Elsevier

Biochimie

Volume 93, Issue 3, March 2011, Pages 533-541
Biochimie

Research paper
A maturase that specifically stabilizes and activates its cognate group I intron at high temperatures

https://doi.org/10.1016/j.biochi.2010.11.008Get rights and content

Abstract

Folding of large structured RNAs into their functional tertiary structures at high temperatures is challenging. Here we show that I-TnaI protein, a small LAGLIDADG homing endonuclease encoded by a group I intron from a hyperthermophilic bacterium, acts as a maturase that is essential for the catalytic activity of this intron at high temperatures and physiological cationic conditions. I-TnaI specifically binds to and induces tertiary packing of the P4–P6 domain of the intron; this RNA–protein complex might serve as a thermostable platform for active folding of the entire intron. Interestingly, the binding affinity of I-TnaI to its cognate intron RNA largely increases with temperature; over 30-fold stronger binding at higher temperatures relative to 37 °C correlates with a switch from an entropy-driven (37 °C) to an enthalpy-driven (55–60 °C) interaction mode. This binding mode may represent a novel strategy how an RNA binding protein can promote the function of its target RNA specifically at high temperatures.

Research highlights

► I-TnaI acts as a maturase on Thermotoga ribozyme. ► I-TnaI binds to the P4–P6 domain of the Thermotoga ribozyme. ► The binding affinity of I-TnaI to its cognate RNA increases with temperatures. ► The binding switches from entropy-driven (37 °C) to enthalpy-driven (55–60 °C).

Introduction

Most functional RNA molecules have complicated tertiary structures, such as ribosomal RNAs, RNase P RNAs, group I intron RNAs and group II intron RNAs [1]. The structural stability of these RNAs is a critical physical property directly related to their biological functions, and is known to be affected by various intrinsic factors including ratio of G–C pairs, interior molecular packing and long-range interactions [2], [3], [4], [5]. The native structure of a thermophilic ribozyme may gain increased stability by folding from less structured intermediates [6], [7]. Meanwhile, ionic conditions at which the RNA folds can greatly affect both its secondary and tertiary structural stability [8], [9], [10]. However, these factors may be inadequate to fully explain the presence of highly structured RNAs in thermophilic and hyperthermophilic organisms living at extremely high temperatures (over 100 °C, for instance), at which RNAs are prone to be thermo-denatured.

Unlike naked RNAs manipulated in test tubes, cellular RNAs are associated with various RNA binding proteins (RBPs) to form ribonucleoproteins (RNPs) immediately after they are synthesized and released from the RNA polymerase. This association is essential for regulating the processing, assembly, metabolism and eventually various biological functions of cellular RNAs [11]; aberrant association may cause diseases [12]. RBP binding is known to stabilize different RNA structures [13]. Previous studies have focused on the specificity of RBPs in recognizing their substrate RNAs [14], [15], [16], [17], [18], [19], but studies on the thermodynamic contribution of the RBP binding to the formation and stability of the structured RNAs are very limited [20], [21]. It is yet largely unknown how RBPs help the structured RNA in thermophiles.

Homing endonucleases are a diverse family of proteins encoded by the mobile genetic elements including introns and inteins. They cleave their target DNA in a highly sequence-specific manner, which leads to the transfer of their host mobile genetic elements [22], [23]. The LAGLIDADG family is the largest of the four homing endonuclease families; each member contains one or two copies of a motif that resembles the consensus ‘LAGLIDADG’ sequence [24], [25]. A few studied LAGLIDADG homing endonucleases encoded by group I introns were shown to bind to their cognate intron RNAs and to possess a maturase activity, i.e. promoting the splicing of the intron [26], [27], [28].

Tna.bL1931 intron is a self-splicing group I intron discovered in the 23S rRNA gene of Thermotoga neapolitana NS-E, a hyperthermophilic bacterium living at up to 90 °C [29]. The insertion located in the terminal loop of P8 encodes a putative LAGLIDADG homing endonuclease that is named as I-TnaI in this study [30]. This work demonstrates that I-TnaI specifically binds to the P4-P6 domain of the Tna.bL1931 intron, with the interaction mode and efficiency of its maturase activity being temperature-dependent. Interestingly, the stronger binding at higher relative to lower temperatures correlates with a switch from an entropy-driven (lower temperatures) to an enthalpy-driven interaction mode. Temperature-dependent hydrophobic interactions may in part contribute to this switch in the interaction mode.

Section snippets

Strain and clones

The T. neapolitana NS-E strain (DSM No.4359) was purchased from DSMZ, the German Resource Centre for Biological Material (Braunschweig). The sequence of the Thermotoga group I intron (AJ556785.1) and the resident i-Tnaph1931b gene (CAD89226.1) coding the putative LAGLIDADG homing endonuclease were available on GenBank. T. neapolitana NS-E was cultured under conditions as previously described [31], and cultures were then diluted in distilled water and subjected to PCR amplification using primers

I-TnaI strongly promotes Tna.bL1931 intron splicing

In order to assay the possible maturase activity of I-TnaI protein encoded by the Tna.bL1931 intron, the protein was expressed in bacteria and purified (Supplementary Methods); its sequence-specific homing endonuclease activity was evident at high temperatures (Supplementary Fig. S1). The precursor RNA of Tna.bL1931 intron containing 70nt and 40nt of 5′ and 3′ rRNA exons, respectively, was labeled during transcription in the presence of [α-32P]GTP. Self-splicing of the gel-purified precursor

Maturase facilitates its cognate intron splicing: binding to the P4–P6 domain is a hallmark

Some homing endonucleases also function as RNA binding maturases [26], but the detailed mechanism of how maturases facilitate their cognate intron splicing is not fully appreciated. Previous study of bI3 maturase has shown that it may bind to the minor groove of the P4–P6 domain of bI3 intron and bring the two ends of the domain together for compact packing [27]. I-AniI maturase binds to multiple sites in the P4–P6 domain of its cognate mitochondrial group I intron from Aspergillus nidulans (A.n

Acknowledgements

We thank Profs. Eric Westhof (CNRS, Strasbourg, France) and Sarah Woodson (Johns Hopkins University) for critical comments, and Prof. Michael J Leibowitz (UMDNJ, New Jersey) for careful reading. We are grateful to Prof. Zheng Tan and Mr. Quan Wang for kind help of the FRET experiment, and to Dr. Keqiong Ye and his lab (NIBS, Beijing) for providing us T7 RNA polymerase and protein purification help during the manuscript revision. This work is supported by the National Natural Science Foundation

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