Mutations affecting pseudoknot control of the replication of B group plasmids

Abstract

The translational initiation region of the mRNA for the replication initiation protein (RepA) of pMU720 is predicted to be sequestered in an inhibitory secondary structure designated stem-loop III. Activation of repA translation requires both the disruption of stem-loop III by ribosomes involved in the translation and termination of the leader peptide RepB and the formation of a pseudoknot, a tertiary RNA structure.Disruption of stem-loop III by site-directed mutagenesis was found to be insufficient to allow high repA expression in the absence of pseudoknot formation, indicating that the pseudoknot acts as an enhancer of repA translation. Furthermore, extending the length of the leader peptide RepB and changing the distance between the pseudoknot and repA Shine-Dalgarno sequence were found to have major elfects on the translation of repA.Plasmid replication in prokaryotes is, in many cases, dependent on a replication initiation protein (Rep), whose expression determines a plasmid's copy number and stability. In the case of pT181 and IncFII plasmids, the regulators of Rep synthesis are small countertranscript RNAs which inhibit Rep expression by binding with their target RNAs. In pT181, binding of the countertranscript RNA to the mRNA for the Rep protein is proposed to cause premature transcriptional termination by altering the folding of the RNA (9, 13). The mechanism by which the countertranscript RNAs of the IncFII plasmids Rl and NR1 regulate Rep expression is not clear. 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. 1). Computer analysis of the folding of RNAII indicates that the translational initiation region (TIR) of repA is sequestered within a secondary structure designated stem-loop III (see Fig. 1 and Fig. 2). 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. One of these sequences lies in the loop of a large structure called stem-loop I (proximal pseudoknot sequence), which is complementary to RNAI (see Fig. 2), and the other involves bases adjacent to the ShineDalgarno (SD) sequence of repA (distal pseudoknot sequence). RNAI is thought to...

The replication of an IncL/M plasmid is subject to antisense control

“…In these plasmids, translation of the mRNA is coupled to, and therefore dependent on, translation of an upstream sequence encoding a small leader peptide. It is thought that the binding of the antisense RNA sterically inhibits translation of the mRNA for the leader peptide, a process which is essential for the translation of the mRNA for the Rep protein (5, 57).In the case of the IncB and IncI 1 plasmids, it has been found that the translation initiation regions (TIR) of the mRNAs encoding for the Rep proteins are very inefficient, not only because they are sequestered within secondary structures but also because they have poor agreement with the consensus sequence for ribosomal binding (4,38,54). Translation of the mRNA for these rep proteins is dependent on the formation of an RNA pseudoknot, which appears to act as a translational enhancer (12,36).…”

“…In the case of the IncB and IncI 1 plasmids, it has been found that the translation initiation regions (TIR) of the mRNAs encoding for the Rep proteins are very inefficient, not only because they are sequestered within secondary structures but also because they have poor agreement with the consensus sequence for ribosomal binding (4,38,54). Translation of the mRNA for these rep proteins is dependent on the formation of an RNA pseudoknot, which appears to act as a translational enhancer (12,36).…”

“…Since the distal pseudoknot sequence normally lies within the paired structure that also includes the rep TIR, formation of pseudoknot requires that this stem-loop structure be disrupted by the ribosomes translating the leader peptide (3,4,37,54). Thus, the antisense RNAs of B and I 1 plasmids control their replication frequency both directly, by competing for bases required for formation of the pseudoknot, and indirectly, by preventing translation of the leader peptide (3,4,12,37,54).In this paper, the region essential for the autonomous replication of the IncL/M plasmid pMU604 has been characterized. On the basis of these studies, it is proposed that (i) an antisense RNA is the primary copy number control element of this plasmid, (ii) the target of the antisense RNA lies within the leader region of the messenger for a Rep protein, (iii) the antisense RNA regulates the expression of rep at the level of translation, and (iv) expression of rep involves both translational coupling and pseudoknot formation.…”

“…The proximal one of these sequences lies within the loop of a stem-loop structure that is the target of the antisense RNA, and the distal sequence adjoins the rep Shine-Dalgarno (SD) sequence. Since the distal pseudoknot sequence normally lies within the paired structure that also includes the rep TIR, formation of pseudoknot requires that this stem-loop structure be disrupted by the ribosomes translating the leader peptide (3,4,37,54). Thus, the antisense RNAs of B and I 1 plasmids control their replication frequency both directly, by competing for bases required for formation of the pseudoknot, and indirectly, by preventing translation of the leader peptide (3,4,12,37,54).…”

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

“…Specifically, they block the translation of a rep leader peptide (RLP) coupled to the translation of rep (5)(6)(7)(8)(9), by binding to the vicinity of the ribosome-binding site (RBS) for the RLP (10 -12). In the case of IncI␣ ColIb-P9 and IncB pMU720 plasmids, the translation of RLP (repY and repB) induces formation of a pseudoknot between the target stem-loop and a sequence preceding the rep (repZ and repA, respectively) RBS, which is required for rep translation (13)(14)(15). The antisense RNA additionally inhibits the pseudoknot formation by binding to the loop of the target stem-loop structure (16).…”