Robert B. Weiss

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.

A nascent peptide is required for ribosomal bypass of the coding gap in bacteriophage T4 gene 60

Abstract Bacteriophage T4 DNA topoisomerase gene 60 contains a 50 nucleotide untranslated region within the coding sequence of its mRNA. Translational bypass of this sequence by elongating ribosomes has been postulated for the mode of synthesis of an 18 kd polypeptide specified by the split coding segments. Ribosome bypass of the untranslated region also occurs when a segment of gene 60 is fused to lacZ and expressed in E. coli. The efficiency of bypass in these gene 60-lacZ fusions approaches 100%. Here, mutations that delete, insert, or substitute nucleotides from gene 60-lacZ fusions are examined. Essential features necessary for high level gap bypass emerging from this analysis are a cis-acting nascent peptide sequence, a short duplication bordering the gap, and a stop codon contained in a stem-loop structure at the 5′ junction of the gap.

Identification and characterization of transcripts from the neurofibromatosis 1 region: the sequence and genomic structure of EVI2 and mapping of other transcripts.

Mapping of the EVI2 gene between the translocation breakpoints of two patients with neurofibromatosis type 1 (NF1), combined with the likely role of its murine homolog in neoplastic disease, implicates EVI2 as a possible candidate for the NF1 gene. We report here the expression of a 1.6-kb EVI2 transcript in normal human brain and peripheral blood mononuclear cells. Sequencing studies predict an EVI2 protein of 232 amino acids that contains an N-terminal signal peptide, an extracellular domain with five potential glycosylation sites, a single hydrophobic transmembrane domain with a leucine zipper, and a hydrophilic cytoplasmic domain. These features are all well-conserved with respect to the mouse Evi-2 protein and are consistent with the hypothesis that EVI2 is a membrane protein that may complex with itself and/or other proteins within the membrane, perhaps to function as part of a cell-surface receptor. In the course of these studies we have also identified three other transcripts (classes of cDNAs) from the NF1 region. Two of these transcripts map between the NF1 translocation breakpoints; the remaining transcript maps just outside this region.

Imaging of fluorescent and chemiluminescent DNA hybrids using a 2-D CCD camera

Fluorescent and chemiluminescent detection of DNA hybrids on polymer membranes has been investigated using a cryogenically cooled, slow readout, two dimensional CCD camera in an imaging mode. The fluorescent background characteristics of commercially available nylon blotting membranes and a polypropylene membrane modified to bind DNA have been studied. The polypropylene membrane exhibits a 15-fold increase in DNA binding, 3-fold less background fluorescence and less background noise than nylon blotting membranes. However the detection limits determined from vacuum slot blots of crosslinked fluoresceinlabelled oligonucleotides show that the improved qualities of the polypropylene support do not result in a lower detection limit. This is mainly due to background noise arising from sources other than the membrane itself during the blotting/washing procedure and to a low signal-to-amount of DNA ratio with the polypropylene membrane. The lowest amount of fluorescein-labelled oligonucleotide detectable is 1.4 femtomol, with a typical exposure time of 10 minutes to image a 6x9 cm area of the membrane. The detection of chemiluminescence was done using a biotin-avidin complex in combination with an enzymatic assay. The assay was carried out after hybridization with biotin-labelled probes on vacuum slot blots with crosslinked target DNA. The detection limit is 0.12 femtomol of DNA target, a result obtained after 30 mm exposure. Further improvements are necessary to image sequencing blots with typically 1 femtomol or less of DNA per band in an acceptable exposure time.