Cis preference of the IS 903 transposase is mediated by a combination of transposase instability and inefficient translation

The transposase protein encoded by the insertion element IS 903 belongs to an unusual class of DNA-binding proteins, termed cis-acting proteins, that act preferentially at their site of synthesis. Previous work had led us to propose that instability of the IS 903 transposase was a major determinant of its cis preference. Here we describe the isolation of two classes of mutations within the transposase gene that increased action in trans. One class specifically increased trans action without increasing the level of transposition when the mutant gene was located in cis to the transposon. In particular, a threonine-to-proline substitution at amino acid 25 (T25P) reduced cis preference about 60-fold. The half-life of this mutant transposase was significantly longer than that of the wild-type transposase, confirming the critical role of protein instability. The second, larger, class of mutations increased the level of transposition both in trans and in cis. The behaviour and location of these mutations were consistent with an increase in gene expression by improving translational initiation. Several of these mutations exerted a disproportionate effect on the action of transposase in trans, implying that translation efficiency may affect more than just the amount of transposase made. Our results indicate that cis preference of the IS 903 transposase is mediated by a combination of transposase instability and inefficient translation initiation.     

Escherichia coli translation initiation factor 3 discriminates the initiation codon in vivo

In a genetic selection designed to isolate Escherichia coli mutations that increase expression of the IS 10 transposase gene ( tnp ), we unexpectedly obtained viable mutants defective in translation initiation factor 3 (IF3). Several lines of evidence led us to conclude that transposase expression, per se , was not increased. Rather, these mutations appear to increase expression of the tnp'–'lacZ gene fusions used in this screen, by increasing translation initiation at downstream, atypical initiation codons. To test this hypothesis we undertook a systematic analysis of start codon requirements and measured the effects of IF3 mutations on initiation from various start codons. Beginning with an efficient translation initiation site, we varied the AUG start codon to all possible codons that differed from AUG by one nucleotide. These potential start codons fall into distinct classes with regard to translation efficiency in vivo : Class I codons (AUG, GUG, and UUG) support efficient translation; Class IIA codons (CUG, AUU, AUC, AUA, and ACG) support translation at levels only 1–3% that of AUG; and Class IIB codons (AGG and AAG) permit levels of translation too low for reliable quantification. Importantly, the IF3 mutations had no effect on translation from Class I codons, but they increased translation from Class II codons 3–5-fold, and this same effect was seen in other gene contexts. Therefore, IF3 is generally able to discriminate between efficient and inefficient codons in vivo , consistent with earlier in vitro observations. We discuss these observations as they relate to IF3 autoregulation and the mechanism of IF3 function.     

IS10 transposase mutations that specifically alter target site recognition.

IS10 inserts preferentially into particular hotspots. We describe here mutations of IS10 transposase, called 'ATS' that confer Altered Target Specificity. These mutations yield a general relaxation in target specificity but do not affect other aspects of transposition. Thus, the preference for specific nucleotide sequences at the target site can be cleanly separated from other steps of the transposition reaction. Eleven ATS mutations identified in a genetic screen occur at only two codons in transposase, one in each of two regions of the protein previously implicated in target site interactions (Patch I and Patch II). Genetic analysis suggests that mutations at the two ATS codons affect the same specific function of transposase, thus raising the possibility that Patch I and Patch II interact. For wild-type IS10, insertion specificity is determined in part by a specific 6 bp consensus sequence and in part by the immediately adjacent sequence context of the target DNA. The ATS mutations do not qualitatively alter the hierarchy with which base pairs are recognized in the consensus sequence; instead, sites selected by ATS transposase exhibit a reduction in the degree to which certain base pairs are preferred over others. Models for the basis of this phenotype are discussed.     

Characterization of Tn10d-Cam: a transposition-defective Tn10 specifying chloramphenicol resistance

Summary We have constructed a small, transposition-defective derivative of the transposon Tn10 that carries the chloramphenicol acetyltransferase gene of pACYC184. This new genetic element, Tn10d-Cam, transposes when Tn10 transposase is provided from a multi-copy plasmid. Transposon insertion mutagenesis of Salmonella typhimurium was performed by using a strain carrying a Tn10d-Cam insertion in an Escherichia coli F' episome as the donor in transductional crosses into recipients that carried a plasmid expressing Tn10 transposase. Tn10d-Cam insertion mutations were also generated by complementation in cis of Tn10d-Cam by a cotransducible Tn10 element that overproduces transposase. Here, transposase was provided only transiently, and the Tn10d-Cam insertion mutations were recovered in a transposase-free strain. Cis complementation was used for mutagenesis of a plasmid target. The site specificity of insertion and the effect of insertions on expression of a downstream gene were investigated, using Tn10d-Cam insertions in a plasmid carrying a segment of the histidine operon.     

The regulatory role of the IS 1-encoded InsA protein in transposition

We show here that the protein InsA, which is encoded by IS 1 and binds specifically to the terminal inverted repeats of this insertion sequence, negatively regulates IS 1 transposition activity. We demonstrate that it inhibits both IS 1-mediated cointegrate formation and transposition of a synthetic IS 1-based transposon (‘omegon’Ω-on). These results also indicate that the Ω-on which does not itself encode IS 1 transposition functions can be complemented in trans, presumably by the copies of IS 1 resident in the Escherichia coli chromosome. Using insA-lacZ gene fusions, we show that at least part of this effect can be explained by the ability of InsA to repress expression of IS 1-encoded genes both in cis or in trans. The experiments involving Ω-on transposition raise the possibility that InsA inhibits transposition directly by competition with the transposase for their cognate site within the ends of IS 1.     

Role of 5′‐ and 3′‐untranslated regions of mRNAs in human diseases

Protein synthesis is often regulated at the level of initiation of translation, making it a critical step. This regulation occurs by both the cis‐regulatory elements, which are located in the 5′‐ and 3′‐UTRs (untranslated regions), and trans‐acting factors. A breakdown in this regulation machinery can perturb cellular metabolism, leading to various physiological abnormalities. The highly structured UTRs, along with features such as GC‐richness, upstream open reading frames and internal ribosome entry sites, significantly influence the rate of translation of mRNAs. In this review, we discuss how changes in the cis‐regulatory sequences of the UTRs, for example, point mutations and truncations, influence expression of specific genes at the level of translation. Such modifications may tilt the physiological balance from healthy to diseased states, resulting in conditions such as hereditary thrombocythaemia, breast cancer, fragile X syndrome, bipolar affective disorder and Alzheimer's disease. This information tends to establish the crucial role of UTRs, perhaps as much as that of coding sequences, in health and disease.     

An insertion sequence unique to Frankia strain ArI5

At the genetic level, understanding of symbiotic nitrogen fixation by Frankia is limited to nif functions that are highly conserved among all organisms. The genetics and biochemistry of nodulation are largely unexplored because of a complete lack of genetic tools. In other bacteria, mobile genetic elements such as insertion sequences (IS) and transposons are commonly used to create mutations and insert new genetic material. We have characterized a 4 kbp segment of DNA from Frankia strain ArI5 that has the hallmarks of a mobile genetic element, inverted repeats flanking a gene encoding a transposase. There are at least six copies of this element in strain ArI5 but none in either strain CcI3 or CpI1. The inverted repeats are 17 nt long and separated by 2156 bp. Within that region are two, overlapping ORFs that each encode a transposase. RT-PCR analysis of RNA from Frankia ArI5 cells conclusively demonstrates the expression of one transposase gene and suggests that both may be transcribed. Numerous attempts to clone the intact IS in E. coli were unsuccessful suggesting that the element may be unstable in this context. A clone containing the complete IS was constructed in E. coli then modified by insertion of the kanamycin (KAN) resistance gene from Tn5. A fragment of DNA including the inverted repeats, transposase genes and KAN gene, was transferred to the suicide vector pJBSD1. The construct, pFRISK, was transformed into E. coli to search for transposition events.     

Characterization of IS1221 from Mycoplasma hyorhinis: expression of its putative transposase in Escherichia coli incorporates a ribosomal frameshift mechanism

Seven complete and two partial copies of IS1221 variants from Mycoplasma hyorhinis and Mycoplasma hyopneumoniae characterized to date have established a consensus IS1221 as a 1513 bp element with unique structural characteristics resembling the IS3 family of bacterial insertion sequences. Each IS1221 copy contains highly conserved 28 bp imperfect terminal inverted repeats and three distinctive internal inverted repeats (LIR, RIR and IIR). IIR is located within the coding region of the element and it is proposed that it plays a critical role in the regulation of putative transposase expression. Consensus IS1221 and one particular copy, G1135.2, contain a single long open reading frame (ORF). Two potential initiation codons are present at nucleotide 46 (AUG46) and nucleotide 397 (AUG397) and both are preceded by strong ribosome-binding sites. Both initiation codons can be used efficiently in an Escherichia coli T7 expression system. The LIR has a negative regulatory effect on translation initiation from AUG46. A -1 translational frameshift event is shown to be involved in expression of the IS1221 ORF and results in the production of 20kDa and 6kDa truncated proteins from the respective upstream initiation codons of the IS1221 ORF. Base substitution and deletion mutations in sequences resembling characterized motifs in documented examples of translational frameshifting resulted in a significant increase in the full-length products and a corresponding decrease in the truncated products from the IS1221 ORF. In contrast to the usual -1 frameshift regulatory event in the IS3 family, which produces a transframe fusion product as the active transposase, IS1221 may have evolved a high-frequency -1 frameshift mechanism that produces a truncated product from the upstream coding domain and thereby results in the regulated low-level production of the full-length presumptive transposase.     

Translational Control of the Picornavirus Phenotype

Picornaviruses are small animal viruses with positive-strand genomic RNA, which is translated using cap-independent internal translation initiation. The key role in this is played by ciselements of the 5"-untranslated region (5"-UTR) and, in particular, by the internal ribosome entry site (IRES). The function of translational ciselements requires both canonical translation initiation factors (eIFs) and additional IRES trans-acting factors (ITAFs). All known ITAFs are cell RNA-binding proteins which play a variety of functions in noninfected cells. Specific features of translational ciselements substantially affect the phenotype and, in particular, tissue tropism and pathogenic properties of picornaviruses. It is clear that, in some cases, the molecular mechanism involved is a change in interactions between viral ciselements and ITAFs. The properties and tissue distribution of ITAFs may determine the biological properties of other viruses that also use the IRES-dependent translation initiation. Since this mechanism is also involved in translation of several cell mRNAs, ITAF may contribute to the regulation of the most important aspects of the living activity in noninfected cells.     

Mechanisms of the initiation of protein synthesis: in reading frame binding of ribosomes to mRNA

The various mechanisms proposed to describe the initiation of protein synthesis are reviewed with a focus on their initiation signals. A characteristic feature of the various mechanisms is that each one of them postulates a distinct initiation signal. The signals of the Shine–Dalgarno (SD), the scanning and the internal ribosome entry site (IRES) mechanisms are all located exclusively in the 5′ leader sequence, whereas, the signal of the cumulative specificity (CS) mechanism includes the entire initiation site (IS). Computer analysis of known E. coli IS sequences showed signal characteristics in the entire model IS consisting of 47 bases, in segments of the 5′ leader and of the protein-coding regions. The proposal that eukaryotic translation actually occurs in two steps is scrutinized. In a first step, initiation factors (eIF4F) interact with the cap of the mRNA, thereby enhancing the accessibility of the IS. In the second step, initiation is by the conserved prokaryotic mechanism in which the ribosomes bind directly to the mRNA without ribosomal scanning. This binding occurs by the proposed process of in reading frame binding of ribosomes to mRNA, which is consistent with the CS mechanism. The basic CS mechanism is able to account for the initiation of translation of leaderless mRNAs, as well as for that of canonical mRNAs. The SD, the scanning and the IRES mechanisms, on the other hand, are inconsistent with the initiation of translation of leaderless mRNAs. Based on these and other observations, it is deemed that the CS mechanism is the universal initiation mechanism.     

Translation acrobatics: how cancer cells exploit alternate modes of translational initiation

Recent work has brought to light many different mechanisms of translation initiation that function in cells in parallel to canonical cap‐dependent initiation. This has important implications for cancer. Canonical cap‐dependent translation initiation is inhibited by many stresses such as hypoxia, nutrient limitation, proteotoxic stress, or genotoxic stress. Since cancer cells are often exposed to these stresses, they rely on alternate modes of translation initiation for protein synthesis and cell growth. Cancer mutations are now being identified in components of the translation machinery and in cis‐regulatory elements of mRNAs, which both control translation of cancer‐relevant genes. In this review, we provide an overview on the various modes of non‐canonical translation initiation, such as leaky scanning, translation re‐initiation, ribosome shunting, IRES‐dependent translation, and m⁶A‐dependent translation, and then discuss the influence of stress on these different modes of translation. Finally, we present examples of how these modes of translation are dysregulated in cancer cells, allowing them to grow, to proliferate, and to survive, thereby highlighting the importance of translational control in cancer.