A 15-17 nucleotide sequence from the gag-pol ribosome frameshift site of HIV-1 directs analogous ribosomal frameshifting in Escherichia coli. Limitation for leucine, which is encoded precisely at the frameshift site, dramatically increased the frequency of leftward frameshifting. Limitation for phenylalanine or arginine, which are encoded just before and just after the frameshift, did not significantly affect frameshifting. Protein sequence analysis demonstrated the occurrence of two closely related frameshift mechanisms. In the first, ribosomes appear to bind leucyl-tRNA at the frameshift site and then slip leftward. This is the 'simultaneous slippage' mechanism. In the second, ribosomes appear to slip before binding aminoacyl-tRNA, and then bind phenylalanyl-tRNA, which is encoded in the left-shifted reading frame. This mechanism is identical to the 'overlapping reading' we have demonstrated at other bacterial frameshift sites. The HIV-1 sequence is prone to frame-shifting by both mechanisms in E. coli.

In cells subjected to moderate aminoacyltRNA limitation, the peptidyl-tRNA-ribosome complex stalled at the ''hungry'' codon can slide well beyond it on the messenger RNA and resume translation further downstream. This behavior is proved by unequivocal amino acid sequence data, showing a protein that lacks the bypassed sequence encoded between the hungry codon and specific landing sites. The landing sites are codons cognate to the anticodon of the peptidyl-tRNA. The efficiency of this behavior can be as high as 10-20% but declines with the length of the slide. Interposition of ''trap'' sites (nonproductive landing sites) in the bypassed region reduces the frequency of successful slides, confirming that the ribosome-peptidyl-tRNA complex passes through the untranslated region of the message. This behavior appears to be quite general: it can occur at the two kinds of hungry codons tested, AUA and AAG; the sliding peptidyltRNA can be any of three species tested, phenylalanine, tyrosine, or leucine tRNA; the peptidyl component can be either of two very different peptide sequences; and translation can resume at any of the three codons tested.Protein synthesis is conventionally pictured as the orderly, sequential addition of amino acids to a growing peptide chain dictated by the sequence of triplets in the messenger RNA. However, alternatives to this linear progression are possible and can occur at surprisingly high frequencies in certain sequence contexts and/or under certain physiological conditions. Limitation for an aminoacyl-tRNA, in particular, raises the probabilities of alternative paths at ''hungry'' codons cognate to the limiting species. The alternative paths described so far include binding of noncognate aminoacyl-tRNA species (reviewed in refs. 1-3) and frameshifting either to the left (Ϫ1) or the right (ϩ1) (reviewed in refs. 4, 5). In addition, peptidyltRNA release, normally a low-frequency event, may occur at hungry codons (6)(7)(8).In this communication, we describe another alternative path the ribosome can follow at a hungry codon: sliding over it and moving several codons downstream, resuming translation further along the message. We encountered this phenomenon in the course of studying ribosome behavior at a sequence conducive to leftward frameshifting. MATERIALS AND METHODSBacteria were cultivated at 37°C under forced aeration in M63-glucose medium (9). To retain comparability with ref. 9, the medium was supplemented with leucine, threonine, arginine, and histidine, even though the strain used in the present experiments is prototrophic. The host was strain C92 (relA Ϫ , spoT Ϫ ) carrying the O6 deletion of the early part of lacZ (10). The deletion, provided to us as an FЈ plasmid by Jon Beckwith (Harvard Medical School, Boston), was introduced into the C92 chromosome by homogenotization. For unknown reasons, identical plasmids yield twice as much ␤-galactosidase in this strain as in the CP78/79 lineage we have used previously (9). This proved useful in purifying material for protein sequenc...

The genus Rhododendron (Ericaceae), which includes horticulturally important plants such as azaleas, is a highly diverse and widely distributed genus of >1,000 species. Here, we report the chromosome-scale de novo assembly and genome annotation of Rhododendron williamsianum as a basis for continued study of this large genus. We created multiple short fragment genomic libraries, which were assembled using ALLPATHS-LG. This was followed by contiguity preserving transposase sequencing (CPT-seq) and fragScaff scaffolding of a large fragment library, which improved the assembly by decreasing the number of scaffolds and increasing scaffold length. Chromosome-scale scaffolding was performed by proximity-guided assembly (LACHESIS) using chromatin conformation capture (Hi-C) data. Chromosome-scale scaffolding was further refined and linkage groups defined by restriction-site associated DNA (RAD) sequencing of the parents and progeny of a genetic cross. The resulting linkage map confirmed the LACHESIS clustering and ordering of scaffolds onto chromosomes and rectified large-scale inversions. Assessments of the R. williamsianum genome assembly and gene annotation estimate them to be 89% and 79% complete, respectively. Predicted coding sequences from genome annotation were used in syntenic analyses and for generating age distributions of synonymous substitutions/site between paralgous gene pairs, which identified whole-genome duplications (WGDs) in R. williamsianum. We then analyzed other publicly available Ericaceae genomes for shared WGDs. Based on our spatial and temporal analyses of paralogous gene pairs, we find evidence for two shared, ancient WGDs in Rhododendron and Vaccinium (cranberry/blueberry) members that predate the Ericaceae family and, in one case, the Ericales order.

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Limitation for aminoacyl-tRNA promotes ribosome frameshifling at certain sites. We have previously demonstrated ribosome frameshifting to the right (3') at an AAG site in one context, and to the left (5') at an AAG site in a different context. Here, we demonstrate that the "rightwing" context is largely specific for frameshifting to the right, and the "leftwing" context is largely specific for frameshifting to the left. Analysis of these context rules, and the conversion of a sequence that promotes leftward frameshifting to one that promotes rigtward frameshifting, demonstrated here, permits us to define a minimal heptanudeotide sequence sufficient for shiftiness in each direction at an AAG codon whose lysyl-tRNA is in short supply.