In the lacI gene of Escherichia coli spontaneous base substituion hotspots occur at 5-methylcytosine residues. The hotspots disappear when the respective cytosines are not methylated. We suggest that the hotspots may result from the spontaneous deamination of 5-methylcytosine to thymine, which is not excised by the enzyme DNA-uracil glycosidase.

Estimates of missense error rates (misreading) during protein synthesis vary from 10 À3 to 10 À4 per codon. The experiments reporting these rates have measured several distinct errors using several methods and reporter systems. Variation in reported rates may reflect real differences in rates among the errors tested or in sensitivity of the reporter systems. To develop a more accurate understanding of the range of error rates, we developed a system to quantify the frequency of every possible misreading error at a defined codon in Escherichia coli. This system uses an essential lysine in the active site of firefly luciferase. Mutations in Lys529 result in up to a 1600-fold reduction in activity, but the phenotype varies with amino acid. We hypothesized that residual activity of some of the mutant genes might result from misreading of the mutant codons by tRNA Lys UUU , the cognate tRNA for the lysine codons, AAA and AAG. Our data validate this hypothesis and reveal details about relative missense error rates of near-cognate codons. The error rates in E. coli do, in fact, vary widely. One source of variation is the effect of competition by cognate tRNAs for the mutant codons; higher error frequencies result from lower competition from low-abundance tRNAs. We also used the system to study the effect of ribosomal protein mutations known to affect error rates and the effect of error-inducing antibiotics, finding that they affect misreading on only a subset of near-cognate codons and that their effect may be less general than previously thought.

▪ Abstract  Errors that alter the reading frame occur extremely rarely during translation, yet some genes have evolved sequences that efficiently induce frameshifting. These sequences, termed programmed frameshift sites, manipulate the translational apparatus to promote non-canonical decoding. Frameshifts are mechanistically diverse. Most cause a −1 shift of frames; the first such site was discovered in a metazoan retrovirus, but they are now known to be dispersed quite widely among evolutionarily diverse species. +1 frameshift sites are much less common, but again dispersed widely. The rarest form are the translational hop sites which program the ribosome to bypass a region of several dozen nucleotides. Each of these types of events are stimulated by distinct mechanisms. All of the events share a common phenomenology in which the programmed frameshift site causes the ribosome to pause during elongation so that the kinetically unfavorable alternative decoding event can occur. During this pause most frameshifts occur because one or more ribosome-bound tRNAs slip between cognate or near-cognate codons. However, even this generalization is not entirely consistent, since some frameshifts occur without slippage. Because of their similarity to rarer translational errors, programmed frameshift sites provide a tool with which to probe the mechanism of frame maintenance.

Ribosomal frameshifting regulates expression of the TYB gene of yeast Ty retrotransposons. We previously demonstrated that a 14 nucleotide sequence conserved between two families of Ty elements was necessary and sufficient to support ribosomal frameshifting. This work demonstrates that only 7 of these 14 nucleotides are needed for normal levels of frameshifting. Any change to the sequence CUU-AGG-C drastically reduces frameshifting; this suggests that two specific tRNAs, tRNA(UAGLeu) and tRNA(CCUArg), are involved in the event. Our tRNA overproduction data suggest that a leucyl-tRNA, probably tRNA(UAGLeu), an unusual leucine isoacceptor that recognizes all six leucine codons, slips from CUU-Leu onto UUA-Leu (in the +1 reading frame) during a translational pause at the AGG-Arg codon induced by the low availability of tRNA(CCUArg), encoded by a single-copy essential gene. Frameshifting is also directional and reading frame specific. Interestingly, frameshifting is inhibited when the "slip" CUU codon is located three codons downstream, but not four or more codons downstream, of the translational initiation codon.

Benzo [a]pyrene diol epoxide (BPDE), the active metabolite of benzo [a]pyrene present in tobacco smoke, is a major cancer-causing compound. To evaluate the effects of BPDE on human breast epithelial cells, we exposed an immortalized human breast cell line, MCF 10A, to BPDE and characterized the gene expression pattern. Of the differential genes expressed, we found consistent activation of DDX3, a member of the DEAD box RNA helicase family. Overexpression of DDX3 in MCF 10A cells induced an epithelial-mesenchymal-like transformation, exhibited increased motility and invasive properties, and formed colonies in soft-agar assays. Besides the altered phenotype, MCF 10A-DDX3 cells repressed E-cadherin expression as demonstrated by both immunoblots and by E-cadherin promoter-reporter assays. In addition, an in vivo association of DDX3 and the E-cadherin promoter was demonstrated by chromatin immunoprecipitation assays. Collectively, these results demonstrate that the activation of DDX3 by BPDE, can promote growth, proliferation and neoplastic transformation of breast epithelial cells.