Raymond F. Gesteland

Autoregulatory frameshifting in decoding mammalian ornithine decarboxylase antizyme.

Abstract Rat antizyme gene expression requires programmed, ribosomal frameshifting. A novel autoregulatory mechanism enables modulation of frameshifting according to the cellular concentration of polyamines. Antizyme binds to, and destabilizes, ornithine decarboxylase, a key enzyme in polyamine synthesis. Rapid degradation ensues, thus completing a regulatory circuit. In vitro experiments with a fusion construct using reticulocyte lysates demonstrate polyamine-dependent expression with a frameshift efficiency of 19% at the optimal concentration of spermidine. The frameshift is +1 and occurs at the codon just preceding the terminator of the initiating frame. Both the termination codon of the initiating frame and a pseudoknot downstream in the mRNA have a stimulatory effect. The shift site sequence, UCC-UGA-U, is not similar to other known frameshift sites. The mechanism does not seem to involve re-pairing of peptidyl-tRNA in the new frame but rather reading or occlusion of a fourth base.

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.

Sequence specificity of aminoglycoside-induced stop codon readthrough: Potential implications for treatment of Duchenne muscular dystrophy

As a result of their ability to induce translational readthrough of stop codons, the aminoglycoside antibiotics are currently being tested for efficacy in the treatment of Duchenne muscular dystrophy patients carrying a nonsense mutation in the dystrophin gene. We have undertaken a systematic analysis of aminoglycoside‐induced readthrough of each stop codon in human tissue culture cells using a dual luciferase reporter system. Significant differences in the efficiency of aminoglycoside‐induced readthrough were observed, with UGA showing greater translational readthrough than UAG or UAA. Additionally, the nucleotide in the position immediately downstream from the stop codon had a significant impact on the efficiency of aminoglycoside‐induced readthrough in the order C > U > A ≥ G. Our studies show that the efficiency of stop codon readthrough in the presence of aminoglycosides is inversely proportional to the efficiency of translational termination in the absence of these compounds. Using the same assay, we analyzed a 33–base pair fragment of the mouse dystrophin gene containing the mdx premature stop codon mutation UAA (A), which is also the most efficient translational terminator. The additional flanking sequences from the dystrophin gene do not significantly change the relatively low‐level aminoglycoside‐induced stop codon readthrough of this stop codon. The implications of these results for drug efficacy in the treatment of individual patients with Duchenne muscular dystrophy or other genetic diseases caused by nonsense mutations are discussed. Ann Neurol 2000;48:164–169

Reading frame switch caused by base-pair formation between the 3' end of 16S rRNA and the mRNA during elongation of protein synthesis in Escherichia coli

Watson‐Crick base pairing is shown to occur between the mRNA and nucleotides near the 3′ end of 16S rRNA during the elongation phase of protein synthesis in Escherichia coli. This base‐pairing is similar to the mRNA‐rRNA interaction formed during initiation of protein synthesis between the Shine and Dalgarno (S‐D) nucleotides of ribosome binding sites and their complements in the 1540‐1535 region of 16S rRNA. mRNA‐rRNA hybrid formation during elongation had been postulated to explain the dependence of an efficient ribosomal frameshift on S‐D nucleotides precisely spaced 5′ on the mRNA from the frameshift site. Here we show that disruption of the postulated base pairs by single nucleotide substitutions, either in the S‐D sequence required for shifting or in nucleotide 1538 of 16S rRNA, decrease the amount of shifting, and that this defect is corrected by restoring complementary base pairing. This result implies that the 3′ end of 16S rRNA scans the mRNA very close to the decoding sites during elongation.

Location and identification of the genes for adenovirus type 2 early polypeptides

Virus-specific RNA was prepared from cells early after adenovirus type 2 infection and fractionated by hybridization to specific fragments of viral DNA. The viral mRNA was used to program cell-free protein synthesis, and the products were analyzed by electrophoresis. The genes for the early polypeptides of apparent molecular weight 44,000, 15,000, 72,000, 15,500, 19,000, and 11,000 daltons were located, respectively, between positions 0-4.1, 4.1-16.7, 58.5-70.7, 75.9-83.4, 89.7-98.6, and 89.7-98.6 of the conventional adenovirus DNA map. The polypeptide of molecular weight 72,000 daltons was shown to be the single-stranded DNA-binding protein described by others. RNAs from three different adeno-transformed cell lines each program the synthesis in vitro of predominantly the 15K polypeptide, as well as variable amounts of the polypeptide of molecular weight 44,000 daltons. The genes for these two polypeptides are located in the portion of DNA known to be required for transformation of rodent cells by adenovirus.

Translation of MuLV and MSV RNAs in nuclease-treated reticulocyte extracts: Enhancement of the gag-pol polypeptide with yeast suppressor tRNA

Abstract The virion RNAs from Moloney murine leukemia virus (MuLV) and Moloney murine sarcoma virus (MSV) were translated in a micrococcal nuclease-treated cell-free system from rabbit reticulocytes. The predominant polypeptides formed from 35S MuLV RNA were 78,000 and 65,000 daltons in molecular weight, and minor components with molecular weights of 180,000,110,000, 52,000 and 40,000 daltons were also observed. The 30S MSV RNA yielded two predominant polypeptides of 62,000 and 43,000 daltons, and minor components about 72,000, 40,000 and 18,000 daltons in molecular weight. The predominant polypeptides generated by both MuLV and MSV RNA were found to be precursors of the core proteins by immunoprecipitation with specific antisera. The 180,000 dalton molecular weight polypeptide encoded by MuLV RNA was immunoprecipitated both by antisera to the core protein (p30) and reverse transcriptase. The major products therefore appear to be Pr65 gag and Pr78 gag ; an important minor product is Pr180 gag-pol . Most of the products of When purified yeast suppressor tRNA was added to the translation mixture directed by 35S MuLV RNA, the amount of the Pr78 gag was reduced, while the yield of the Pr180 gag-pol was enhanced. Amber suppressor tRNA was about 3 times as effective as ochre suppressor tRNA and nonsuppressor tRNA. This pattern of suppression was also seen for an established amber mutation (UAG) in the synthetase gene of Qβ (Qβ aml), suggesting that it is a UAG codon which terminates synthesis of Pr78 gag . In the MSV system, the amber suppressor tRNA, and to a lesser extent the ochre suppressor tRNA, markedly increased the synthesis of the 72,000 dalton molecular weight polypeptide with a slight reduction of the 62,000 dalton protein. Since read-through between the core protein and reverse transcriptase genes occurs to a low level both in vivo and in vitro and can be enhanced in vitro by amber suppressor tRNA, these results suggest that a suppression mechanism may control the relative amounts of core protein and reverse transcriptase synthesized from 35S mRNA. Such a mechanism might be used more generally by mRNAs from mammalian cells.

Ribosome gymnastics-Degree of difficulty 9.5, style 10.0

John F. Atkins:? Robert B. Weiss, and Raymond F. Gesteland’ * Howard Hughes Medical Institute and Department of Human Genetics University of Utah Salt Lake City, Utah 84132 TDepartment of Biochemistry University College Cork Ireland Prolonged and sophisticated investigations into ribosome structure and function are being rewarded with a new un- derstanding of normal decoding. Ribosomal RNA plays a much more active role than previously thought (Dahlberg, 1989; Moazed and Noller, 1989). Sites have been identi- fied for decoding, peptidyl transferase, and termination, though none are yet as firmly established as that for initia- tion. Recent biochemical data have brought widespread acceptance of the existence of an exit (E) site in addition to the classical A and P sites. Finally, the proposal for a reciprocating motion of the two subunits, involving inter- mediate hybrid sites during the translocation process, has suggested a comforting rationale for ribosome design. However, just as ribosome functioning is beginning to come more into focus, it has been found that ribosomes are capable of unexpected gymnastic feats that, even classical ribosomologists are coming to accept, will throw light on the great areas of darkness remaining (Dahlberg, 1989). These nonstandard decoding events involve ribo- somal hopping, frameshifting, and reading through stop codons, all at unexpectedly high levels using surprising mechanisms. These possibilities mean that the protein sequence cannot always be simply deduced from the se- quence of the mature message. Hopping During translational elongation, the paired codon and an- ticodon can sometimes disengage at certain sequences, allowing the mRNA to slip with respect to the ribosome- peptidyl-tRNA complex. The anticodon may then re-pair with a now-nearby similar codon, so that synthesis con- tinues downstream. On a run of 4 identical bases the reen- gagement may occur at a codon 1 base removed from the original in-frame codon, with a resultant frameshift. “Slip- ping” of this type is part of the mechanism for many of the examples of -1 or +l frameshifting described below. If the shift occurs over a considerable distance without intermediate pairing, however, the ribosome hops down the mRNA. Hopping requires a “takeoff site” codon and a similar sequence acting as a “landing site” immediately 5’ of the codon for the next amino acid on the resumption of synthesis. Hopping was first encountered over short dis- tances by inserting test sequences early in the lacZ gene of Escherichia coli (Weiss et al., 1987). For instance, CUU UAG CUA (Leu stop Leu) was decoded with an efficiency of 1% as a single leucine from the 9 nucleotides. Hopping was also detected when the takeoff and landing sites over- lapped, as in the sequence WUA (Weiss et al., 1987; O’Connor et al., 1989). At about the same time, tRNA mu- tants were isolated that increased the hopping at certain sites (Falahee et al., 1988; Hughes 1989), and hop- ping was detected over as many three stop codons, al- beit at decreased efficiency. The mutants have an extra base in a tRNA anticodon (O’Connor et al., 1989) that somehow promotes hopping. One inference to be made from their study is that there may be good reasons why almost all natural tRNAs have seven-membered anticod- on loops! Even with these precedents, the discovery of high level, natural, programmed hopping was a surprise. The 50 nucleotides that separate codon 46 from 47 in the mature message of phage T4 topoisomerase subunit gene 60 are bypassed by the translation appara- tus with an efficiency approaching 100% (Huang et al., 1988). Several key features required for ribosomal bypass of this coding gap have been defined utilizing variants generated as gene 60-/acZ fusions (Weiss et al., 199Oa). The analogy low level and tRNA suppressor-mediated hopping is supported by a strict requirement for a matched set of codons at the takeoff and landing sites. As is the case with all high level unusual ribosomal frameshift or readthrough sites, the interesting question is how message conspires with the translation apparatus to in- crease the efficiency and scope of these events. In the gene 60 case, there are at least four distinct ele- ments that contribute significantly to the bypass. Three of these elements are located at the coding gap: the matched codon set defining its borders, a stop at the 5’junction of gap contained within a short stem- loop structure, and an optimal 50 nucleotide spacing separating the 5’ and 3’ junctions. The fourth, most surprising, feature is a stringent requirement for specific amino acid sequence in the nascent peptide translated from the 46 codons preceding coding gap. This na- scent peptide enables the ribosome that has just synthe- sized it to bypass the coding gap, although its mode of ac- tion is undefined. This nascent-chain effect adds another example to an expanding list of interesting translation events mediated through the nascent protein chain, such as signal recognition particle arrest of elongation (Wolin and Walter, 1988) autoregulated instability of 8-tubulin mRNA (Yen et al., 1989) and regulation of the carbamoyl- phosphate synthetase A gene, CPA7, in Saccharomyces cerevisiae (Werner et al., 1987). Another example of high level natural hopping could be in the carA gene of Pseudomonas aeruginosa, which en- codes the small subunit of carbamoyl-phosphate synthe- tase (Wang and Abdelal, 1990). Two sets of codons that could potentially act as the takeoff and landing sites occur at nucleotides 9 to 15 and 21 27 downstream of the start codon. In contrast to the gene 60 case, untranslated 12 nucleotides do not contain a stop codon. Since this putative example has just been found, the critical features are unknown, but cannot fail to be interesting.

Presence and location of modified nucleotides in Escherichia coli tmRNA: structural mimicry with tRNA acceptor branches

Escherichia coli tmRNA functions uniquely as both tRNA and mRNA and possesses structural elements similar to canonical tRNAs. To test whether this mimicry extends to post‐transcriptional modification, the technique of combined liquid chromatography/ electrospray ionization mass spectrometry (LC/ESIMS) and sequence data were used to determine the molecular masses of all oligonucleotides produced by RNase T1 hydrolysis with a mean error of 0.1 Da. Thus, this allowed for the detection, chemical characterization and sequence placement of modified nucleotides which produced a change in mass. Also, chemical modifications were used to locate mass‐silent modifications. The native E.coli tmRNA contains two modified nucleosides, 5‐methyluridine and pseudouridine. Both modifications are located within the proposed tRNA‐like domain, in a seven‐nucleotide loop mimicking the conserved sequence of T loops in canonical tRNAs. Although tmRNA acceptor branches (acceptor stem and T stem–loop) utilize different architectural rules than those of canonical tRNAs, their conformations in solution may be very similar. A comparative structural and functional analysis of unmodified tmRNA made by in vitro transcription and native E.coli tmRNA suggests that one or both of these post‐transcriptional modifications may be required for optimal stability of the acceptor branch which is needed for efficient aminoacylation.

Readthrough of dystrophin stop codon mutations induced by aminoglycosides

We report the translational readthrough levels induced by the aminoglycosides gentamicin, amikacin, tobramycin, and paromomycin for eight premature stop codon mutations identified in Duchennes and Beckers muscular dystrophy patients. In a transient transfection reporter assay, aminoglycoside treatment results show that one stop codon mutation is suppressed significantly better (up to 10% stop codon readthrough) than the others; five show lower but statistically significant suppression (<2% stop codon readthrough); and two appear refractory to aminoglycoside treatment. Readthrough levels do not substantially vary between different sources of gentamicin, and, for this set of mutations, the efficiency of termination at the premature stop codon mutation does not appear to correlate with disease severity.

Conservation of polyamine regulation by translational frameshifting from yeast to mammals

Regulation of ornithine decarboxylase in vertebrates involves a negative feedback mechanism requiring the protein antizyme. Here we show that a similar mechanism exists in the fission yeast Schizosaccharomyces pombe. The expression of mammalian antizyme genes requires a specific +1 translational frameshift. The efficiency of the frameshift event reflects cellular polyamine levels creating the autoregulatory feedback loop. As shown here, the yeast antizyme gene and several newly identified antizyme genes from different nematodes also require a ribosomal frameshift event for their expression. Twelve nucleotides around the frameshift site are identical between S.pombe and the mammalian counterparts. The core element for this frameshifting is likely to have been present in the last common ancestor of yeast, nematodes and mammals.