We identified a 1,845-base-pair sequence that contains essential information for the autonomous replication and regulation of the 93-kilobase-pair Inclot group ColIb-P9 plasmid. Biochemical and genetic analyses revealed that this sequence specifies at least two structural genes, designated repZ MATERIALS AND METHODSBacteria, phages, and plasmids. The bacterial strains, phages, and plasmids used are listed in Table 1. E. coli P3478 (polAI) (9) was used to isolate the replication fragments of ColIb-P9. Strain PR13 (31) was used to prepare fraction I in in vitro protein synthesis. Strains W3110, W3110 (X ind), and C600 (supE) were used as the hosts for phage A. JM103 (23) was used as a host for M13 phages. XCH10, a composite replicon of the A phage and the ColIb-P9 plasmid, was constructed by cloning a 3.0-kb EcoRI-BglII ColIb-P9 fragment into the unique EcoRI site of XVIII (25)

The autonomous replication region of plasmid ColIb-P9 contains repZ encoding the RepZ replication protein, and inc and repY as the negative and positive regulators of repZ translation, respectively. inc encodes the antisense Inc RNA, and repY is a short open reading frame upstream of repZ. Translation of repY enables repZ translation by inducing formation of a pseudoknot containing stem-loop I, which base pairs with the sequence preceding the repZ start codon. Inc RNA inhibits both repY translation and formation of the pseudoknot by binding to the loop I. To investigate control of repY expression by Inc RNA, we isolated a number of mutations that express repY in the presence of Inc RNA. One class of mutations delete a part of another stem-loop (II), which derepresses repY expression by initiating translation at codon 10 (GUG), located within this structure. Point mutations in stem-loop II can also derepress repY translation, and the introduction of compensatory basechanges restores control of repY translation. These results not only indicate that suppressing a cryptic start codon by secondary structure is important for maintaining the translational control of repZ but also demonstrate that the position of start site for repY translation is critical for its control by Inc RNA. Thus, Inc RNA controls repY translation by binding in the vicinity of the start codon, in contrast to the control of repZ expression at the level of loop-loop interaction.The initiation phase of translation is rate-limiting in protein synthesis and often the target of control of gene expression (1). It is well established that the selection of translational start sites in prokaryotes is stimulated by base pairing between the 3Ј-end of the small ribosomal RNA (16 S rRNA) and the Shine Dalgarno (SD) 1 sequence in the mRNA, located ϳ7 bases upstream of the initiation codon (2). The ribosome-binding site (RBS) of the mRNA contains the SD sequence and the initiation codon (3). Translational control can be achieved by sequestering or opening the RBS, either by binding of trans-acting factors (RNA or protein) or by modulating the higher-ordered structure of the mRNA (2). We have been studying the various aspects of translational control found in the replication control system in plasmid ColIb-P9 (4).The replication of the ColIb-P9 plasmid (93 kilobases, IncI␣ group) depends on expression of the replication initiator protein RepZ, encoded by its autonomous replication region (5), shown schematically in Fig. 1A. The level of repZ expression is strictly controlled at the translational level by the actions of positive and negative regulatory elements, repY (6) and inc (5, 7), respectively. repY is a short open reading frame encoding 29 amino acids, the 3Ј-end of which overlaps with the 5Ј-end of repZ (6) (see Fig. 1A). An amber mutation of repY codon 11 (rep57) did not allow repZ to be translated, indicating that repY translation is required for repZ translation (6). The process of repY translation and its termination, rather than the RepY polypeptide...

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