Dale Lindsley

The function of a ribosomal frameshifting signal from human immunodeficiency virus‐1 in Escherichia coli

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 phenylaianine 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 closeiy 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 amlnoacyl‐tRNA, and then bind phenylaianyl‐tRNA, which is encoded in the left‐shifted reading frame. This mechanism is identicai 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.

Ribosome bypassing elicited by tRNA depletion

Ribosome bypassing refers to the ability of the ribosome::peptidyl‐tRNA complex to slide down the message without translation to a site several or dozens of nucleotides downstream and resume protein chain elongation there. The product is an isoform of a protein with a ‘coding’ gap corresponding to the region of the message which was bypassed. Previous work showed that ribosome bypassing was strongly stimulated at ‘hungry’ codons calling for a tRNA whose aminoacylation was limited. We have now used the ‘minigene’ phenomenon to ascertain whether depletion of the pool of specific isoacceptors has a similar effect. High level expression of plasmid‐borne minigenes results in the sequestration as peptidyl‐tRNA of tRNA cognate to the last triplet of the minigene, thereby limiting protein synthesis for lack of the tRNA in question. We find that induction of a minigene ending in AUA stimulates bypassing at an AUA codon, but not in a control sequence with AGA at the test position; induction of a minigene ending in AGA stimulates bypassing at the latter but not the former. Induction of the AUA minigene also stimulates both leftward and rightward frameshifting at ‘shifty’ sequences containing an AUA codon. The normal, background frequency of bypassing at an AUA codon is markedly reduced by increasing the cellular level of the tRNA which reads the codon. Thus, the frequency of bypassing can be increased or decreased by lowering or raising the concentration of a relevant tRNA isoacceptor. These observations suggest that the occurrence of ribosome bypassing reflects the length of the pause at a given codon.

Evidence that the bypassing ribosome travels through the coding gap

In translational bypassing, a peptidyl-tRNA::ribosome complex skips over a number of nucleotides in a messenger sequence and resumes protein chain elongation after a “landing site” downstream of the bypassed region. The present experiments demonstrate that the complex “scans” processively through the bypassed region. This conclusion rests on three observations. (i) When two potential “landing sites” are present, the protein sequence of the product shows that virtually all ribosomes land at the first and virtually none at the second. (ii) In such a sequence with two landing sites, the presence of a terminator triplet in phase in the coding region immediately after the first landing site drastically reduces the efficiency of bypassing. (iii) Internally complementary sequences that can form a stable stemloop in the bypassed region significantly reduce the efficiency of bypassing. We analyze bypassing from a given “takeoff” site to “landing sites” at different distances downstream so as to derive estimates of the frequency of ribosome takeoff and of the stability of the bypassing complex.

Somatic-Cell Mutation Monitoring System Based on Human Hemoglobin Mutants

The system described in this chapter was developed as a means of detecting rare red cells, in genetically normal (HbA/HbA) individuals, that are heterozygous for an abnormal hemoglobin. It is assumed, first, that mutations arise spontaneously in human hemopoietic stem cells, as they do in gametal stem cells, and second, that somatic mutations of globin-chain genes do not diminish the viability of affected stem cells. The latter assumption is a reasonable one, since phenotypic expression of such mutations occurs very late in hemopoietic cell differentiation. It is expected that as a result of these stem cell mutations, lines of stem cells containing the mutant globin genes are established and produce erythrocytes heterozygous for structurally abnormal globin chains. Development of appropriate methods of screening blood samples should then permit detection and enumeration of red cells that contain an abnormal hemoglobin as a result of somatic mutation in a stem cell.

Influence of the relA gene on ribosome frameshifting.

Abstract. We have examined the influence of genotype at the relA locus on the kinetics of leftward (or –1) frameshifting at a variety of codons calling for a limiting aminoacyl-tRNA species. We used lacZ left-frameshift reporter constructs carrying the sequence U UUC xyz, where xyz was each of three triplets coding for three different amino acids; we slowed the ribosomes at each of these by limiting for the amino acid or for the aminoacyl-tRNA. In all cases, limitation stimulated leftward frameshifting. In all cases, the stimulation was greater in relA mutant cells than in their wild-type relA+ counterparts. In the latter genotype, the increased frameshifting was constant from the start of the limitation regime. This was also true of the relA mutant strain during limitation for lysine-tRNA or for leucine; however, during limitation for isoleucine-tRNA (or for isoleucine) the mutant showed a gradual, progressive increase in frameshifting, suggesting an indirect effect. We suggest that gradual accumulation of undermodified tRNAs, which is characteristic of the relA response, is involved. However, the specific modification involved is unknown. It is not queosine: analysis of a tgt mutant that is completely defective in queosine modification showed no increase in leftward frameshifting on the reporter which showed the larger, gradual increase during the relA response to isoleucine-tRNA limitation.

Ribosome bypassing at serine codons as a test of the model of selective transfer RNA charging

Recently, a model of the flux of amino acids through transfer RNAs (tRNAs) and into protein has been developed. The model predicts that the charging level of different isoacceptors carrying the same amino acid respond very differently to variation in supply of the amino acid or of the rate of charging. It has also been shown that ribosome bypassing is specifically stimulated at ‘hungry’ codons calling for an aminoacyl‐tRNA in short supply. We have constructed two reporters of bypassing, which differ only in the identity of the serine codon subjected to starvation. The stimulation of bypassing as a function of starvation differed greatly between the two serine codons, in good agreement with the quantitative predictions of the model.