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Biomedical subjects

N M Wills

Publications and source records attributed to N M Wills.

11 recordsLinked to original sources
PubMed2000

Nonlinearity in genetic decoding: homologous DNA replicase genes use alternatives of transcriptional slippage or translational frameshifting.

The tau and gamma subunits of DNA polymerase III are both encoded by a single gene in Escherichia coli and Thermus thermophilus. gamma is two-thirds the size of tau and shares virtually all its amino acid sequence with tau. E. coli and T. thermophilus have evolved very different mechanisms for setting the approximate 1:1 ratio between tau and gamma. Both mechanisms put ribosomes into alternate reading frames so that stop codons in the new frame serve to make the smaller gamma protein. In E. coli, approximately 50% of initiating ribosomes translate the dnaX mRNA conventionally to give tau, but the other 50% shift into the -1 reading frame at a specific site (A AAA AAG) in the mRNA to produce gamma. In T. thermophilus ribosomal frameshifting is not required: the dnaX mRNA is a heterogeneous population of molecules with different numbers of A residues arising from transcriptional slippage on a run of nine T residues in the DNA template. Translation of the subpopulation containing nine As (or +/- multiples of three As) yields tau. The rest of the population of mRNAs (containing nine +/- nonmultiples of three As) puts ribosomes into the alternate reading frames to produce the gamma protein(s). It is surprising that two rather similar dnaX sequences in E. coli and T. thermophilus lead to very different mechanisms of expression.

Bacterial Proteins↗
PubMed1999

Structural studies of the RNA pseudoknot required for readthrough of the gag-termination codon of murine leukemia virus.

Retroviruses, such as murine leukemia virus (MuLV), whose gag and pol genes are in the same reading frame but separated by a UAG stop codon, require that 5-10 % of ribosomes decode the UAG as an amino acid and continue translation to synthesize the Gag-Pol fusion polyprotein. A specific pseudoknot located eight nucleotides 3' of the UAG is required for this redefinition of the UAG stop codon. The structural probing and mutagenic analyses presented here provide evidence that loop I of the pseudoknot is one nucleotide, stem II has seven base-pairs, and the nucleotides 3' of stem II are important for function. Stem II is more resistant to single-strand-specific probes than stem I. Sequences upstream of the UAG codon allow formation of two competing structures, a stem-loop and the pseudoknot.

Aldehydes↗
PubMed1997

Folding of an mRNA pseudoknot required for stop codon readthrough: effects of mono- and divalent ions on stability.

Unfolding of an mRNA pseudoknot that induces ribosome suppression of the gag gene stop codon in Moloney murine leukemia virus has been studied by UV hyperchromicity and calorimetry. The pseudoknot melts in two steps, corresponding to its two helical stems. The total enthalpy of denaturation is approximately 170 kcal/mol, approximately the value expected for the secondary structure. At low salt concentrations (<50 mM KCl) the unfolding transitions are not two-state, but they approach two-state behavior at higher salt concentrations. The structure is preferentially stabilized by smaller alkali metal ions (Li+ > Na+ > K+ > Rb+ > Cs+) and by NH4+; the same preferences are exhibited by one of the stems in the context of a hairpin. Divalent metal ions are not required to fold the pseudoknot but do stabilize it further. To examine divalent ion effects over a wide concentration range, urea was used to lower the RNA unfolding temperature and was shown not to affect characteristics of the pseudoknot unfolding in other respects. The pseudoknot binds divalent ions somewhat more tightly than a hairpin but shows only weak selectivity for different size ions. It is suggested that a region of "intermediate" divalent ion binding affinity, in between highly ligated specific sites and purely delocalized ion binding in character, is created by the pseudoknot fold but that nonspecific, delocalized ion binding contributes at least half the free energy of pseudoknot stabilization by Mg2+.

Animals↗
PubMed1997

Reported translational bypass in a trpR'-lacZ' fusion is accounted for by unusual initiation and +1 frameshifting.

I. Benhar and H. Engelberg-Kulka reported that a 55 nucleotide translational bypass occurs in decoding a fusion of the Escherichia coli tryptophan repressor, trpR, and lacZ genes. The start of the bypass occurred in the trpR gene and coding resumed in the lacZ gene. It was considered that bypassing likely occurred in expression of trpR itself to produce an additional 10 kDa product which may be biologically important. We report here that bypass is undetectable in the same and related trpR'-lacZ' fusions. The beta-galactosidase activity derived from the fusions is accounted for by unusual internal initiation and +1 frameshifting, both of which occur in the lacZ part of the fusion. The 10 kDa product reportedly encoded by the trpR gene was not detectable to a level of 1% of the full-length 12 kDa tryptophan repressor product, at least when expressed from a T7 promoter.

Amino Acid Sequence↗
PubMed1996

Reading two bases twice: mammalian antizyme frameshifting in yeast.

Programmed translational frameshifting is essential for the expression of mammalian ornithine decarboxylase antizyme, a protein involved in the regulation of intracellular polyamines. A cassette containing antizyme frameshift signals is found to direct high-level (16%) frameshifting in yeast, Saccharomyces cerevisiae. In contrast to +1 frameshifting in the mammalian system, in yeast the same frame is reached by -2 frameshifting. Two bases are read twice. The -2 frameshifting is likely to be mediated by slippage of mRNA and re-pairing with the tRNA in the P-site. The downstream pseudoknot stimulates frameshifting by 30-fold compared with 2.5-fold in reticulocyte lysates. When the length of the spacer between the shift site and the pseudoknot is extended by three nucleotides, +1 and -2 frameshifting become equal.

Amino Acid Sequence↗
PubMed1994

Pseudoknot-dependent read-through of retroviral gag termination codons: importance of sequences in the spacer and loop 2.

Retroviruses whose gag and pol genes are in the same reading frame depend upon approximately 5% read-through of the gag UAG termination codon to make the gag-pol polyprotein. For murine leukemia virus, this read-through is dependent on a pseudoknot located eight nucleotides 3' of the UAG. Other retroviruses whose gag and pol genes are in the same frame can potentially form similar pseudoknots 3' of their UAG codons. Beyond the similar secondary structures, there is strong sequence conservation in the spacer region and in loop 2 of the pseudoknots. The detrimental effects of substitutions of several of these conserved spacer and loop 2 nucleotides in the murine leukemia virus sequence show their importance for the read-through process. The importance of specific nucleotides in loop 2 of the pseudoknot contrasts with the flexibility of sequence in loop 2 of the most intensively studied frameshift-promoting pseudoknot which occurs in infectious bronchitis virus. Two nucleotides in loop 2 of the murine leukemia virus pseudoknot, which were shown to be important by mutagenic analysis, display hypersensitivity to the single-strand specific nuclease, S1. They are likely to be particularly accessible or are in an unusually reactive conformation.

Base Sequence↗
PubMed1994

rRNA-mRNA base pairing stimulates a programmed -1 ribosomal frameshift.

Base pairing between the 3' end of 16S rRNA and mRNA is shown to be important for the programmed -1 frameshifting utilized in decoding the Escherichia coli dnaX gene. This pairing is the same as the Shine-Dalgarno pairing used by prokaryotic ribosomes in selection of translation initiators, but for frameshifting the interaction occurs within elongating ribosomes. For dnaX -1 frameshifting, the 3' base of the Shine-Dalgarno sequence is 10 nucleotides 5' of the shift site. Previously, Shine-Dalgarno rRNA-mRNA pairing was shown to stimulate the +1 frameshifting necessary for decoding the release factor 2 gene. However, in the release factor 2 gene, the Shine-Dalgarno sequence is located 3 nucleotides 5' of the shift site. When the Shine-Dalgarno sequence is moved to the same position relative to the dnaX shift site, it is inhibitory rather than stimulatory. Shine-Dalgarno interactions by elongating ribosomes are likely to be used in stimulating -1 frameshifting in the decoding of a variety of genes.

Base Composition↗
PubMed1991

Evidence that a downstream pseudoknot is required for translational read-through of the Moloney murine leukemia virus gag stop codon.

Approximately 5% of the ribosomes translating the gag gene of murine leukemia viruses read through the UAG terminator and translate the in-frame pol gene to produce the gag-pol fusion polyprotein, the sole source of the pol gene products. We show that a pseudoknot located eight nucleotides 3' of the UAG codon in the Moloney murine leukemia virus is required for read-through. This requirement is markedly different from that known to be involved in other cases of read-through but surprisingly similar to some stimulatory sequences known to promote ribosomal frameshifting.

Animals↗
PubMed1990

Guanine tracts enhance sequence directed DNA bends.

Synthetic DNA fragments were constructed to determine the effect of G tracts, in conjunction with periodically spaced A tracts, on DNA bends. Relative length measurements showed that the G tracts spaced at the half helical turn enhanced the DNA bend. When the G tract was interrupted with a thymine or shortened to one or two guanines, the relative lengths decreased. If the G tract was replaced with either an A tract or a T tract, the bend was cancelled. Replacement with a C tract decreased the relative length to that of a thymine interruption suggesting that bend enhancement due to G tracts requires A tracts on the same strand.

Base Sequence↗
PubMedDate not supplied

Upstream stimulators for recoding.

Recent progress in elucidation of 5' stimulatory elements for translational recoding is reviewed. A 5' Shine-Dalgarno sequence increases both +1 and -1 frameshift efficiency in several genes; examples cited include the E. coli prfB gene encoding release factor 2 and the dnaX gene encoding the gamma and tau subunits of DNA polymerase III holoenzyme. The spacing between the Shine-Dalgarno sequence and the shift site is critical in both the +1 and -1 frameshift cassettes; however, the optimal spacing is quite different in the two cases. A frameshift in a mammalian chromosomal gene, ornithine decarboxylase antizyme, has recently been reported; 5' sequences have been shown to be vital for this frameshift event. Escherichia coli bacteriophage T4 gene 60 encodes a subunit of its type II DNA topoisomerase. The mature gene 60 mRNA contains an internal 50 nucleotide region that appears to be bypassed during translation. A 16 amino acid domain of the nascent peptide is necessary for this bypass to occur.

Animals↗