Regulation of gene expression in restriction-modification system Eco29kI

Nagornykh, Maxim; Zakharova, Marina; Protsenko, A. S.; Bogdanova, Ekaterina; Solonin, Alexander S.; Severinov, Konstantin

doi:10.1093/nar/gkr055

Cited by 14 publications

(11 citation statements)

References 19 publications

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“…Hypothetically, it might happen due to a pronounced secondary structure of mRNA in the region corresponding to R.Eco29kI ORF or, as it was reported personally by M. Nagornykh, to transcription initiation from an alternative promoter closely preceding M.Eco29kI ORF [13]. …”

Section: Resultsmentioning

confidence: 99%

Fused eco29kIR- and Mgenes coding for a fully functional hybrid polypeptide as a model of molecular evolution of restriction-modification systems

2011

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BackgroundThe discovery of restriction endonucleases and modification DNA methyltransferases, key instruments of genetic engineering, opened a new era of molecular biology through development of the recombinant DNA technology. Today, the number of potential proteins assigned to type II restriction enzymes alone is beyond 6000, which probably reflects the high diversity of evolutionary pathways. Here we present experimental evidence that a new type IIC restriction and modification enzymes carrying both activities in a single polypeptide could result from fusion of the appropriate genes from preexisting bipartite restriction-modification systems.ResultsFusion of eco29kIR and M ORFs gave a novel gene encoding for a fully functional hybrid polypeptide that carried both restriction endonuclease and DNA methyltransferase activities. It has been placed into a subclass of type II restriction and modification enzymes - type IIC. Its MTase activity, 80% that of the M.Eco29kI enzyme, remained almost unchanged, while its REase activity decreased by three times, concurrently with changed reaction optima, which presumably can be caused by increased steric hindrance in interaction with the substrate. In vitro the enzyme preferentially cuts DNA, with only a low level of DNA modification detected. In vivo new RMS can provide a 102-fold less protection of host cells against phage invasion.ConclusionsWe propose a molecular mechanism of appearing of type IIC restriction-modification and M.SsoII-related enzymes, as well as other multifunctional proteins. As shown, gene fusion could play an important role in evolution of restriction-modification systems and be responsible for the enzyme subclass interconversion. Based on the proposed approach, hundreds of new type IIC enzymes can be generated using head-to-tail oriented type I, II, and III restriction and modification genes. These bifunctional polypeptides can serve a basis for enzymes with altered recognition specificities. Lastly, this study demonstrates that protein fusion may change biochemical properties of the involved enzymes, thus giving a starting point for their further evolutionary divergence.

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Section: Resultsmentioning

confidence: 99%

Fused eco29kIR- and Mgenes coding for a fully functional hybrid polypeptide as a model of molecular evolution of restriction-modification systems

2011

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“…The second mechanism of coordinated expression of R-M system seems to apply to bicistronic systems where the REase gene precedes the MTase, as in EcoRI and Eco29kI ( 21 – 24 ). In these cases antisense RNAs produced from oppositely-oriented promoters negatively regulate REase and MTase expression ( 23 , 24 ). Weakening or knocking out an antisense promoter results in enhancement of REase expression to the point of toxic enzyme accumulation and cell death ( 23 ).…”

Section: Introductionmentioning

confidence: 99%

Natural C-independent expression of restriction endonuclease in a C protein-associated restriction-modification system

2015

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Restriction–modification (R-M) systems are highly prevalent among bacteria and archaea, and appear to play crucial roles in modulating horizontal gene transfer and protection against phage. There is much to learn about these diverse enzymes systems, especially their regulation. Type II R-M systems specify two independent enzymes: a restriction endonuclease (REase) and protective DNA methyltransferase (MTase). Their activities need to be finely balanced in vivo. Some R-M systems rely on specialized transcription factors called C (controller) proteins. These proteins play a vital role in the temporal regulation of R-M gene expression, and function to indirectly modulate the horizontal transfer of their genes across the species. We report novel regulation of a C-responsive R-M system that involves a C protein of a poorly-studied structural class - C.Csp231I. Here, the C and REase genes share a bicistronic transcript, and some of the transcriptional auto-control features seen in other C-regulated R-M systems are conserved. However, separate tandem promoters drive most transcription of the REase gene, a distinctive property not seen in other tested C-linked R-M systems. Further, C protein only partially controls REase expression, yet plays a role in system stability and propagation. Consequently, high REase activity was observed after deletion of the entire C gene, and cells bearing the ΔC R-M system were outcompeted in mixed culture assays by those with the WT R-M system. Overall, our data reveal unexpected regulatory variation among R-M systems.

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“…Analyzing other R-M systems can further test relation of the system features with the simple dynamical properties, where the main obstacle is that their transcription regulation is generally not well studied. In particular, investigating up to now poorly understood linear R-M systems, which have different architecture compared to the convergent and the divergent systems studied here, and which do not encode C proteins – but may exhibit control by antisense RNAs or at the level of translation initiation efficiency - may be particularly useful [ 43 , 44 ]. As a further outlook, it will be interesting investigating if properties of other bacterial immune systems, such as recently discovered CRISPR/Cas systems [ 45 ], can also be explained by similar dynamical properties [ 34 ].…”

Section: Discussionmentioning

confidence: 99%

Understanding key features of bacterial restriction-modification systems through quantitative modeling

et al. 2017

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BackgroundRestriction-modification (R-M) systems are rudimentary bacterial immune systems. The main components include restriction enzyme (R), which cuts specific unmethylated DNA sequences, and the methyltransferase (M), which protects the same DNA sequences. The expression of R-M system components is considered to be tightly regulated, to ensure successful establishment in a naïve bacterial host. R-M systems are organized in different architectures (convergent or divergent) and are characterized by different features, i.e. binding cooperativities, dissociation constants of dimerization, translation rates, which ensure this tight regulation. It has been proposed that R-M systems should exhibit certain dynamical properties during the system establishment, such as: i) a delayed expression of R with respect to M, ii) fast transition of R from “OFF” to “ON” state, iii) increased stability of the toxic molecule (R) steady-state levels. It is however unclear how different R-M system features and architectures ensure these dynamical properties, particularly since it is hard to address this question experimentally.ResultsTo understand design of different R-M systems, we computationally analyze two R-M systems, representative of the subset controlled by small regulators called ‘C proteins’, and differing in having convergent or divergent promoter architecture. We show that, in the convergent system, abolishing any of the characteristic system features adversely affects the dynamical properties outlined above. Moreover, an extreme binding cooperativity, accompanied by a very high dissociation constant of dimerization, observed in the convergent system, but absent from other R-M systems, can be explained in terms of the same properties. Furthermore, we develop the first theoretical model for dynamics of a divergent R-M system, which does not share any of the convergent system features, but has overlapping promoters. We show that i) the system dynamics exhibits the same three dynamical properties, ii) introducing any of the convergent system features to the divergent system actually diminishes these properties.ConclusionsOur results suggest that different R-M architectures and features may be understood in terms of constraints imposed by few simple dynamical properties of the system, providing a unifying framework for understanding these seemingly diverse systems. We also provided predictions for the perturbed R-M systems dynamics, which may in future be tested through increasingly available experimental techniques, such as re-engineering R-M systems and single-cell experiments.Electronic supplementary materialThe online version of this article (doi:10.1186/s12918-016-0377-x) contains supplementary material, which is available to authorized users.

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Regulation of gene expression in restriction-modification system Eco29kI

Cited by 14 publications

References 19 publications

Fused eco29kIR- and Mgenes coding for a fully functional hybrid polypeptide as a model of molecular evolution of restriction-modification systems

Fused eco29kIR- and Mgenes coding for a fully functional hybrid polypeptide as a model of molecular evolution of restriction-modification systems

Natural C-independent expression of restriction endonuclease in a C protein-associated restriction-modification system

Understanding key features of bacterial restriction-modification systems through quantitative modeling

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