In vitro RNA transcription by the New Jersey serotype of vesicular stomatitis virus. I. Characterization of the mRNA species.

The in vitro RNA synthesis by the virion-associated RNA polymerase of vesicular stomatitis virus (VSV), New Jersey serotype, was compared with that of the serologically distinct Indiana serotype of VSV. The New Jersey serotype of VSV synthesized five distinct mRNA species in vitro, three of which were smaller than the corresponding species synthesized by the Indiana serotype of VSV. These included the mRNA's coding for the G, M, and NS proteins. By hybridization experiments, virtually no sequence homology was detected between the mRNA's of the two serotypes. Despite this lack of overall homology, the 12 to 18S mRNA species of both serotype contained a common 5'-terminal hexanucleotide sequence, G(5')ppp(5')A-A-C-A-G. The signicance of this finding in light of specific interactions between the two serotypes of VSV in vivo is discussed.     

The major RNA in prosomes of HeLa cells and duck erythroblasts is tRNA(Lys,3).

Two-dimensional gel electrophoresis of HeLa cell prosomal RNAs, 3'-end labeled by RNA ligase, revealed one prominent spot. Determination of a partial sequence at the 3'-end indicated full homology to the 18 nucleotides at the 3'-end of tRNA(Lys,3) from rabbit, the bovine and the human species. An oligonucleotide complementary to the 3'-end of tRNA(Lys,3) hybridized on Northern blots with prosomal RNA from both HeLa cells and duck erythroblasts. In two-dimensional PAGE, the major pRNA of HeLa cells co-migrated with bovine tRNA(Lys,3). Reconstitution of the CCA 3'-end of RNA from both human and duck prosomes, by tRNA-nucleotidyl-transferase, confirmed the tRNA character of this type of RNA. Furthermore, it revealed at least one additional tRNA band about 85 nt long among the prosomal RNA from both species. Finally, confirming an original property of prosomal RNA, we show that in vitro synthesized tRNA(Lys,3) hybridizes stably to duck globin mRNA, and to poly(A)(+)- and poly(A)(-)-RNA from HeLa cells.     

Characterization of ChpBK, an mRNA interferase from Escherichia coli

Escherichia coli contains a number of antitoxin-toxin modules on its chromosome, which are responsible for cell growth arrest and possible cell death. ChpBK is a toxin encoded by the ChpBIK antitoxin-toxin module. This module consists of a pair of genes, chpBI and chpBK encoding antitoxin ChpBI and toxin ChpBK, respectively. ChpBK consists of 116 amino acid residues, and its sequence shows 35% identity and 52% similarity to MazF, another E. coli toxin. MazF has been shown to be a sequence-specific (ACA) endoribonuclease that cleaves cellular mRNAs and effectively blocks protein synthesis and is thus termed as an mRNA interferase. Here we demonstrate that ChpBK is another mRNA interferase in E. coli whose induction effectively blocks cell growth in a manner similar to that of MazF. The protein synthesis as judged by incorporation of [35S]methionine was, however, reduced by only 60% upon ChpBK induction. We demonstrate that ChpBK is a new sequence-specific endoribonuclease that cleaves mRNAs both in vivo and in vitro at the 5'-or3'-side of the A residue in ACY sequences (Y is U, A, or G). The ChpBK cleavage of a synthetic RNA substrate generated a 2',3'-cyclic phosphate group at the 3'-end of the 5'-end product and a 5'-OH group at the 5'-end of the 3'-end product in a manner identical to that of MazF.