Coupling of rRNA transcription and ribosomal assembly in vivo. Formation of active ribosomal subunits in Escherichia coli requires transcription of rRNA genes by host RNA polymerase which cannot be replaced by bacteriophage T7 RNA polymerase.
Transcription of a plasmid-located rrnB operon and the corresponding formation of ribosomes in vivo were studied using either T7 RNA polymerase or host RNA polymerase as transcriptase. The 23 S rRNA gene on the plasmid carried an A1067-->T mutation, which confers resistance against the drug thiostrepton. The proportion of particles containing plasmid-borne 23 S rRNA versus chromosome-borne rRNA was quantified with a precision of better than 10% by scanning sequence autoradiograms around nucleotide 1067. The activity of these particles was determined in the presence of thiostrepton which exclusively abolishes the activity of chromosomal wild-type ribosomes. When the plasmid rrnB operon was transcribed with phage T7 RNA polymerase, up to 80% of the rRNA synthesis was plasmid-directed (pulse labelling) in the late induction phase, most of which (about 85%) became degraded. The cells accumulated 50 S particles with plasmid-borne intact rRNA that was hardly found in 70 S ribosomes, i.e. particles harbouring plasmid-borne rRNA did not enter the pool of active ribosomes. The particles with plasmid-derived rRNAs were also practically inactive in protein synthesis in vitro. However, the rRNA was functional as shown by reconstitution analysis. The same patterns were found at various expression levels of the plasmid rrnB operon, indicating that not the overproduction of rRNA but rather the T7 transcriptase was responsible for the observed effects. However, when the plasmid rrnB operon was transcribed with host RNA polymerase, growth was not affected upon induction, the 30 S to 50 S to 70 S ratios in the cell were not altered, both 50 S subunits and 70 S ribosomes contained large amounts of plasmid-borne rRNA, and the particles with plasmid-derived rRNA were active in vitro. When the induction of rRNA transcription by T7 RNA polymerase was performed at 25 degrees C instead of 37 degrees C, an almost normal pattern was observed. Inactive 50 S particles did not accumulate, and large amounts of plasmid-borne rRNA were found in the pool of 70 S ribosomes. Lowering the induction temperature reduces the transcription rate by T7 RNA polymerase, which is five times faster at 37 degrees C than the host polymerase. The results suggest that the formation of active ribosomal subunits in vivo requires a fine adaptation of the transcription rate of rRNAs and the assembly process, underlining the importance of a coupling between rRNA transcription and ribosome assembly in vivo. T7 RNA polymerase cannot replace the host RNA polymerase in this process at 37 degrees C.
Max-Planck-Institut für Molekulare Genetik, Abteilung Wittmann, Berlin, Germany.
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Lewicki
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Margus
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Remme
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Nierhaus
D017123:Bacteriophage T7 / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D002499:Centrifugation, Density Gradient; D003001:Cloning, Molecular; D004274:DNA, Recombinant; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D008969:Molecular Sequence Data; D010957:Plasmids; D012336:RNA, Ribosomal, 16S / Q000235:genetics / Q000378:metabolism; D012338:RNA, Ribosomal, 23S / Q000235:genetics* / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism*; D014158:Transcription, Genetic; D014764:Viral Proteins
D004274:DNA, Recombinant; D012336:RNA, Ribosomal, 16S; D012338:RNA, Ribosomal, 23S; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases