Biomedical subjects

Raymond F Gesteland

Publications and source records attributed to Raymond F Gesteland.

10 recordsLinked to original sources

PubMed2003

Programmed translational -1 frameshifting on hexanucleotide motifs and the wobble properties of tRNAs.

Programmed -1 ribosomal frameshifting, involving tRNA re-pairing from an AAG codon to an AAA codon, has been reported to occur at the sequences CGA AAG and CAA AAG. In this study, using the recoding region of insertion sequence IS3, we have investigated the influence on frameshifting in Escherichia coli of the first codon of this type of motif by changing it to all other NNA codons. Two classes of NNA codons were distinguished, depending on whether they favor or limit frameshifting. Their degree of shiftiness is correlated with wobble propensity, and base 34 modification, of their decoding tRNAs. A more flexible anticodon loop very likely makes the tRNAs with extended wobble more prone to liberate the third codon base, A, for re-pairing of tRNALys in the -1 frame.

Base Sequence↗

PubMed2003

Deletion of selenoprotein P alters distribution of selenium in the mouse.

Selenoprotein P (Se-P) contains most of the selenium in plasma. Its function is not known. Mice with the Se-P gene deleted (Sepp(-/-)) were generated. Two phenotypes were observed: 1) Sepp(-/-) mice lost weight and developed poor motor coordination when fed diets with selenium below 0.1 mg/kg, and 2) male Sepp(-/-) mice had sharply reduced fertility. Weanling male Sepp(+/+), Sepp(+/-), and Sepp(-/-) mice were fed diets for 8 weeks containing <0.02-2 mg selenium/kg. Sepp(+/+) and Sepp(+/-) mice had similar selenium concentrations in all tissues except plasma where a gene-dose effect on Se-P was observed. Liver selenium was unaffected by Se-P deletion except that it increased when dietary selenium was below 0.1 mg/kg. Selenium in other tissues exhibited a continuum of responses to Se-P deletion. Testis selenium was depressed to 19% in mice fed an 0.1 mg selenium/kg diet and did not rise to Sepp(+/+) levels even with a dietary selenium of 2 mg/kg. Brain selenium was depressed to 43%, but feeding 2 mg selenium/kg diet raised it to Sepp(+/+) levels. Kidney was depressed to 76% and reached Sepp(+/+) levels on an 0.25 mg selenium/kg diet. Heart selenium was not affected. These results suggest that the Sepp(-/-) phenotypes were caused by low selenium in testis and brain. They strongly suggest that Se-P from liver provides selenium to several tissues, especially testis and brain. Further, they indicate that transport forms of selenium other than Se-P exist because selenium levels of all tissues except testis responded to increases of dietary selenium in Sepp(-/-) mice.

Animals↗

PubMed2003

RECODE 2003.

The RECODE database is a compilation of translational recoding events (programmed ribosomal frameshifting, codon redefinition and translational bypass). The database provides information about the genes utilizing these events for their expression, recoding sites, stimulatory sequences and other relevant information. The Database is freely available at http://recode.genetics.utah.edu/.

Animals↗

PubMed2003

Maintenance of the correct open reading frame by the ribosome.

During translation, a string of non-overlapping triplet codons in messenger RNA is decoded into protein. The ability of a ribosome to decode mRNA without shifting between reading frames is a strict requirement for accurate protein biosynthesis. Despite enormous progress in understanding the mechanism of transfer RNA selection, the mechanism by which the correct reading frame is maintained remains unclear. In this report, evidence is presented that supports the idea that the translational frame is controlled mainly by the stability of codon-anticodon interactions at the P site. The relative instability of such interactions may lead to dissociation of the P-site tRNA from its codon, and formation of a complex with an overlapping codon, the process known as P-site tRNA slippage. We propose that this process is central to all known cases of +1 ribosomal frameshifting, including that required for the decoding of the yeast transposable element Ty3. An earlier model for the decoding of this element proposed 'out-of-frame' binding of A-site tRNA without preceding P-site tRNA slippage.

Anticodon↗

PubMed2002

Artificial neural network prediction of antisense oligodeoxynucleotide activity.

An mRNA transcript contains many potential antisense oligodeoxynucleotide target sites. Identification of the most efficacious targets remains an important and challenging problem. Building on separate work that revealed a strong correlation between the inclusion of short sequence motifs and the activity level of an oligo, we have developed a predictive artificial neural network system for mapping tetranucleotide motif content to antisense oligo activity. Trained for high-specificity prediction, the system has been cross-validated against a database of 348 oligos from the literature and a larger proprietary database of 908 oligos. In cross- validation tests the system identified effective oligos (i.e. oligos capable of reducing target mRNA expression to <25% that of the control) with 53% accuracy, in contrast to the <10% success rates commonly reported for trial-and-error oligo selection, suggesting a possible 5-fold reduction in the in vivo screening required to find an active oligo. We have implemented a web interface to a trained neural network. Given an RNA transcript as input, the system identifies the most likely oligo targets and provides estimates of the probabilities that oligos targeted against these sites will be effective.

Binding Sites↗

PubMed2002

Recoding: translational bifurcations in gene expression.

During the expression of a certain genes standard decoding is over-ridden in a site or mRNA specific manner. This recoding occurs in response to special signals in mRNA and probably occurs in all organisms. This review deals with the function and distribution of recoding with a focus on the ribosomal frameshifting used for gene expression in bacteria.

Animals↗

PubMed2002

Release factor 2 frameshifting sites in different bacteria.

The mRNA encoding Escherichia coli polypeptide chain release factor 2 (RF2) has two partially overlapping reading frames. Synthesis of RF2 involves ribosomes shifting to the +1 reading frame at the end of the first open reading frame (ORF). Frameshifting serves an autoregulatory function. The RF2 gene sequences from the 86 additional bacterial species now available have been analyzed. Thirty percent of them have a single ORF and their expression does not require frameshifting. In the approximately 70% that utilize frameshifting, the sequence cassette responsible for frameshifting is highly conserved. In the E. coli RF2 gene, an internal Shine-Dalgarno (SD) sequence just before the shift site was shown earlier to be important for frameshifting. Mutagenic data presented here show that the spacer region between the SD sequence and the shift site influences frameshifting, and possible mechanisms are discussed. Internal translation initiation occurs at the shift site, but any functional role is obscure.

DNA Mutational Analysis↗

PubMed2002

Influence of the stacking potential of the base 3' of tandem shift codons on -1 ribosomal frameshifting used for gene expression.

Translating ribosomes can shift reading frame at specific sites with high efficiency for gene expression purposes. The most common type of shift to the -1 frame involves a tandem realignment of two anticodons from pairing with mRNA sequence of the form X XXY YYZ to XXX YYY Z where the spaces indicate the reading frame. The predominant -1 shift site of this type in eubacteria is A AAA AAG. The present work shows that in Escherichia coli the identity of the 6 nt 3' of this sequence can be responsible for a 14-fold variation in frameshift frequency. The first 3' nucleotide has the primary effect, with, in order of decreasing efficiency, U > C > A > G. This effect is independent of other stimulators of frameshifting. It is detected with other X XXA AAG sequences, but not with several other heptameric -1 shift sites. Pairing of E. coli tRNALYS with AAG is especially weak at the third codon position. We propose that strong stacking of purines 3' of AAG stabilizes pairing of tRNALys, diminishing the chance of codon:anticodon dissociation that is a prerequisite for the realignment involved in frameshifting.

Anticodon↗

PubMed2002

Comparative studies of frameshifting and nonframeshifting RNA pseudoknots: a mutational and NMR investigation of pseudoknots derived from the bacteriophage T2 gene 32 mRNA and the retroviral gag-pro frameshift site.

Mutational and NMR methods were used to investigate features of sequence, structure, and dynamics that are associated with the ability of a pseudoknot to stimulate a -1 frameshift. In vitro frameshift assays were performed on retroviral gag-pro frameshift-stimulating pseudoknots and their derivatives, a pseudoknot from the gene 32 mRNA of bacteriophage T2 that is not naturally associated with frameshifting, and hybrids of these pseudoknots. Results show that the gag-pro pseudoknot from human endogenous retrovirus-K10 (HERV) stimulates a -1 frameshift with an efficiency similar to that of the closely related retrovirus MMTV. The bacteriophage T2 mRNA pseudoknot was found to be a poor stimulator of frameshifting, supporting a hypothesis that the retroviral pseudoknots have distinctive properties that make them efficient frameshift stimulators. A hybrid, designed by combining features of the bacteriophage and retroviral pseudoknots, was found to stimulate frameshifting while retaining significant structural similarity to the nonframeshifting bacteriophage pseudoknot. Mutational analyses of the retroviral and hybrid pseudoknots were used to evaluate the effects of an unpaired (wedged) adenosine at the junction of the pseudoknot stems, changing the base pairs near the junction of the two stems, and changing the identity of the loop 2 nucleotide nearest the junction of the stems. Pseudoknots both with and without the wedged adenosine can stimulate frameshifting, though the identities of the nucleotides near the stem1/stem2 junction do influence efficiency. NMR data showed that the bacteriophage and hybrid pseudoknots are similar in their local structure at the junction of the stems, indicating that pseudoknots that are similar in this structural feature can differ radically in their ability to stimulate frameshifting. NMR methods were used to compare the internal motions of the bacteriophage T2 pseudoknot and representative frameshifting pseudoknots. The stems of the investigated pseudoknots are similarly well ordered on the time scales to which nitrogen-15 relaxation data are sensitive; however, solvent exchange rates for protons at the junction of the two stems of the nonframeshifting bacteriophage pseudoknot are significantly slower than the analogous protons in the representative frameshifting pseudoknots.

Bacteriophage T4↗

PubMed2002

Computational identification of putative programmed translational frameshift sites.

MOTIVATION: In an effort to identify potential programmed frameshift sites by statistical analysis, we explore the hypothesis that selective pressure would have rendered such sites underabundant and underrepresented in protein-coding sequences. We developed a computer program to compare the frequencies of k-length subsequences of nucleotides with the frequencies predicted by a zero order Markov chain determined by the codon bias of the same set of sequences. The program was used to calculate and evaluate the distribution of 7-base oligonucleotides in the 6000+ putative protein-coding sequences of S. cerevisiae preliminary to the laboratory testing of the most highly underrepresented oligos for frameshifting efficiency. RESULTS: Among the most significant results is the finding that the heptanucleotides CUU-AGG-C and CUU-AGU-U, sites of the programmed +1 translational frameshifts required for the production in yeast of actin filament-binding protein ABP140 and telomerase subunit EST3, respectively, rank among the least represented of phase I heptanucleotides in the coding sequences of S. cerevisiae. Laboratory experiments demonstrated that other underrepresented heptanucleotides identified by the program, for example GGU-CAG-A, are also prone to significant translational frameshifting, suggesting the possibility that genes containing other underrepresented heptamers may also encode transframe products. AVAILABILITY: The program is available for download from http://www.gesteland.genetics.utah.edu/freqAnalysis SUPPLEMENTARY INFORMATION: Complete results from the analysis of S. cerevisiae are available on http://www.gesteland.genetics.utah.edu/freqAnalysis

Algorithms↗