Replication control of plasmid R1: disruption of an inhibitory RNA structure that sequesters the repA ribosome-binding site permits tap-independent RepA synthesis.
The replication frequency of plasmid R1 is controlled by an antisense RNA, CopA, that inhibits the synthesis of the replication initiator protein, RepA, at the post-transcriptional level. This inhibition is indirect and affects translation of a leader peptide reading frame (tap). Translation of tap is required for repA translation (Blomberg et al., 1992). Here we asked whether an RNA stem-loop sequestering the repA ribosome-binding site blocks tap translation-independent repA expression. Destabilization of this structure resulted in tap-independent RepA synthesis, concomitant with a loss of CopA-mediated inhibition; thus, CopA acts at the level of tap translation. Structure probing of RepA mRNAs confirmed that the introduced mutations induced a local destabilization in the repA ribosome-binding site stem-loop. An increased spacing between the repA Shine-Dalgarno region and the start codon permitted even higher repA expression. In Incl alpha/IncB plasmids, an RNA pseudoknot acts as an activator for rep translation. We suggest that the regulatory pathway in plasmid R1 does not involve an activator RNA pseudoknot.
Blomberg
P
P
Department of Microbiology, Uppsala University, Sweden.
Engdahl
H M
HM
Malmgren
C
C
Romby
P
P
Wagner
E G
EG
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D004265:DNA Helicases; D004261:DNA Replication; D004268:DNA-Binding Proteins; D015964:Gene Expression Regulation, Bacterial; D008957:Models, Genetic; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009690:Nucleic Acid Conformation; D010455:Peptides / Q000235:genetics* / Q000502:physiology; D014176:Protein Biosynthesis; D011506:Proteins; D011815:R Factors / Q000235:genetics*; D016372:RNA, Antisense / Q000737:chemistry / Q000502:physiology*; D012329:RNA, Bacterial / Q000235:genetics*; D016364:Reading Frames; D012270:Ribosomes / Q000378:metabolism*; D016415:Sequence Alignment; D015534:Trans-Activators
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D010455:Peptides; D011506:Proteins; D016372:RNA, Antisense; D012329:RNA, Bacterial; D015534:Trans-Activators; C045264:replication initiator protein; D004265:DNA Helicases