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88 a.a.
Key reference
DOI no: 10.1038/sj.emboj.7600260 EMBO J 23:2468-2477 (2004) PubMed id: 15192703
A new alpha-helical extension promotes RNA binding by the dsRBD of Rnt1p RNAse III. N.Leulliot, S.Quevillon-Cheruel, M.Graille, H.van Tilbeurgh, T.C.Leeper, K.S.Godin, T.E.Edwards, S.T.Sigurdsson, N.Rozenkrants, R.J.Nagel, M.Ares, G.Varani. ABSTRACT
Rnt1 endoribonuclease, the yeast homolog of RNAse III, plays an important role in the maturation of a diverse set of RNAs. The enzymatic activity requires a conserved catalytic domain, while RNA binding requires the double-stranded RNA-binding domain (dsRBD) at the C-terminus of the protein. While bacterial RNAse III enzymes cleave double-stranded RNA, Rnt1p specifically cleaves RNAs that possess short irregular stem-loops containing 12-14 base pairs interrupted by internal loops and bulges and capped by conserved AGNN tetraloops. Consistent with this substrate specificity, the isolated Rnt1p dsRBD and the 30-40 amino acids that follow bind to AGNN-containing stem-loops preferentially in vitro. In order to understand how Rnt1p recognizes its cognate processing sites, we have defined its minimal RNA-binding domain and determined its structure by solution NMR spectroscopy and X-ray crystallography. We observe a new carboxy-terminal helix following a canonical dsRBD structure. Removal of this helix reduces binding to Rnt1p substrates. The results suggest that this helix allows the Rnt1p dsRBD to bind to short RNA stem-loops by modulating the conformation of helix alpha1, a key RNA-recognition element of the dsRBD.
Selected figure(s)
Figure 4.
Figure 4 Structure of loop 2. The 2F[o] -F[c] electron density map is contoured at 1. The flexible loop 2 in the NMR structure (Figure 3A) is stabilized in the X-ray structure by intermolecular interactions (indicated in blue sticks).
Figure 6.
Figure 6 (A) RNA-free Rnt1p medium-dsRBD structure in the same orientations used in the surface representations. (B) A region of positive electrostatic potential on the surface of the Rnt1p dsRBD crystal structure (blue patches) coincides with the RNA-binding surface of the protein. (C) Residues that shift upon RNA binding identify the RNA-binding surface of Rnt1p (red-coded residues). (D) Residues located close to the AGGA tetraloop as identified in the spin-labeling experiments (green-coded residues). The tetraloop interaction site is precisely determined by the spin label experiment and coincides with helix1, loop 1 and the C-terminus of helix
The above figures are reprinted from an Open Access publication published by Macmillan Publishers Ltd: EMBO J (2004, 23, 2468-2477) copyright 2004. Figures were selected by an automated process.
Literature references that cite this PDB file's key reference
PubMed id Reference
17991894 M.Catala, M.Tremblay, E.Samson, A.Conconi, and S.Abou Elela (2008).
Deletion of Rnt1p alters the proportion of open versus closed rRNA gene repeats in yeast.Mol Cell Biol, 28, 619-629.
18158302 P.Comella, F.Pontvianne, S.Lahmy, F.Vignols, N.Barbezier, A.Debures, E.Jobet, E.Brugidou, M.Echeverria, and J.Sáez-Vásquez (2008).
Characterization of a ribonuclease III-like protein required for cleavage of the pre-rRNA in the 3'ETS in Arabidopsis.Nucleic Acids Res, 36, 1163-1175.
17473849 B.M.Lunde, C.Moore, and G.Varani (2007).
RNA-binding proteins: modular design for efficient function.Nat Rev Mol Cell Biol, 8, 479-490.
17703207 T.E.Edwards, and S.T.Sigurdsson (2007).
Site-specific incorporation of nitroxide spin-labels into 2'-positions of nucleic acids.Nat Protoc, 2, 1954-1962.
16809288 B.J.Fenner, W.Goh, and J.Kwang (2006).
Sequestration and protection of double-stranded RNA by the betanodavirus b2 protein.J Virol, 80, 6822-6833.
15987808 A.K.Henras, M.Sam, S.L.Hiley, H.Wu, T.R.Hughes, J.Feigon, and G.F.Chanfreau (2005).
Biochemical and genomic analysis of substrate recognition by the double-stranded RNA binding domain of yeast RNase III.RNA, 11, 1225-1237.
16257978 J.Ohlson, M.Ensterö, B.M.Sjöberg, and M.Ohman (2005).
A method to find tissue-specific novel sites of selective adenosine deamination.Nucleic Acids Res, 33, e167.
15853796 K.Y.Chang, and A.Ramos (2005).
The double-stranded RNA-binding motif, a versatile macromolecular docking platform.FEBS J, 272, 2109-2117.
15643449 R.Stefl, L.Skrisovska, and F.H.Allain (2005).
RNA sequence- and shape-dependent recognition by proteins in the ribonucleoprotein particle.EMBO Rep, 6, 33-38.
15853794 Y.Chen, and G.Varani (2005).
Protein families and RNA recognition.FEBS J, 272, 2088-2097. The most recent references are shown first. Citation data come partly from CiteXplore and partly from an automated harvesting procedure. Note that this is likely to be only a partial list as not all journals are covered by either method. However, we are continually building up the citation data so more and more references will be included with time.
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