Recognition of Nucleotide Sequences

Yarus, Michael

doi:10.1146/annurev.bi.38.070169.004205

Cited by 108 publications

(33 citation statements)

References 94 publications

(132 reference statements)

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“…Similar results have been reported for other noncovalent nucleic acid-protein complexes on cellulose nitrate filters (26).…”

Section: Resultssupporting

confidence: 89%

“…2). By contrast, high concentrations of salt interfere with filter-binding assays for noncovalent nucleic acid-protein complexes containing proteins such as RNA polymerase (28) and the lac repressor (29) as well as with other types of filter-binding assays (26). Hence, the adenovirus complex can be assayed under conditions that disrupt noncovalent complexes.…”

Section: Resultsmentioning

confidence: 99%

“…Because all adenovirus proteins bind, at least qualitatively, to filters (not shown), it is likely that many other proteins may bind as well (26,30). Thus, the filter-binding assay may prove useful to detect both naturally occurring as well as artificially induced covalent DNA-protein complexes.…”

Section: Resultsmentioning

confidence: 99%

See 2 more Smart Citations

Filter-binding assay for covalent DNA-protein complexes: adenovirus DNA-terminal protein complex.

Coombs¹,

Pearson²

1978

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

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A rapid, simple, and quantitative filter-binding assay using glass fiber filters has been developed to detect the covalent adenovirus DNA-terminal protein complex. The Each of these assays, however, suffers with respect to quantitation or ease of use or both. Our laboratory has now developed a rapid, simple, and quantitative filter-binding assay for the adenovirus DNA-terminal protein complex. In this paper we show that the adenovirus complex or terminal restriction endonuclease fragments of the complex are quantitatively retained (>98%) on glass fiber filters under conditions such that protein-free DNA is not (<0.1%). The adenovirus complex binds maximally to filters at NaCI concentrations greater than 200 mM. This means that the adenovirus complex can be assayed even in the presence of other proteins that interact ionically with DNA. It is likely that the filter-binding assay will prove useful for detecting and quantitating covalent nucleic acid-protein complexes from a wide range of organisms.The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. Filter-Binding Assay. The standard filter-binding assay used 2.4-cm Whatman GF/C glass fiber filters held in a commercially available stainless steel filter holder. Filters were wetted with 1-2 ml of the sample buffer to establish a hydrostatic head, and the samples (up to 1 ml) were applied freely. The filter was rinsed with an additional 2 ml of the appropriate buffer and sucked dry. In some cases (for example, 1 M sodium phosphate buffer) the filter was rinsed last with 1 ml of TEN buffer prior to suction drainage. Both the filter and the filtrate were assayed for radioactivity to determine the percentage DNA bound to the filter. Miniature filters (3.5 mm diameter), called "microdot filters," were cut from stock GF/C filters. Microdot filters were mounted in a special holder constructed from a 1-ml polystyrene pipet (detailed instructions for constructing the holder are available upon request). The microdot filter facilitates handling of volumes less than 0.1 ml. Bound DNA-protein complexes could be eluted from the microdot filter in 10-25 ,l of solutions containing detergents or proteolytic enzymes. During elution, the liquid was carefully passed down and up through the filter several times by use of suction and pressure. The solution was removed from above the filter with a micropipet.Enzymes. EcoRI (23,24). (Some restriction enzyme preparations contain nonionic detergents-for example, Triton X-100-that may artifactually reduce binding of DNA-protein complexes to filters.) Pronase (grade B, Calbiochem) and Proteinase K (EM Biochemicals) were incubated at 370C for 2 hr at a concentration of 10 mg/ml before use.Radioactivity Determinations. Dried filters were assayed by liquid scintillation spectrometry using a toluene/2,5-diphenyloxazole/1,4 bis[2-(5-phenyloxazolyl)]benzene mixture (18). 32P radioactivity was determined by Cerenkov ra...

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“…Similar results have been reported for other noncovalent nucleic acid-protein complexes on cellulose nitrate filters (26).…”

Section: Resultssupporting

confidence: 89%

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Filter-binding assay for covalent DNA-protein complexes: adenovirus DNA-terminal protein complex.

Coombs¹,

Pearson²

1978

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

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“…This negative result implies that the only possible specific recognition sites are in the outer minor groove. However, the pseudo-operator of the fl-galactosidase gene has an A-T rather than G-C base pair at position 17 pears free of repressor. Conversely, the N7 of guanine is protected by repressor against alkylation with dimethyl sulfate (5).…”

Section: T-g-t-g-g-a-a-t-t-g-t-g-a-g-c-g-g-a-t-a-a-c-a-a-t-t A-c-a-c-mentioning

confidence: 99%

How lac repressor recognizes lac operator.

Goeddel¹,

Yansura²,

Caruthers³

1978

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

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Nucleotide analogs were substituted for unmodified nucleotides at specific sites in the lac operator sequence by a combination of chemical and enzymatic procedures. The nitrocellulose filter assay was used to-study the interactions of these modified operators with wild-type (SQ) and tight-binding (QX86) lac repressors. These studies iicat directly the 5 methyl of thymine and the 2 amino of ganine as important operator-repressor contact sites. Furthermore, when these findings are combined with published results from other laboratories, a model for the lac operator-Iac repressor interaction can be derived. Two important postulates follow from this model. (il The repressor interacts at specific and defined sites with the N7 of fuanine, the 5 methyl of thymine, the 2 amino of guanine, and the central major groove of the operator.(ii) The repressor binds to one side of the operator. DNA-protein interactions are of fundamental importance in a large variety of molecular processes, yet are not well understood biochemically (1,2). We are currently studying the lac operator-repressor system in order to determine the mechanisms by which proteins recognize and interact with specific DNA sequences.The lac repressor protein binds tightly to the lac operator, a unique sequence in the Escherichia coli chromosome. An examination of operator-constitutive )Oc) mutants has identified eight base pairs that are involved in binding to repressor (8. 4). Gilbert et al. (5) have shown that the binding of repressor to operator specifically protects four guanines and three adenines against methylation with dimethyl sulfate. At the same time, the methylation of two guanines and one adenine is enhanced. (Dimethyl sulfate methylates double-stranded DNA at the N7 of guanine and the N3 of adenine exposed, respectively, in the major and minor grooves.) Crosslinking experiments have identified thymine residues that contact repressor in the major groove (6). Thus the lac repressor appears to interact with operator DNA in both the major and minor grooves. All these sets of data are shown in Fig. 1.One primary objective of research from this laboratory is to decipher those elempts of the lac operator bases that stabilize the repressor-operator (RO) complex. The approach outlined in this paper involves insertion of nucleic acid base analogs at specific sites in the DNA, followed by analysis of how these analogs affect the stability of the RO interaction. These results, when used in conjunction with the above sets of data, have allowed us to deduce several lac operator sites that are recognized by lac repressor.MATERIALS AND METHODS Syntheses of unmodified and various modified lac operator DNAs have either been described (7-11) or remain to be published. Nitrocellulose filter binding assays were performed as described previously (9,12,13). Wild-type (SQ) repressor was purified by a published procedure (9). Tight-binding (QX86) repressor was provided by J. Sadler. RESULTSThe 26 operator duplexes listed in Table 1 were prepared by a combination...

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“…2 A, B). The tRNA was mainly cleaved a t one position in the dihydrouridine loop which gave the fragments Phe [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18] and Phe 21-76 [15]. A small amount of additional splitting at position 57 yielded half molecules (Phe 21-57) and quarter molecules (Phe 58-76) [15].…”

Section: Partial Digestions Of Trnaphe Complexesmentioning

confidence: 99%

Complexes of Aminoacyl‐tRNA Synthetases with tRNAs as Studied by Partial Nuclease Digestion

Hörz

Zachau

1973

European Journal of Biochemistry

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Complexes of tRNAPhe and tRNAser with their cognate aminoacyl tRNA synthetases have been exposed to a number of nucleases under conditions of partial digestion in order to gain information on the topography of the complexes. tRNAPhe was found to be strongly protected by yeast phenylalanyl tRNA synthetase a t both nuclease-sensitive sites, the dihydrouridine loop and the anticodon. tRNASer, on the other hand, was selectively shielded by yeast seryl tRNA synthetase a t one of the two nuclease targets, the G-C-C-A terminus, whereas splitting at the other target, the anticodon, was not affected a t all by the synthetase.There was complete specificity of shielding : tRNAs were only protected by their respective synthetases. The complexes were sufficiently stable to allow an analysis of protection not only at pH 5.5 but also a t pH 7.0. Phenylalanyl tRNA synthetase protected one mole of tRNAPhe, seryl tRNA synthetase two moles of tRNAser against nuclease attack. Denatured tRNAPhe did not bind to phenylalanyl tRNA synthetase.Two effects of ligands on complex stability have been observed. The analog phenylalaninol-AMP increased the protection by phenylalanyl tRNA synthetase of tRNAPhe but not of PhetRNAPhe; ATP abolished the shielding of the G-C-C-A terminus of tRNASer by seryl tRNA synthetase.A number of tRNAPhe and tRNAser fragments was studied. It was concluded that the terminal 7 nucleotides in tRNAPhe are not required for complex formation. As expected, combinations of homologous tRNA half molecules resembled the intact tRNAs in the degree of protection.When the half molecules were tested separately or in heterologous combinations, phenylalanyl tRNA synthetase afforded some protection for the pG-half molecule of tRNAPhe and seryl tRNA synthetase for the C-C-A-half molecule of tRNASer.The interactions between aminoacyl tRNA synthetases and their cognate tRNAs have been the subject of considerable work over the past several Enzymes. Ti-RNAase or ribonucleate guanidine nucleotido-2'-transferase (EC 2.7.7.26) ; T2-RNAase or ribonucleate nucleotido-2'-transferase (cyclizing) (EC 2.7.7.17) ; N1-RNAase (EC 2.7.7.-) ; pancreatic RNAase or ribonucleate pyrimidme-nucleotido-2'-transferase (cyclizing) (EC 2.7.7.16); phenylalanyl tRNA synthetase (EC 6.1.1.1.-); seryl tRNA synthetase (EC 6.1.1.11).Definitim. A,,,~,,,, unit, the quantity of material contained in 1 ml of a solution which has an absorbance of 1 at 260 (280) nm, when measured in a 1 cm pathlength cell; RB value, ratio of distance migrated by a compound to distance migrated by bromphenolblue.

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Recognition of Nucleotide Sequences

Cited by 108 publications

References 94 publications

Filter-binding assay for covalent DNA-protein complexes: adenovirus DNA-terminal protein complex.

Filter-binding assay for covalent DNA-protein complexes: adenovirus DNA-terminal protein complex.

How lac repressor recognizes lac operator.

Complexes of Aminoacyl‐tRNA Synthetases with tRNAs as Studied by Partial Nuclease Digestion

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