John Abelson

Mechanism of action of a yeast RNA ligase in tRNA splicing

Abstract The yeast endonuclease and ligase activities that carry out the splicing of tRNA precursors in vitro have been physically separated. The properties of a partially purified ligase fraction were examined. The ligase requires a divalent cation and a nucleoside triphosphate as cofactor. The product of ligation is a 2′-phosphomonoester, 3′,5′-phosphodiester linkage. The phosphate in the newly formed phosphodiester bond comes from the γ position of ATP, while the 2′ phosphate is derived from the RNA substrate. An adenylylated enzyme intermediate was identified by incorporation of label from α- 32 P-ATP. Adenylylation was reversed by pyrophosphate, releasing ATP, whereas ligation was accompanied by release of AMP. Polynucleotide kinase and cyclic phosphodiesterase activities copurify with the adenylylated protein and may be required for the tRNA splicing reaction.

Mutations in the lactose operon caused by bacteriophage Mu

Abstract Infection of Escherichia coli by baoteriophage Mu leads to a 100-fold increase in the frequency of lac − mutations in the surviving lysogens. The Mu-induced lac Z mutants were mapped and the mutants were located in 11 of 16 genetic segments of the lac Z gene. Some 13% of the Mu-induced lac Z gene mutants were total deletions of the gene. The Mu prophage is closely linked to the site of mutation in the mappable mutants but is unlinked to the lac Z deletions. It is suggested that the lac Z deletions are caused by a Mu-induced recombination system whereas the mappable mutations are caused by Mu prophage integration into the lac Z gene. Induction of the lac operon leads to a decrease in the frequency of Mu-induced insertion mutations but it does not affect the frequency of deletions.

Bacteriophage T4 Transfer RNA

Two T4-coded nonsense suppressors, psu+a and psu+b, have been isolated and characterized. Both were isolated as pseudo-wild type revertants of phage strains which carry multiple amber mutations. psu+a is an amber suppressor which occurs at a frequency of 10-11 to 10-12 and is indistinguishable from wild type phage in its growth on both B and K strains of E. coli bacteria. psu+b may be either an amber or on an ochre suppressor which occurs at a frequency of 10-7 - 10-10 and makes small plaques on B strains but grows very poorly or not at all on K strains. Phage with the characteristics of psu+a occure in populations of psu+b phage at a frequency of 10-4. Both suppressors insert serine in response to the amber codon at an efficiency of about 45%. psu+a and psu+b map less than 0.3 map units apart and are located between genes e and 57 about 8 map units from gene e. On the basis of their initial frequencies of appearance and the frequency of psu+b mutation to psu+a, we speculate that psu+a is derived from the wild type ser-tRNA by two base changes in the anticodon and that psu+b is a one-base-change intermediate. 151 independent suppressor-negative derivatives of psu+b phage have been isolated and characterized. They fall into two complementation groups. One, designated mb (modifier of psu+b phenotype), is unlinked to psu+b and has been located about 10 map units from rII. The second group, designated psu+b, is made up of deletions and single base changes which affect sites within 0.2 map units of the original mutation. Those psu-b mutants which still contain the original mutation, psub, have been mapped relative to psub and each other by a series of two-factor and intragenic three-factor crosses. 32P-labeles tRNA from mb, psu-b and wild type infected cells have been compared by polyacrylamide gel electrophoresis. In mb-infected cells several of the tRNA species are missing, while in psu-b-infected cells only the ser-tRNA is clearly absent. These studies suggest there are only two phage genes which are essential for the production of functional ser-tRNA. One is the structural gene for the ser-tRNA and the second plays an undefined role which affects several tRNAs. E. coli cells infected with phage strains carrying a large deletion of gene e or gene psu+b, are missing most if not all of the phage tRNAs normally present in wild type infected cells. By DNA-RNA hybridization we have demonstrated that the DNA corresponding to the missing tRNAs is absent. Thus the genes for these tRNAs must be clustered in the same region of the genome as the ser-tRNA gene. We have been able to locate and to define a maximum size for the cluster by physically mapping the deletions of genes e and psu+b by examination of heteroduplex DNA in the electron microscope. That such deletions can be isolated indicates that the phage-specific tRNAs from this cluster are dispensable.

Cell-Free Circularization of Viroid Progeny RNA by an RNA Ligase from Wheat Germ

Linear, potato spindle tuber viroid RNA has been used as a substrate for an RNA ligase purified from wheat germ. Linear viroid molecules are efficiently converted to circular molecules (circles) which are indistinguishable by electrophoretic mobility and two-dimensional oligonucleotide pattern from viroid circles extracted from infected plants. In light of recent evidence for multimeric viroid replication intermediates, cleavage followed by RNA ligation by a cellular enzyme may (i) be a normal step in the viroid life cycle and (ii) may also reflect cellular events.

In vitro transcription and processing of a yeast tRNA gene containing an intervening sequence.

A gene for Saccharomyces cerevisiae tRNATrp has been sequenced which contains an intervening sequence of 34 bp (H. S. Kang and J. Abelson, unpublished results). The mutant yeast strain ts-136 accumulates a precursor to tRNATrp which contains mature ends and is colinear with the tRNATrp gene. A nuclear extract from Xenopus oocytes is capable of supporting transcription of the tRNATrp gene contained on plasmid pBR313. The products are precursor tRNAs which contain the intervening RNA sequence. The Xenopus extract accurately splices the precursor transcript to mature-sized tRNATrp.

Mutant tyrosine tRNA of altered amino acid specificity

Each set of tRNA molecules accepting a particular amino acid are recognized and acylated by an amino acyl tRNA synthetase. This enzyme must recognize some feature in this set of tRNA molecules which distinguishes them from all other tRNAs. In this communication we describe the isolation and initial characterization of a set of E. coli tyr tRNA mutants that have altered amino acid specificity. These mutants should eventually lead to an understanding of which features of the tRNA are recognized by the tyrosine tRNA synthetase. Sequence analysis of one of the mutants (SU+III~-“~) has revealed that a single base change (A82 + G) near the amino acid acceptor end of the molecule is sufficient to alter its specificity.

Bacteriophage T4 transfer RNA: II. Mutants of T4 defective in the formation of functional suppressor transfer RNA

Abstract 151 independent suppressor-negative derivatives of a T4-coded nonsense suppressor, psub+, have been isolated and characterized. They fall into two complementation groups. One, designated mb (modifier of psub+ phenotype), is unlinked to psub+ and has been located about 10 map units from rII. The second group, designated psub−, includes deletions and single base changes which affect sites within 0.2 map units of the original mutation. The secondary mutations in those psub− mutants which still contain the original mutation, psub, have been positioned within the gene for the serine-transfer RNA by a series of two- and threefactor genetic crosses. 32P-labeled tRNA from mb, psub− and wild-type infected cells has been compared by polyacrylamide gel electrophoresis. In mb-infected cells several of the tRNA species are missing, while in psub−-infected cells only the ser-tRNA is clearly absent. These studies suggest there are only two phage genes which are essential for the production of functional ser-tRNA. One is the structural gene for the ser-tRNA and the second plays an undefined role which affects several tRNAs.

DNA sequence of a T4 transfer RNA gene cluster

Abstract Bacteriophage T4 codes for eight tRNAs, whose genes are tightly clustered between genes e and 57. The clustering of the transfer RNA genes suggested that these tRNAs are synthesized in a single transcriptional unit. Transcriptional experiments in vitro and experiments using the λ-T4 hybrid in vivo have supported this notion and located a T4 tRNA promoter. Detailed biochemical pathways of parts of the T4 tRNA processing are known. Maturation of the 3′ end precedes that of the 5′ end, and the 3′ ends of three dimeric precursors are matured in different ways by tRNA nucleotidyltransferase and ribonuclease BN. All T4 tRNAs require RNAase P for their 5′ maturation. Monomeric and dimeric precursors accumulate in the absence of RNAase P. How these monomeric and dimeric precursors are synthesized from their primary transcript, however, is poorly understood. Using the λ-T4 hybrid, carrying tRNA genes, and a fine restriction map of the tRNA gene cluster, we have determined the DNA sequence of the tRNA gene cluster, hoping that understanding of gene organization may suggest how the tRNAs are processed from their primary transcript. The DNA sequence of the tRNA gene cluster indicates that the monomeric and dimeric precursors are generated from their primary transcript by single endonucleolytic cleavages. We propose a model for the maturation of T4 tRNAs by predicting such an endonuclease (or endonucleases). The proposed endonucleolytic activities exist in the host. This model answers various questions about processing and reveals the unique features of T4 tRNA processing.

Bacteriophage Mu integration: On the mechanism of Mu-induced mutations☆

Abstract Infection of Escherichia coli with bacteriophage Mu leads to an increase in the mutation frequency in a wide variety of genes. Taylor (1963) has proposed that these mutations are caused by the integration of the Mu prophage into the gene. The experiments reported in this paper show that the Mu prophage is genetically located at or near the site of the Mu-induced mutation in accord with Taylors proposal. A Mu-induced chl-D mutation between the λ prophage attachment site and the gal operon prevents λ from transducing gal genes. It is possible to isolate λ-transducing particles from lysates of this strain which carry portions of the Mu prophage. These particles are packaged in λ capsids. Further aspects of Mu mutagenesis are discussed.

In vitro construction of bacteriophage λ and plasmid DNA molecules containing DNA fragments from bacteriophage T4

Restriction endonucleases EcoRI and HindIII generated fragments of T4 cytosine-containing DNA were inserted into bacteriophage vector lambdagtSuIII and plasmid vectors pMB9 and pBR313. Resulting clones were screened for hybridization with 32P labeled T4 tRNA. Recombinant bacteriophages and plasmids were isolated which contained a T4 fragment coding for T4 RNA species 1 and 2 and T4 tRNA Arg. Selected lambda-T4 hybrid bacteriophages were grown to high titer and their DNA analyzed by gel electrophoresis.