Cross-Linking of Proteins to Nucleic Acids by Ultraviolet Light

Shetlar, Martin D.

doi:10.1007/978-1-4684-3641-9_4

Cited by 100 publications

(59 citation statements)

References 211 publications

(278 reference statements)

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“…It has been shown that UV crosslinking at 254 nm generates a covalent bond between the side chains of UV-crosslinkable amino acids (methionine, tyrosine, histidine, phenylalanine, leucine, and cysteine) and the uridine base of RNA when both of the reacting groups lie within atomic distance. 30,31 The autoradiogram image showed that 5'-end-labeled p-BR13 was crosslinked to both the mutant and wild-type proteins (Fig. 8A).…”

Section: ©2 0 1 1 L a N D E S B I O S C I E N C E D O N O T D I S Tmentioning

confidence: 99%

Upregulation of RNase E activity by mutation of a site that uncompetitively interferes with RNA binding

Moore

Shin

et al. 2011

RNA Biology

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Escherichia coli RNase e contains a site that selectively binds to RNAs containing 5′-monophosphate termini, increasing the efficiency of endonucleolytic cleavage of these RNAs. Random mutagenesis of N-Rne, the N-terminal catalytic region of RNase e, identified a hyperactive variant that remains preferentially responsive to phosphorylation at 5' termini. Biochemical analyses showed that the mutation (Q36R), which replaces glutamine with arginine at a position distant from the catalytic site, increases formation of stable RNA-protein complexes without detectably affecting the enzyme's secondary or tertiary structure. studies of cleavage of fluorogenic substrate and eMsA experiments indicated that the Q36R mutation increases catalytic activity and RNA binding. however, UV crosslinking and mass spectrometry studies suggested that the mutant enzyme lacks an RNA binding site present in its wild-type counterpart. Two substrate-bound tryptic peptides, 65 hGFLpLK 71 -which includes amino acids previously implicated in substrate binding and catalysisand 24 LYDLDIespGheQK 37 -which includes the Q36 locus-were identified in wild-type enzyme complexes, whereas only the shorter peptide was observed for complexes containing Q36R. Our results identify a novel RNase e locus that disparately affects the number of substrate binding sites and catalytic activity of the enzyme. We propose a model that may account for these surprising effects.Upregulation of RNase E activity by mutation of a site that uncompetitively interferes with RNA binding

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Section: ©2 0 1 1 L a N D E S B I O S C I E N C E D O N O T D I S Tmentioning

confidence: 99%

Upregulation of RNase E activity by mutation of a site that uncompetitively interferes with RNA binding

Moore

Shin

et al. 2011

RNA Biology

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“…The reaction mixtures were then supplemented with heparin to 0.8 mg/ml, incubated for 10 min at 30°C, and irradiated as described previously (2) for 15 min on a Fotodyne Prep I transilluminator (X = 300 nm, 7 mW/cm2) at room temperature. (Unlike unsubstituted nucleic acid which cross-links most efficiently to protein at 254 nm, 4-S-U-substituted nucleic acid cross-links most efficiently at a longer wavelength [1,2,7,8,26].) Samples were then diluted twofold with water and treated with a final concentration of 1 ,ug of RNase A per ,ul and 10 U of RNase Ti per ,ul for 15 min at 30°C.…”

mentioning

confidence: 99%

The first pre-rRNA-processing event occurs in a large complex: analysis by gel retardation, sedimentation, and UV cross-linking.

Kass

Sollner‐Webb

1990

Mol. Cell. Biol.

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The first processing event that mouse pre-rRNA undergoes occurs within the external transcribed spacer and is efficiently reproduced in vitro. Analysis with nondenaturing polyacrylamide gels revealed the formation of heparin-resistant complexes of retarded electrophoretic mobility on the substrate rRNA. The specificity of these complexes was demonstrated by their elimination due to competition with processing-competent, but not with processing-incompetent, rRNAs. Furthermore, complex formation, like the processing cleavage, required only 28 nucleotides of rRNA sequence adjacent to the processing site but was stimulated by additional downstream conserved sequences. These processing complexes formed in a time-dependent manner, and once assembled, they were stable to challenge by competitor rRNA and remained on the processed rRNA. Their sedimentation coefficient was -20S. UV cross-linking studies with 4-thiouridine-substituted rRNA have identified six polypeptides, 52 to 250 kilodaltons, that are specifically bound to the rRNA processing substrate.

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“…Changes within the potential RNA binding site had varying effects depending on whether aromatic or charged residues were altered. The triple charged mutant (Arg 20 , Arg 22 , Lys 24 to Leu) yielded a protein with partial activity in splicing assays (Fig. 5), in contrast to the triple aromatic mutant (Tyr 21 , Tyr 23 , and Phe 25 to Leu), which was completely defective in splicing (Fig.…”

Section: Discussionmentioning

confidence: 98%

“…Tyr 21 and Tyr 23 Are Essential for Stimulation of CatalysisPreliminary splicing experiments with the triple charged mutant (Arg 20 , Arg 22 , Lys 24 changed to leucine) and three point mutants, Y21L (tyrosine at position 21 changed to leucine), Y23L and F25L showed varying degrees of activity. These mutants were characterized further by a series of time course experiments that measured their initial rates of splicing.…”

Section: Identification Of Cbp2 Peptides That Contact Intronmentioning

confidence: 99%

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An RNA Binding Motif in the Cbp2 Protein Required for Protein-stimulated RNA Catalysis

Tirupati

Shaw

Lewin

1999

Journal of Biological Chemistry

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The fifth and terminal intron of yeast cytochrome b pre-mRNA (a group I intron) requires a protein encoded by the nuclear gene CBP2 for splicing. Because catalysis is intrinsic to the RNA, the protein is believed to promote formation of secondary and tertiary structure of the RNA, resulting in a catalytically competent intron. In vitro, this mitochondrial intron can be made to selfsplice or undergo protein-facilitated splicing by varying the Mg 2؉ and monovalent salt concentrations. This twocomponent system, therefore, provides a good model for understanding the role of proteins in RNA folding. A UV cross-linking experiment was initiated to identify RNA binding sites on Cbp2 and gain insights into Cbp2-intron interactions. A 12-amino acid region containing a presumptive contact site near the amino terminus was targeted for mutagenesis, and mutant proteins were characterized for RNA binding and stimulation of splicing. Mutations in this region resulted in partial or complete loss of function, demonstrating the importance of this determinant for stimulation of RNA splicing. Several of the mutations that severely reduced splicing did not significantly shift the overall binding isotherm of Cbp2 for the precursor RNA, suggesting that contacts critical for activity are not necessarily reflected in the dissociation constant. This analysis has identified a unique RNA binding motif of alternating basic and aromatic residues that is essential for protein facilitated splicing.The terminal intron (intron 5) of the yeast gene encoding cytochrome b (COB) can self-splice in vitro at high concentrations of Mg 2ϩ (1, 2), but the protein encoded by the nuclear CBP2 gene is essential for splicing at physiological concentrations of Mg 2ϩ (3). Although this group IA intron possesses the conserved secondary and tertiary structures found in all group I introns, it varies in important ways from the prototype Tetrahymena rRNA intron (4). In particular, the internal guide sequence that establishes the substrate specificity starts 220 nucleotides downstream of the 5Ј splice junction, rather than the usual 14 -20 nucleotides found in most other group I introns. The intron is AU-rich, and requires higher levels of Mg 2ϩ for stabilization of the active structure than the GC-rich Tetrahymena group I intron. Like other group IA introns, the fifth intron of COB pre-mRNA (bI5) is also devoid of the peripheral RNA element, P5abc, that is important for stabilizing the catalytic core of the Tetrahymena rRNA intron (5). It is therefore conceivable that Cbp2 compensates for this RNA structure and stabilizes its RNA partner by contributing substantial binding energy in a manner similar to the CYT-18 protein of Neurospora (6, 7). UV cross-linking, chemical, and enzymatic modification studies indicate that Cbp2 contacts bI5 at multiple sites in the catalytic core and peripheral RNA elements and stimulates the formation of the catalytically active structure (8, 9). Based on these and kinetic studies, Weeks and Cech (10) proposed that Cbp2 serves as a ter...

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Cross-Linking of Proteins to Nucleic Acids by Ultraviolet Light

Cited by 100 publications

References 211 publications

Upregulation of RNase E activity by mutation of a site that uncompetitively interferes with RNA binding

Upregulation of RNase E activity by mutation of a site that uncompetitively interferes with RNA binding

The first pre-rRNA-processing event occurs in a large complex: analysis by gel retardation, sedimentation, and UV cross-linking.

An RNA Binding Motif in the Cbp2 Protein Required for Protein-stimulated RNA Catalysis

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