Mutations affecting translational coupling between the rep genes of an IncB miniplasmid

Abstract

The nature of translational coupling between repB and repA, the overlapping rep genes of the IncB plasmid pMU720, was examined. Mutations in the start codon of the promoter proximal gene, repB, reduced the efficiency of translation of both rep genes. Moreover, there was no independent initiation of repA translation in the absence of repB translation. The position of the repB stop codon was crucial for the efficient expression of repA, with the wild-type positioning being optimal. Translational coupling was found to be totally dependent on the formation of a pseudoknot structure. A model which invokes formation of a pseudoknot to facilitate initiation of repA is proposed.

Mutations affecting pseudoknot control of the replication of B group plasmids

“…The minimal medium used Construction of the lacZ fusion plasmids. Because there are no convenient sites in pMU720 that would allow the movement of DNA fragments into the lacZ fusion plasmids pMU525 and pMU2385 (Table 1) to create repA fusions, polymerase chain reaction was used to generate a fragment with appropriate restriction enzyme sites as described previously (15). The fragment consisted of nucleotides (nt) 1 to 789 of pMU720 ( Fig.…”

“…However, it is thought that they indirectly regulate Rep expression by sterically inhibiting the translation of a leader peptide. Translation of the Rep protein is believed to be dependent on the translation of the leader peptide, and the two genes are said to be translationally coupled (4, 23).The replication frequency of the B group miniplasmid pMU720 is thought to be dependent on the expression of the repA gene, which is negatively regulated primarily at the posttranscriptional level by a small countertranscript RNA, RNAI (14,15). RNAI is transcribed from the opposite strand of, and is complementary to, the leader region of the mRNA coding for repA (RNAII) (see Fig.…”

“…The replication frequency of the B group miniplasmid pMU720 is thought to be dependent on the expression of the repA gene, which is negatively regulated primarily at the posttranscriptional level by a small countertranscript RNA, RNAI (14,15). RNAI is transcribed from the opposite strand of, and is complementary to, the leader region of the mRNA coding for repA (RNAII) (see Fig.…”

“…It is postulated that stem-loop III inhibits ribosome access to the repA TIR. Previous studies have revealed that for repA to be expressed, stem-loop III must be disrupted by the translation and termination of a small leader peptide RepB, and a pseudoknot has to form (15). Pseudoknot formation is essential for the translation of repA and involves pairing between complementary sequences in RNAII.…”

“…RNAI is thought to regulate the translation of repA primarily by pairing with stem-loop I to form an RNA-RNA duplex. The major consequence of duplex formation for repA expression is the sequestering of the proximal bases required * Corresponding author. for the formation of the pseudoknot, although this duplex formation also interferes with the access of ribosomes to the repB TIR (15). In support of its primary role in the inhibition of pseudoknot formation is the recent finding that the initial site of RNAI-RNAII interaction in pMU720 involves three of the four proximal bases essential for the formation of the pseudoknot (20).…”