wild type cells. This accumulation would appear to be a consequence of the requirement for the generation of the C-C-A sequence prior to RNase P cleavage. The enzymic specificity of RNase P in vitro therefore reflects the in vivo pathway for serine tRNA biosynthesis, where the C-C-A sequence is synthesized while the serine tRNA sequence is still a part of the large precursor RNA.

Base Sequence↗
PubMed1975

A novel function of Escherichia coli transfer RNA nucleotidyltransferase. Biosynthesis of the C-C-A sequence in a phage T4 transfer RNA precursor.

The biosynthesis of the phage T4-coded proline and serine transfer RNA species proceeds through a precursor RNA containing both tRNA sequences. Neither tRNA sequence in the precursor RNA contains the 3'-terminal C-C-A common to all mature tRNAs. Seidman and McClain ((1975) Proc. Natl. Acad. Sci. U. S. A. 72, 1491-1495) have proposed that the C-C-A sequence is added to serine tRNA while it is still part of the large precursor RNA. In the present work, I show that, in vitro, a purified preparation of Escherichia coli tRNA nucleotidyltransferase (EC 2.7.7.25) accurately synthesized 3'-terminal C-C-A in the serine tRNA portion of the precursor RNA. This result establishes a role of tRNA nucleotidyltransferase in the biosynthesis of the phage T4 serine tRNA. The finding that tRNA nucleotidyltransferase utilizes the large precursor RNA as a substrate represents a novel function of the enzyme.

Adenine Nucleotides↗
PubMed1975

A mutant of escherichia coli defective in removing 3' terminal nucleotides from some transfer RNA precursor molecules.

The conversion of precursor RNA into bacteriophage T4 proline and serine transfer RNAs includes two steps for the enzymatic removal of nucleotides from the 3' ends of RNA chains. Neither of these steps occur following infection of a mutant of Escherichia coli that was previously shown to block the suppressor function of T4 serine transfer RNA. Cell-free extracts of this mutant are furthermore deficient in a wild type enzyme activity that removes nucleotides from the 3' ends of one of the RNA chains described above. The relation of this enzyme to other 3' ribonucleases is not known. We subsequently examined the mutant for its ability to support the biosynthesis of other bacteriophage transfer RNAs. In one instance that is analogous to the proline-serine precursor RNA, maturation of the precursor RNA was blocked during infection of mutant cells. In another instance, precursor RNA maturation was normal, even though this involved the removal of 3'nucleotides. These observations point to the possible existence of at least two 3' ribonucleases for the biosynthesis of transfer RNAs.

Base Sequence↗
PubMed1974

Isomeric phenylalanyl-tRNAs. Position of the aminoacyl moiety during protein biosynthesis.

The preparation of phenylalanyl-tRNA terminating in 3'-deoxyadenosine has been achieved by incubation of abbreviated tRNA (tRNA-CpC(OH)) with 3'-deoxyadenosine 5'-diphosphate and polynucleotide phosphorylase (EC 2.7.7.8), followed by aminoacylation. The isomeric phenylalanyl-tRNA terminating in 2'-deoxyadenosine was constructed by incubation of tRNA-CpC(OH) with 2'-deoxy-3'-O-phenylalanyladenosine 5'-diphosphate and polynucleotide phosphorylase. While tRNA is aminoacylated at the 2'-position, only the 3'-aminoacyl-tRNA is active as a peptide acceptor in the peptidyltransferase reaction. Both modified tRNAs were bound to the A-site as efficiently as unmodified tRNA, but neither was so efficient at P-site binding or as an acceptor in the peptidyltransferase reaction. Neither of the modified tRNAs acted as a donor in the peptidyltransferase reaction.

Amino Acyl-tRNA Synthetases↗