In vitro Transcription and Translation Directed by Caulobacter crescentus CB15 Nucleoids

Abstract

Envelope-associated nucleoids isolated from Caulobacter crescentus contained DNA-dependent RNA polymerase and nascent RNA. In the presence of nucleotide substrates, and under suitable conditions, RNA chain elongation took place at similar rates on both fast sedimenting (FS) and slow sedimenting (SS) nucleoids. Reinitiation of transcription did not occur in vitro except when exogenous holoenzyme was added to the assay system. Three major classes of RNA were synthesized in vitro. The two larger species were heterodisperse and had electrophoretic mobilities equivalent to 16-20S and 23-35S. The third class contained RNA in the region of 3-4S. The high molecular weight components were absent from RNA synthesized on FS nucleoid templates whereas the 3-4S fraction was much increased. Caulobacter crescentus nucleoids were also examined for the presence of mRNA in a protein-synthesizing system derived from Escherichia coli cells. The activity of heterologous mRNA was detectable only after the conversion of E. coli cell lysates into an mRNA-dependent translation system by treatment with micrococcal nuclease. Incorporation of [35S]methionine into trichloroacetic acid-insoluble material reflected synthesis of bona fide template-specific polypeptides by several criteria showing that SS and FS envelope-associated nucleoids contained biologically active mRNAs. Since the nucleoids were transcriptionally inactive during protein synthesis, the assay reflected the presence of mRNAs produced at different stages in the Caulobacter cell cycle. The translational capacity of the nucleoids increased markedly after transcription indicating that some RNA synthesized in vitro was mRNA. The specific activity of transcribed nucleoids as templates for protein synthesis supported the contention that the predominant RNAs synthesized in vitro were rRNAs and their precursors.