The Plasmid ColIb-P9 Antisense Inc RNA Controls Expression of the RepZ Replication Protein and Its Positive Regulator repYwith Different Mechanisms

Abstract

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...

The replicon of pSW800 from Pantoea stewartii The GenBank accession number for the sequence of the minimal replicon of pSW800 is AF310258.

“…In these plasmids, and in pSW800, repB (repY) typically overlaps with repA (repZ). This unusual organization seems crucial for the translation of Rep, since mutation of the termination codon or generation of a nonsense mutation in repB in pUM604, pUM720 and repY in ColIb-P9 significantly reduces the expression of the Rep protein (Asano et al, 1999 ;Athanasopoulos et al, 1995 ;Siemering et al, 1993). These studies suggest that, in translation coupling, ribosome translation of the upstream gene has to terminate at an exact location so that the ribosome can translate the downstream gene directly without leaving the mRNA (Athanasopoulos et al, 1995).…”

“…Plasmids in the IncB, IncIα and IncL\M groups, which are regulated post-transcriptionally by antisense RNA, are typical examples (Asano et al, 1999 ;Athanasopoulos et al, 1995 ;Siemering et al, 1993). In these plasmids the gene encoding the Rep protein overlaps with an upstream gene that is less than 90 bp in length (Asano & Mizobuchi, 1998b) and contains a ribosome-binding sequence (RBS) hidden in a stem-loop structure preventing the translation of Rep (Asano et al, 1999 ;Athanasopoulos et al, 1995 ;Siemering et al, 1993). Although the peptide translated from the upstream gene is unimportant for plasmid replication, the translation process is believed to destablize the stemloop structure (Praszkier et al, 1992).…”

Stealth regulation: biological circuits with small RNA switches

“…In IncB and other plasmids, a pseudoknot in the upstream ORF (RepB) is needed for translation of the RepA protein. Antisense RNA inhibits translation of RepB, and translation of this ORF is necessary for pseudoknot formation and, therefore, for RepA translation (Wilson et al 1994;Asano et al 1999). Why are these upstream sequences and extra ORFs in the regulatory circuit?…”

Structural Analysis of Late Intermediate Complex Formed between Plasmid ColIb-P9 Inc RNA and Its Target RNA

“…Inc RNA-The plasmid ColIb-P9 Inc RNA controls translation of repY and repZ with different mechanisms (12). By using Inc RNA 54 lacking the 5Ј leader of Inc RNA and Ps 14 , a part of RepZ mRNA complementary to loop I, we previously showed that the initial loop-loop base pairing between Inc RNA and the target in the repZ mRNA leader can compete with formation of the pseudoknot required for repZ translation (16).…”