Exploration of the L18 binding site on 5S RNA by deletion mutagenesis

Gewirth, D.T.; Moore, Peter B.

doi:10.1093/nar/16.22.10717

Cited by 14 publications

(5 citation statements)

References 25 publications

(27 reference statements)

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“…A key indicator of the reasonableness of the approach was the existence of mutant 5S rRNAs, which, despite their failure to incorporate into ribosomes, still accumulated to high levels in cells. It had previously been reported (33) that a 5S rRNA containing a duplication of nucleotides 25 to 72 accumulates in vivo, as did an rRNA with a deletion of positions 35 to 46 (15). We ourselves had observed many V. proteolyticus point mutations that resulted in the same phenotype.…”

Section: Discussionsupporting

confidence: 51%

A novel approach for monitoring genetically engineered microorganisms by using artificial, stable RNAs

Pitulle

Hedenstierna

Fox

1995

Appl Environ Microbiol

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Further improvements in technology for efficient monitoring of genetically engineered microorganisms (GEMs) in the environment are needed. Technology for monitoring rRNA is well established but has not generally been applicable to GEMs because of the lack of unique rRNA target sequences. In the work described herein, it is demonstrated that a deletion mutant of a plasmid-borne Vibrio proteolyticus 5S rRNA gene continues to accumulate to high levels in Escherichia coli although it is no longer incorporated into 70S ribosomes. This deletion construct was subsequently modified by mutagenesis to create a unique recognition site for the restriction endonuclease BstEII, into which new sequences could be readily inserted. Finally, a novel 17-nucleotide identifier sequence from Pennisetum purpureum was embedded into the construct to create an RNA identification cassette. The artificial identifier RNA, expressed from this cassette in vivo, accumulated in E. coli to levels comparable to those of wild-type 5S rRNA without being seriously detrimental to cell survival in laboratory experiments and without entering the ribosomes. These results demonstrate that artificial, stable RNAs containing sequence segments remarkably different from those present in any known rRNA can be designed and that neither the deleted sequence segment nor ribosome incorporation is essential for accumulation of an RNA product.

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Section: Discussionsupporting

confidence: 51%

A novel approach for monitoring genetically engineered microorganisms by using artificial, stable RNAs

Pitulle

Hedenstierna

Fox

1995

Appl Environ Microbiol

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“…Another application may be choice of sequences for structural studies with x-ray and NMR methods. Sequences with clearly defined optimal structures will be preferred over those with fluxional character (47). Performance of predictions with various parameter sets also indicates parameters requiring further study.…”

Section: Resultsmentioning

confidence: 99%

Improved predictions of secondary structures for RNA.

Jaeger

Turner

Zuker

1989

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

774

541

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The accuracy of computer predictions of RNA secondary structure from sequence data and free energy parameters has been increased to roughly 70%. Performance is judged by comparison with structures known from phylogenetic analysis. The algorithm also generates suboptimal structures. On average, the best structure within 10% of the lowest free energy contains roughly 90% of phylogenetically known helixes. The algorithm does not include tertiary interactions or pseudoknots and employs a crude model for singlestranded regions. The only favorable interactions are base pairing and stacking of terminal unpaired nucleotides at the ends of helixes. The excellent performance is consistent with these interactions being the primary interactions determining RNA secondary structure.RNA is important for functions such as catalysis, RNA splicing, regulation of transcription and translation, and transport of proteins across membranes (1). Many RNA sequences are known. Determination of secondary structures, however, is difficult. Thermodynamics has been applied to predict RNA secondary structure from sequence (2, 3), but with modest success. Predictions of suboptimal structures make the method more useful (4). We report combining several recent advances to improve predictions of RNA secondary structures. Three advances are incorporated in this work. (i) New methods for synthesizing RNA make it possible to obtain model systems with a large variety of sequence (5, 6). This technique has led to measurements of improved parameters and the realization that non-base-paired nucleotides contribute sequence-dependent interactions that stabilize secondary structure (7, 8). (ii) A computer algorithm has been developed that allows incorporation of non-base-paired interactions in the prediction of optimal and suboptimal secondary structures (9). (iii) Several RNA secondary structures have been determined by phylogeny (10-15). Comparison of predicted and known structures allows optimization of parameters that have not been measured. Resultant predictions appear sufficiently reliable to aid planning and interpretation of experiments on RNA. MATERIALS AND METHODSThermodynamic Parameters. When possible, free energy increments at 370C, AG037, were taken from experiments in 1 M NaCl. For fully base-paired regions, experiments on dGCATGC indicate that 1 M NaCl mimics solutions containing 1-100 mM Mg2+ in the presence of 0.15-1 M NaCl (16).Relatively few experiments are available for loop structures, and, therefore, little is known about interactions determining loop stability. This situation forced approximations. (i) Jacobson-Stockmayer theory (17) was used to extrapolate the length dependence of AG37 for bulge, hairpin, and internal loops: AG0(n) = AG(nmax) + 1.75 RTln (n/nmax).For this equation n is the number of unpaired nucleotides in the loop, nma is the maximum-length loop for which experimental data is available, R is the gas constant (1.987 cal mol'lK-l; 1 cal = 4.184 J), and T is the temperature in K (310.15 K for 370C). (ii) When...

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“…38, 39 One is a hydrophobic patch; similar to what is seen in amicyanin (Figure 6), which is a region of nonpolar residues around the solvent accessible, exposed His ligand of copper. The other, which is unique to plastocyanin is an acidic patch which was thought to interact with a basic Lys-rich region on the surface of cytochrome f. 38, 40–42 No crystal structure of a complex of plastocyanin with a redox partner protein is available but the solution structure of the complex between plastocyanin and cytochrome f was determined by NMR. 43 This study confirmed the interaction between residues in the lysine-rich domain on cytochrome f with the acidic patch of plastocyanin, and interactions between the hydrophobic patch of plastocyanin and hydrophobic residues around the heme of cytochrome f. Given the close proximity of the copper and heme redox sites of the two proteins at the interface of the hydrophobic patches, this interaction is thought to be directly related to mediation of interprotein electron transfer.…”

Section: What Dictates the Specificity For The Redox Partner Proteinsmentioning

confidence: 99%

Cupredoxins—A study of how proteins may evolve to use metals for bioenergetic processes

Choi

Davidson

2011

Metallomics

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Cupredoxins are small proteins that contain type I copper centers, which are ubiquitous in nature. They function as electron transfer shuttles between proteins. This review of the structure and properties of native cupredoxins, and those modified by site-directed mutagenesis, illustrates how these proteins may have evolved to specifically bind copper, develop recognition sites for specific redox partners, tune redox potential for a particular function, and allow for efficient electron transfer through the protein matrix. This is relevant to the general understanding of the roles of metals in energy metabolism, respiration and photosynthesis.

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Exploration of the L18 binding site on 5S RNA by deletion mutagenesis

Cited by 14 publications

References 25 publications

A novel approach for monitoring genetically engineered microorganisms by using artificial, stable RNAs

A novel approach for monitoring genetically engineered microorganisms by using artificial, stable RNAs

Improved predictions of secondary structures for RNA.

Cupredoxins—A study of how proteins may evolve to use metals for bioenergetic processes

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