Interaction between the antisense and target RNAs involved in the regulation of IncB plasmid replication

Siemering, Kirby; Praszkier, Judyta; Pittard, A. J.

doi:10.1128/jb.175.10.2895-2906.1993

Cited by 44 publications

(59 citation statements)

References 54 publications

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“…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).…”

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confidence: 79%

Mutations affecting pseudoknot control of the replication of B group plasmids

1993

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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...

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confidence: 79%

Mutations affecting pseudoknot control of the replication of B group plasmids

1993

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“…4). Bases in the interior loops of the upper stem have been shown to be as critical to the interaction between the antisense molecules and their targets as bases in the hairpin loops (43), so it is not surprising that despite the similarity in their hairpin loops, the IncL/M and IncK plasmids are compatible. Similarly, the differences in the hairpin loops and the interior loops of the upper stems of the respective antisense RNAs are sufficient to account for the observation that pMU604 is also compatible with both IncB and FII plasmids (9).…”

Section: Discussionmentioning

confidence: 99%

“…Since the proximal octanucleotide sequence is located in the loop of the stem-loop structure thought to be the target of RNAI, it is likely that formation of a complex between this structure and RNAI will sequester the proximal octanucleotide sequence and thus prevent formation of the pseudoknot. This is the case in plasmids pMU720 and ColIb-P9, where it has been shown that the bases in the loop of the target of RNAI that are involved in the initial kissing interaction with the antisense RNA are also the ones that are crucial for the formation of the pseudoknot (3,37,43). Thus, in broad terms, the mechanism by which RNAI controls the expression of repA of pMU604 appears to closely resemble that described for plasmids pMU720 and ColIb-P9 (3, 4, 12, 37, 54, 55).…”

Section: Discussionmentioning

confidence: 99%

“…Both antisense and target RNAs are highly structured molecules capable of assuming stable stem-andloop configurations. These structural motifs are just as important as their primary sequences for efficient and specific interaction between antisense and target molecules (6,10,32,34,43,47,52). Because the small RNAs act in trans, they are also able to affect the replication of other plasmids showing the same specificity of replication control.…”

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

1995

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A 2,385-bp sequence that contains the information for the autonomous replication of the IncL/M plasmid pMU604 was characterized. Genetic analyses revealed that the replicon specifies at least four structural genes, designated repA, repB, repC, and rnaI. The repA gene encodes a protein with a molecular weight of 40,861 which probably functions as an initiator for replication. The functions of the proteins of the repB and repC genes are unclear; however, mutations in the start codon of repB reduced the expression of both repB and repA, indicating that these two genes are translationally coupled. The rnaI gene encodes a small antisense RNA of about 75 to 77 bases and is responsible for the incompatibility phenotype, thus implicating its role as the main copy number determinant. RNAI exerts its effect in trans to repress the expression of repA at the posttranscriptional level. Furthermore, two complementary sequences of 8 bases, with the potential to interact and form a putative pseudoknot structure, were identified in the leader region of the repA mRNA. Base-pairing between the two complementary sequences was shown to be critical for efficient repA expression. A model for the regulation of pMU604 replication involving both translational coupling and pseudoknot formation is proposed.Plasmids that employ a system of copy number control that involves inhibitor molecules binding to target sequences generally regulate their replication via a small antisense RNA molecule. The target of this inhibitory RNA is the complementary region of a large RNA transcript, whose product is essential for replication. Both antisense and target RNAs are highly structured molecules capable of assuming stable stem-andloop configurations. These structural motifs are just as important as their primary sequences for efficient and specific interaction between antisense and target molecules (6,10,32,34,43,47,52). Because the small RNAs act in trans, they are also able to affect the replication of other plasmids showing the same specificity of replication control. The ability to interact with the replication machinery of other plasmids means that antisense RNAs act as incompatibility determinants (29).Although a number of plasmids use antisense RNA to regulate the frequency with which they initiate replication, the particular molecular mechanism differs for each system. For example, replication of the ColE1 plasmids is regulated by an antisense RNA, which by binding to a preprimer RNA alters its folding and prevents its subsequent processing to form a primer for the initiation of DNA synthesis (19,24,49,50). The antisense RNAs of the pT181, FII, IncI 1 and IncB plasmids inhibit the expression of the genes coding for the essential replication initiation proteins (Rep) (13,30,31,35,42,46). In pT181, binding of the antisense alters the folding of the rep mRNA, resulting in transcriptional attenuation of the message (31). Control of copy number in the FII plasmids R1 and NR1 involves the hybridization of the antisense RNA with its complementary sequ...

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“…Previous work has demonstrated the functional importance of interior loop structures within antisense RNA mechanisms that repress translation (Asano et al 1991;Siemering et al 1993;Hjalt and Wagner 1995a,b;Kolb et al 2001b). In these systems, antisense binding initiates through a kissing hairpin interaction with structures on the sense target mRNA, proceeding to a stable four-way junction complex that ultimately results in translation inhibition (Siemering et al 1994;Kolb et al 2001a,b).…”

Section: Introductionmentioning

confidence: 99%

Using in-cell SHAPE-Seq and simulations to probe structure–function design principles of RNA transcriptional regulators

Takahashi

Watters

Gasper

et al. 2016

RNA

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Antisense RNA-mediated transcriptional regulators are powerful tools for controlling gene expression and creating synthetic gene networks. RNA transcriptional repressors derived from natural mechanisms called attenuators are particularly versatile, though their mechanistic complexity has made them difficult to engineer. Here we identify a new structure-function design principle for attenuators that enables the forward engineering of new RNA transcriptional repressors. Using in-cell SHAPE-Seq to characterize the structures of attenuator variants within Escherichia coli, we show that attenuator hairpins that facilitate interaction with antisense RNAs require interior loops for proper function. Molecular dynamics simulations of these attenuator variants suggest these interior loops impart structural flexibility. We further observe hairpin flexibility in the cellular structures of natural RNA mechanisms that use antisense RNA interactions to repress translation, confirming earlier results from in vitro studies. Finally, we design new transcriptional attenuators in silico using an interior loop as a structural requirement and show that they function as desired in vivo. This work establishes interior loops as an important structural element for designing synthetic RNA gene regulators. We anticipate that the coupling of experimental measurement of cellular RNA structure and function with computational modeling will enable rapid discovery of structure-function design principles for a diverse array of natural and synthetic RNA regulators.

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Interaction between the antisense and target RNAs involved in the regulation of IncB plasmid replication

Cited by 44 publications

References 54 publications

Mutations affecting pseudoknot control of the replication of B group plasmids

Mutations affecting pseudoknot control of the replication of B group plasmids

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

Using in-cell SHAPE-Seq and simulations to probe structure–function design principles of RNA transcriptional regulators

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