Ribosomal -1 frameshifting during decoding of Bacillus subtilis cdd occurs at the sequence CGA AAG

During translation of the Bacillus subtilis cdd gene, encoding cytidine deaminase (CDA), a ribosomal -1 frameshift occurs near the stop codon, resulting in a CDA subunit extended by 13 amino acids. The frequency of the frameshift is approximately 16%, and it occurs both when the cdd gene is expressed from a multicopy plasmid in Escherichia coli and when it is expressed from the chromosomal copy in B. subtilis. As a result, heterotetrameric forms of the enzyme are formed in vivo along with the dominant homotetrameric species. The different forms have approximately the same specific activity. The cdd gene was cloned in pUC19 such that the lacZ' gene of the vector followed the cdd gene in the -1 reading frame immediately after the cdd stop codon. By using site-directed mutagenesis of the cdd-lacZ' fusion, it was shown that frameshifting occurred at the sequence CGA AAG, 9 bp upstream of the in-frame cdd stop codon, and that it was stimulated by a Shine-Dalgarno-like sequence located 14 bp upstream of the shift site. The possible function of this frameshift in gene expression is discussed.     

Identification of the site of translational frameshifting required for production of the transposase encoded by insertion sequence IS 1.

Summary Previous genetic analyses indicated that translational frameshifting in the –1 direction occurs within the run of six adenines in the sequence 5-TTAAAAAACTC-3 at nucleotide positions 305–315 in IS 1, where the two out-of-phase reading frames insA and B-insB overlap, to produce transposase with a polypeptide segment Leu-Lys-Lys-Leu at residues 84–87. IS 1 mutants with a 1 by insertion, which encode mutant transposases with an amino acid substitution within the polypeptide segment at residues 84–87, did not efficiently mediate cointegration, except for an IS 1 mutant which encodes a mutant transposase with a Leu-Arg-Lys-Leu segment instead of Leu-LysLys-Leu. An IS 1 mutant with the DNA segment 5-CTTAAAAACTC-3 at positions 305–315 carrying the termination codon TAA in the B-insB reading frame could still mediate cointegration, indicating that codon AAA for Lys corresponding to second, third and fourth positions in the run of adenines is the site of frameshifting. The -galactosidase activity specified by several IS 1- lacZ fusion plasmids, in which B-insB is in-frame with lacZ, showed that the region 292–377 is sufficient for frameshifting. The protein produced by frameshifting from the IS 1-lacZ plasmid in fact contained the polypeptide segment Leu - Lys - Lys - Leu encoded by the DNA segment 5-TTAAAAAACTC-3, indicating that –1 frameshifting does occur within the run of adenines.     

A sequence required for -1 ribosomal frameshifting located four kilobases downstream of the frameshift site

Programmed ribosomal frameshifting allows one mRNA to encode regulate expression of, multiple open reading frames (ORFs). The polymerase encoded by ORF 2 of Barley yellow dwarf virus (BYDV) is expressed via minus one (-1) frameshifting from the overlapping ORF 1. Previously, this appeared to be mediated by a 116 nt RNA sequence that contains canonical -1 frameshift signals including a shifty heptanucleotide followed by a highly structured region. However, unlike known -1 frameshift signals, the reporter system required the zero frame stop codon and did not require a consensus shifty site for expression of the -1 ORF. In contrast, full-length viral RNA required a functional shifty site for frameshifting in wheat germ extract, while the stop codon was not required. Increasing translation initiation efficiency by addition of a 5' cap on the naturally uncapped viral RNA, decreased the frameshift rate. Unlike any other known RNA, a region four kilobases downstream of the frameshift site was required for frameshifting. This included an essential 55 base tract followed by a 179 base tract that contributed to full frameshifting. The effects of most mutations on frameshifting correlated with the ability of viral RNA to replicate in oat protoplasts, indicating that the wheat germ extract accurately reflected control of BYDV RNA translation in the infected cell. However, the overall frameshift rate appeared to be higher in infected cells, based on immunodetection of viral proteins. These findings show that use of short recoding sequences out of context in reporter constructs may overlook distant signals. Most importantly, the remarkably long-distance interaction reported here suggests the presence of a novel structure that can facilitate ribosomal frameshifting.     

Evidence for a role of translational frameshifting in the expression of transposition activity of the bacterial insertion element IS1

The bacterial insertion element IS1 contains two essential open reading frames, insA and insB, arranged in tandem. We have introduced a number of site-specific mutations into the region including the 3'-terminal region of insA, the region between insA and insB, and the ATG codon at the start of insB. Relative transposition activities of mutant and wild-type elements were determined using a modified in vivo cointegration assay. The results support the hypothesis that a translational (-1) frameshift occurring in the 3'-terminal region of insA and linking insB to insA is responsible for the synthesis of the active IS1 transposition enzyme. Further results with IS1 elements containing internal deletions are in agreement with a role of the normally terminated insA product as an inhibitor of transposition.     

IS1 insertion generates duplication of a nine base pair sequence at its target site.

Three independent integrations of the E. coli insertion sequence, IS1, into the gal operon have been analyzed. DNA sequences of portions of the wild-type galT gene which act as the target sites for these insertions, as well as the corresponding gal/IS1 junctions, are reported. Two features are particularly noteworthy. First, similar sequences appearing in inverted orientation consitute the ends of IS1: 18 of the terminal 23 base pairs at each end are identical. Second, in all three insertions, a 9 base pair segment found once in the wild-type sequence at the site of insertion is duplicated and appears in the same orientation at each end of the inserted element. The sequence of this 9 base pair repeat is different for each insertion analyzed. No homology between the inverted repeat sequences at the ends of IS1 and the sequences of the target sites is observed. Models for the mechanism of IS1 insertion are proposed.     

A three-way junction and constituent stem-loops as the stimulator for programmed -1 frameshifting in bacterial insertion sequence IS911

Several signals are required for the programmed frameshifting in translation of IS911 mRNA. These include a Shine Dalgarno (SD)-like sequence, a slippery sequence of six adenine residues and a guanine residue (A6G) and a 3' secondary structure. The structure of the mRNA containing these elements was investigated using chemical and enzymatic probing. The probing data show that the 3' structure is a three-way junction of stems. The function of the three-way junction was investigated by mutagenesis. Disrupting the stability of the structure greatly affects frameshifting and transposition levels as tested by separate in vivo assays. Structural probing and thermal melting profiles indicate that the disrupted three-way junctions have altered structures.     

Immunity to repeated transposition of the insertion sequence IS21

The ability of pBR325 derivatives carrying a copy of IS21-element to accept the second copy of this element from plasmid pRP19.6, a temperature-sensitive for replication mutant of RPI containing the duplicated IS21 was studied. It was shown that the frequency of IS21 transposition into plasmids pBR32S::IS21 differing by localization IS21 was lower by two orders of magnitude as compared to that of pBR325. The restriction endonuclease analysis revealed that the insertion of the second copy of IS21 resulted in the formation of pBR325 derivatives carrying the tandem repeated copies of IS21. It was also shown that the plasmids pBR325::IS21 were capable of increasing the frequency of pRP19.6 insertion into the bacterial chromosome from 3-9 to 200-300 times depending on IS21 localization. On the basis of the results obtained and literature data the possible mechanism of the transposition immunity is discussed.     

Anatomy of a preferred target site for the bacterial insertion sequence IS903

Like many transposons the bacterial insertion sequence IS903 was thought to insert randomly. However, using both genetic and statistical approaches, we have derived a target site for IS903 that is used 84% of the time. Computational and genetic analyses of multiple IS903 insertion sites predicted a preferred target consisting of a 21 bp palindromic pattern centered on the 9 bp target duplication generated during transposition. Here we show that targeting can be dissected into four components: the 5 bp flanking sequences, the most important sequences required for site-specific insertion; the 7 bp palindromic core within the target duplication; the dinucleotide pair at the transposon-target junction; and the local DNA context. Finally, using a substrate with multiple target sites we show that a target site is more likely found by a local bind-and-slide model and not by extended DNA tracking.     

Linearization and transposition of circular molecules of insertion sequence IS3.

IS3 transposase has been shown to promote production of characteristic circular and linear IS3 molecules from the IS3-carrying plasmid; IS3 circles have the entire IS3 sequence with terminal inverted repeats, IRL and IRR, which are separated by a three base-pair sequence originally flanking either end in the parental plasmid, whereas linear IS3 molecules have three nucleotide overhangs at their 5' ends. Here, we showed that a plasmid carrying an IS3 derivative, which is flanked by different sequences at both ends, generated IS3 circles and linear IS3 molecules owing to the action of transposase. Cloning and sequencing analyses of the linear molecules showed that each had the same 5'-protruding three nucleotide overhanging sequences at both ends, suggesting that the linear molecules were not generated from the parental plasmid by the two double-strand breaks at both end regions of IS3. The plasmid carrying IS3 with a two base-pair mutation in the terminal dinucleotide, which would be required for transposase to cleave the 3' end of IS3, could still generate linear molecules as well as circles. Plasmids bearing an IS3 circle were cleaved by transposase and gave linear molecules with the same 5'-protruding three nucleotide overhanging sequences. These show that the linear molecules are generated from IS3 circles via a double-strand break at the three base-pair intervening sequence. Plasmids carrying an IS3 circle with the two base-pair end mutation still were cleaved by transposase, though with reduced efficiencies, suggesting that IS3 transposase has the ability to cleave not only the 3' end of IS3, but a site three nucleotides from the 5' end of IS3. IS3 circles also were shown to transpose to the target plasmids. The end mutation almost completely inhibited this transposition, showing that the terminal dinucleotides are important for the transfer of the 3' end of IS3 to the target as well as for the end cleavage.     

Intramolecular transposition of insertion sequence IS91 results in second-site simple insertions

A series of plasmids carrying an IRL-kan-IRR transposable cassette, in which IRL and IRR are the left- and right-terminal sequences of IS91, have been constructed. These cassettes could be complemented for transposition with similar efficiency when IS91 transposase was provided either in cis or in trans. A total of 87% of IS91 transposition products were simple insertions of the element, while the remaining 13% were plasmid fusions and co-integrates. When transposase expression was induced from an upstream lac promoter, transposition frequency increased approximately 100-fold. An open reading frame (ORF) present upstream of the transposase gene, ORF121, could be involved in target selection, as mutations affecting this ORF were altered in their insertion specificity. Intramolecular rearrangements were analysed by looking at transposition events disrupting a chloramphenicol resistance gene (cat ) located outside the transposable cassette. Plasmid instability resulting from insertion of an extra copy of IRL-kan-IRR within the cat gene was observed; transposition products contained a second copy of the cassette inserted either as a direct or as an inverted repeat. No deletion or inversion of the intervening DNA was observed. These results could be explained as a consequence of intramolecular transposition of IS91 according to a model of rolling-circle transposition.     

Terminal inverted repeats of insertion sequence IS30 serve as targets for transposition.

In the present study, we demonstrate that the terminal inverted repeats of the Escherichia coli insertion sequence IS30 are functional target sites for the transposition of the (IS30)2 dimer, which represents an intermediate structure in the transposition of IS30. Comparative analysis of various target regions revealed that the left and right ends differ in their "attractivity." In our experiments, the joined left and right ends, i.e., the (IS30)2 intermediate structure, was found to be the most preferred target. It was also shown that flanking sequences can influence the target activity of the terminal repeats. The functional part of the target region was localized in the inverted repeats by means of mutational analysis, and it corresponds to the binding site of IS30 transposase. Insertion of 1 bp into the right inverted repeat resulted in unusual target duplication accompanied by gene conversion. The choice of the terminal inverted repeats as targets in transposition leads to the reconstruction of the (IS30)2 structure, which may induce a cascade of further rearrangements. Therefore, this process can play a role in the evolution of the genome.     

IS4 is still found at its chromosomal site after transposition to galT

Summary IS4-DNA has been hybridized to separated DNA fragments of E. coli K12 strain M28 and to three mutants caused by transposition of IS4 to galT. The parental strain shows one band hybridizing to IS4 representing one copy of IS4 in the chromosome. The mutants have this copy retained and show in addition a second band corresponding to the IS4 copy in galT. The experiments support the hypothesis that transposition of IS4 is accompanied by replication of the element.     

Programmed translational frameshifting and initiation at an AUU codon in gene expression of bacterial insertion sequence IS911.

The proteins expressed by insertion sequence IS911, a member of the widespread IS3 family of elements, have been analyzed. The results indicate that three major species are produced from two consecutive reading frames. A protein of Mr 11,500, ORFA, is synthesized from an upstream reading frame. A larger protein, ORFAB, uses the same initiation codon and is produced by a -1 programmed translational frameshift between orfA and a downstream frame, orfB, whose amino acid sequence shows significant homology with retroviral integrase proteins. The orfB frame is also expressed independently in two alternative forms: the first uses a rare AUU initiation codon in the orfB phase whereas the second appears to initiate in the orfA phase and is produced by a -1 frameshift mechanism similar to that used in ORFAB expression. A specific IS911 integration reaction using a minimal active junction composed of 51 base-pairs of the right inverted repeat and a flanking phase lambda sequence resembling a second end in inverted orientation has been developed to analyze the functions of these proteins by transcomplementation in vivo. The orfA and orfB frames are shown to be essential and production of ORFAB is shown to stimulate integration in this system, suggesting that this fusion protein is the IS911 transposase.     

Two frameshift products involved in the transposition of bacterial insertion sequence IS629

IS629 is 1,310 bp in length with a pair of 25-bp imperfect inverted repeats at its termini. Two partially overlapping open reading frames, orfA and orfB, are present in IS629, and two putative translational frameshift signals, TTTTG (T4G) and AAAAT (A4T), are located near the 3'-end of orfA. With the lacZ gene as the reporter, both T4G and A4T motifs are determined to be a -1 frameshift signal. Two peptides representing the two transframe products designated OrfAB' and OrfAB, are identified by a liquid chromatography-tandem mass spectrometric approach. Results of transposition assays show that OrfAB' is the transposase and that OrfAB aids in the transposition of IS629. Pulse-chase experiments and Escherichia coli two-hybrid assays demonstrate that OrfAB binds to and stabilizes OrfAB', thus increasing the transposition activity of IS629. This is the first transposable element in the IS3 family shown to have two functional frameshifted products involved in transposition and to use a transframe product to regulate transposition.     

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.