Conformation-controlled hydrolysis of polyribonucleotides by sequential basic polypeptides

Barbier, Bernard; Brack, Andrè

doi:10.1021/ja00035a053

Cited by 88 publications

(37 citation statements)

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“…Sci. USA 96 (1999) 10013 dinary (13)(14)(15)(16)(17). Unlike these other ribonuclease mimics, the ZFY-6 peptide requires no intercalating moieties or other conjugates to enhance binding to the substrate.…”

Section: Discussionmentioning

confidence: 99%

Highly efficient endonucleolytic cleavage of RNA by a Cys ₂ His ₂ zinc-finger peptide

Lima

Crooke²

1999

Proc. Natl. Acad. Sci. U.S.A.

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We have identified a 30-aa peptide that efficiently cleaves single-stranded RNA. The peptide sequence corresponds to a single zinc finger of the human maleassociated ZFY protein; a transcription factor belonging to the Cys 2 His 2 family of zinc-finger proteins. RNA cleavage was observed only in the absence of zinc. Coordination with zinc resulted in complete loss of ribonuclease activity. The ribonuclease active structure was determined to be a homodimeric form of the peptide. Dimerization of the peptide occurred through a single intermolecular disulfide between two of the four cystines. The observed hydrolytic activity was singlestranded RNA-specific. Single-stranded DNA, doublestranded RNA and DNA, and 2-methoxy-modified sequences were not degraded by the peptide. The peptide specifically cleaved pyrimidines within single-stranded RNA and the dinucleotide sequence 5-pyr-A-3 was preferred. The RNA cleavage products consisted of a 3 phosphate and 5 hydroxyl. The initial rates of cleavage (V 0 ) observed for the finger peptide were comparable to rates observed for human ribonucleases, and the catalytic rate (K cat ) was comparable to rates observed for the group II intron rybozymes. The pH profile exhibited by the peptide is characteristic of general acid-base catalytic mechanisms observed with other ribonucleases. These observations raise interesting questions about the potential biological roles of zinc-finger proteins.ZFY is a human male-associated transcription factor containing zinc-finger domains and is believed to participate in spermatogenesis and the regulation of male embryogenesis (1-3). A single zinc-finger element, generally 30 aa in length, has been shown to form an independent structural unit through the tetrahedral coordination of the zinc ion with either cysteine thiolates or histidine imidazoles (4-5). The zinc-finger domain of ZFY was shown to consist of finger elements in which the zinc ion was coordinated by two cysteines and two histidines to form the canonical ␤␤␣ finger structure (4, 6). These finger elements were arranged within the ZFY protein as alternating two-finger repeats. The odd-numbered domains were shown to fit the general zinc-finger consensus sequence, whereas the even numbered domains exhibited a unique zinc-finger sequence (6). The solution structure of two 30-aa peptides representing the odd (ZFY-switch) and even (ZFY-6) numbered zinc fingers showed that these peptides exhibited measurably different higher order structures (7).Although the metal bound structures of the ZFY finger peptides have been extensively characterized, the structures of the finger peptides in the absence of zinc are less well understood. Weiss et al. (8) showed that a progressive reduction in metal coordination with the ZFY-6 finger peptide resulted in a loss in ␣-helix formation. A similar loss in ␣-helix formation was observed for the oxidized form of the peptide in the absence of zinc. We have observed that in the absence of zinc and at concentrations Ͼ1 mM, the ZFY-6 peptide formed a str...

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Section: Discussionmentioning

confidence: 99%

Highly efficient endonucleolytic cleavage of RNA by a Cys ₂ His ₂ zinc-finger peptide

Lima

Crooke²

1999

Proc. Natl. Acad. Sci. U.S.A.

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“…5). Studies with synthetic peptides containing leucines and lysines showed that stability of an a-helix was enhanced by a spacing that placed leucine residues consecutively at the same position in the helix (25). In contrast, random placement of the leucine and lysine residues did not support formation of an a-helix (Table 2).…”

Section: Discussionmentioning

confidence: 99%

Subunit interaction in the CCAAT-binding heteromeric complex is mediated by a very short alpha-helix in HAP2.

Xing

Zhang

Olesen

et al. 1994

Proc. Natl. Acad. Sci. U.S.A.

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We did the domain of HAP2 that mediates subunit asiation In the heteromeric CCAAT-binding complex, first by genetic mutational analysis and then by structural studies. The mutational data suggest that a very short region in HAP2 mediates protein-protein association and that the structure of this domain is likely to be an a-helix. The CD analyses ofa 15-residue synthetic oligopeptide covering this region confirm this surmise. The oligopeptide indeed formed an unusuafly thermal stable a-helix in aqueous solution. Eight amino acids that lie along one face of this helix, including three arginines, are found to be critical for protein-protein association. The partner that interacts with this helical motif is likely to be another subunit in the HAP complex, since the CCAATbinding factor is shown to contain one molecule of HAP2. Our results suggest that very short regions in proteins can encode precise structures and mediate stable and specific proteinprotein recognition and interactions.The specificity and stability of protein-protein interactions are critical determinants in the assembly of macromolecular complexes consisting of protein subunits. One well-studied example of these complexes involves the transcription factors Fos and Jun (for review, see refs. 1 and 2). Jun-Jun homodimers and Fos-Jun heterodimers will form readily and are active in cells, whereas Fos-Fos dimers do not form (3-5). Inspection of a family of transcription factors with DNA-binding domains like those of Fos and Jun led to the leucine zipper hypothesis (6).A heteromeric transcriptional activator that was identified early on is the yeast (Saccharomyces cerevisiae) CCAATbox binding factor HAP2/3/4 (7-9). Both HAP2 and HAP3were present in a gel-shift complex with a CCAAT boxcontaining probe (9), and HAP2 and HAP3 remained stably associated in the absence of DNA (8) vivo (10, 14).Here we describe the dissection of the SAD of HAP2 by genetic mutational and biophysical structural analyses. Our findings delimit the region and pinpoint which residues in HAP2 are critical for its function. The mutational data suggest that a very short region in HAP2 is likely to form an a-helix which mediates assembly. Our findings show that 8 aa along one face of this helix dictate the specificity and at least part of the stability of interaction between HAP2 and its interacting partner. Further, a 15-aa synthetic peptide covering this region showed a typical a-helical circular dichroism (CD) spectrum. We conclude that very short peptide regions can encode precise structures and mediate stable and specific protein-protein recognition and interactions in macromolecular complexes. MATERIALS AND METHODSStrains, Plasmids, and f-Galactodase Assays. The S. cerevisiae strain BWG 1-7a (MAT a leu2-3,112 his4-519 adel-100 ura3-52) or isogenic derivatives were used in all experiments described. HAP2 complementation was assayed by using strain JO1-la (14). Vectors pRB1155 (16), pJO323 (14), and pLGA265UP1 (17); plasmid pXY3236 (10); and the assay for /3-galactosidase ac...

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“…23,24 The metal ion-dependent phosphoryl transfer mechanism 25 can be ruled out due to the independence of LZ catalytic activity on divalent ions. 7 In the case of acid-base catalysis, the active site of LZ-GCN4 may be comprised of E8, D9, or E12 as a catalytic base and R3, K5 or K10 as a catalytic acid.…”

Section: Putative Reaction Mechanismmentioning

confidence: 99%

Structural basis of RNA binding by leucine zipper GCN4

Nikolaev¹,

Pervushin²

2012

Protein Science

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Recently, we showed that leucine zipper (LZ) motifs of basic leucine zipper (bZIP) transcription factors GCN4 and c-Jun are capable of catalyzing degradation of RNA (Nikolaev et al., PLoS ONE 2010; 5:e10765). This observation is intriguing given the tight regulation of RNA turnover control and the antiquity of bZIP transcription factors. To support further mechanistic studies, herein, we elucidated RNA binding interface of the GCN4 leucine zipper motif from yeast. Solution NMR experiments showed that the LZ-RNA interaction interface is located in the first two heptads of LZ moiety, and that only the dimeric (coiled coil) LZ conformation is capable of binding RNA. Site-directed mutagenesis of the LZ-GCN4 RNA binding interface showed that substrate binding is facilitated by lysine and arginine side chains, and that at least one nucleophilic residue is located in proximity to the RNA phosphate backbone. Further studies in the context of full-length bZIP factors are envisaged to address the biological relevance of LZ RNase activity.

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Conformation-controlled hydrolysis of polyribonucleotides by sequential basic polypeptides

Cited by 88 publications

References 1 publication

Highly efficient endonucleolytic cleavage of RNA by a Cys ₂ His ₂ zinc-finger peptide

Highly efficient endonucleolytic cleavage of RNA by a Cys ₂ His ₂ zinc-finger peptide

Subunit interaction in the CCAAT-binding heteromeric complex is mediated by a very short alpha-helix in HAP2.

Structural basis of RNA binding by leucine zipper GCN4

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Conformation-controlled hydrolysis of polyribonucleotides by sequential basic polypeptides

Cited by 88 publications

References 1 publication

Highly efficient endonucleolytic cleavage of RNA by a Cys 2 His 2 zinc-finger peptide

Highly efficient endonucleolytic cleavage of RNA by a Cys 2 His 2 zinc-finger peptide

Subunit interaction in the CCAAT-binding heteromeric complex is mediated by a very short alpha-helix in HAP2.

Structural basis of RNA binding by leucine zipper GCN4

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Highly efficient endonucleolytic cleavage of RNA by a Cys ₂ His ₂ zinc-finger peptide

Highly efficient endonucleolytic cleavage of RNA by a Cys ₂ His ₂ zinc-finger peptide