Gabriel Guarneros

The pair of arginine codons AGA AGG close to the initiation codon of the lambda int gene inhibits cell growth and protein synthesis by accumulating peptidyl-tRNAArg4.

To analyse the mechanism by which rare codons near the initiation codon inhibit cell growth and protein synthesis, we used the bacteriophage lambda int gene or early codon substitution derivatives. The lambda int gene has a high frequency of rare ATA, AGA and AGG codons; two of them (AGA AGG) located at positions 3 and 4 of the int open reading frame (ORF). Escherichia coli pth (rap) cells, which are defective in peptidyl‐tRNA hydrolase (Pth) activity, are more susceptible to the inhibitory effects of int expression as compared with wild‐type cells. Cell growth and Int protein synthesis were enhanced by overexpression of Pth and tRNAArg4 cognate to AGG and AGA but not of tRNAIle2a specific for ATA. The increase of Int protein synthesis also takes place when the rare arginine codons AGA and AGG at positions 3 and 4 are changed to common arginine CGT or lysine AAA codons but not to rare isoleucine ATA codons. In addition, overexpression of int in Pth defective cells provokes accumulation of peptidyl‐tRNAArg4 in the soluble fraction. Therefore, cell growth and Int synthesis inhibition may be due to ribosome stalling and premature release of peptidyl‐tRNAArg4 from the ribosome at the rare arginine codons of the first tandem, which leads to cell starvation for the specific tRNA.

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.

RNaselll activation of bacteriophage λ N synthesis

The bacteriophage λ gene product is one of the first genes expressed during phage development. N protein allows the expression of other phage genes by altering the transcription elongation process so as to prevent transcription termination. We have found that N levels may be modulated soon after induction or infection. Using N‐lacZ fusions, we determined that cells containing RNaselll have at least a fourfold greater expression than cells defective for RNaselll. This effect is exerted at the post‐transcriptional level. RNaselll processes an RNA stem structure in the N‐ leader RNA. Removal of the stem structure by deletion increases λ expression and prevents further stimulation by RNaselll. The base of this stable stem is adjacent to the λ ribosome binding site. We present a model for control of N synthesis in which this stable stem inhibits ribosome access to the N mRNA.

Mutations of bacteriophage lambda that define independent but overlapping RNA processing and transcription termination sites.

Bacteriophage lambda int gene expression is regulated differentially from transcripts originated at the pL and pI promoters. Transcripts initiated at pI terminate at the site tI and express int gene product efficiently. Polymerases starting at pL do not terminate at tI, due to the antiterminating activity of lambda N protein. The pL transcripts are unable to express Int protein efficiently because sib, a control site overlapping tI in the unterminated RNA, is processed by host RNase III. We have isolated lambda sib- mutants by their inability to inhibit int expression from pL transcripts. sib mutations were genetically mapped to the left of the lambda attachment site, and do not structurally alter this site for recombination. Several sib mutations do alter the nucleotide sequence of the overlapping sib and tI sites. The lambda sib- mutants tested prevent RNA processing but do not affect transcription termination in vivo.

A short DNA sequence from λ phage inhibits protein synthesis in Escherichia coli rap

The Escherichia coli rap mutant inhibits vegetative growth of bacteriophage lambda. Phage mutations termed bar, which overcome the rap defect, have been mapped to three genetic loci in the pL operon. Plasmids with a lambda wild-type bar DNA segment cloned downstream from an active promoter cannot be maintained in rap mutant bacteria. The viability of a rap mutant strain decreases rapidly after induction of transcription through bar regions present on plasmids. Under these (restrictive) conditions the expression of plasmid-encoded beta-lactamase and plasmid DNA replication are arrested, but plasmid RNA synthesis continues for several hours. Analysis of protein extracts from E. coli rap cells containing bar plasmids revealed that both plasmid and bacterial protein synthesis are inhibited under restrictive conditions. In addition, unlike other RNAs tested, the chemical half-life of bar RNA increases 3.5-fold relative to the half-life of bar RNA under permissive conditions. We propose that transcription through the bar region, or the accumulation of bar RNA, results in an irreversible defect in cellular mRNA translation. This defect eventually kills the rap cells, and thus prevents bar plasmid maintenance.

Increased bar minigene mRNA stability during cell growth inhibition

Bacteriophage lambda is unable to grow vegetatively on Escherichia coli mutants defective in peptidyl‐tRNA hydrolase (Pth) activity. Mutations which allow phage growth on the defective host have been located at regions named bar in the lambda genome. Expression of wild‐type bar regions from plasmid constructs results in inhibition of protein synthesis and lethality to Pth‐defective cells. Two of these wild‐type bar regions, barI+ and barII+, contain minigenes with similar AUG–AUA–stop codon sequences preceded by different Shine–Dalgarno (SD) and spacer regions. The induced expression of barI+ and barII+ regions from plasmid constructs resulted in similar patterns of protein synthesis inhibition and cell growth arrest. Therefore, these deleterious effects may stem from translation of the transcripts containing the minigene two‐codon ‘ORF’ (open reading frame). To test for this possibility, we assayed the effect of point mutations within the barI minigene. The results showed that a base pair substitution within the SD and the two‐codon ‘ORF’ sequences affected protein synthesis and cell growth inhibition. In addition, mRNA stability was altered in each mutant. Higher mRNA stability correlated with the more toxic minigenes. We argue that this effect may be caused by ribosome protection of the mRNA in paused complexes as a result of deficiency of specific tRNA.

Minigene-like inhibition of protein synthesis mediated by hungry codons near the start codon

Rare AGA or AGG codons close to the initiation codon inhibit protein synthesis by a tRNA-sequestering mechanism as toxic minigenes do. To further understand this mechanism, a parallel analysis of protein synthesis and peptidyl-tRNA accumulation was performed using both a set of lacZ constructs where AGAAGA codons were moved codon by codon from +2, +3 up to +7, +8 positions and a series of 3–8 codon minigenes containing AGAAGA codons before the stop codon. β-Galactosidase synthesis from the AGAAGA lacZ constructs (in a Pth defective in vitro system without exogenous tRNA) diminished as the AGAAGA codons were closer to AUG codon. Likewise, β-galactosidase expression from the reporter +7 AGA lacZ gene (plus tRNA, 0.25 μg/μl) waned as the AGAAGAUAA minigene shortened. Pth counteracted both the length-dependent minigene effect on the expression of β-galactosidase from the +7 AGA lacZ reporter gene and the positional effect from the AGAAGA lacZ constructs. The +2, +3 AGAAGA lacZ construct and the shortest +2, +3 AGAAGAUAA minigene accumulated the highest percentage of peptidyl-tRNAArg4. These observations lead us to propose that hungry codons at early positions, albeit with less strength, inhibit protein synthesis by a minigene-like mechanism involving accumulation of peptidyl-tRNA.

Evidence of bar minigene expression and tRNA2Ile sequestration as peptidyl-tRNA2Ile during lambda bacteriophage development.

Lambda bacteriophage development is impaired in Escherichia coli cells defective for peptidyl (pep)-tRNA hydrolase (Pth). Single-base-pair mutations (bar(-)) that affect translatable two-codon open reading frames named bar minigenes (barI or barII) in the lambda phage genome promote the development of this phage in Pth-defective cells (rap cells). When the barI minigene is cloned and overexpressed from a plasmid, it inhibits protein synthesis and cell growth in rap cells by sequestering tRNA(2)(Ile) as pep-tRNA(2)(Ile). Either tRNA(2)(Ile) or Pth may reverse these effects. In this paper we present evidence that both barI and barII minigenes are translatable elements that sequester tRNA(2)(Ile) as pep-tRNA(2)(Ile). In addition, overexpression of the barI minigene impairs the development even of bar(-) phages in rap cells. Interestingly, tRNA or Pth may reestablish lambda phage development. These results suggest that lambda bar minigenes are expressed and tRNA(2)(Ile) is sequestered as pep-tRNA(2)(Ile) during lambda phage development.

Regulation Of λ N-Gene Expression

The N gene product of phage λ is a positive regulatory factor for the transcription of other λ genes during phage development. It acts by modifying the RNA polymerase transcription complex. In the absence of N function, RNA polymerase initiates at the early λ promoters, p L and p R,but terminates transcription shortly after initiation at terminators, t L1 and t R1 respectively. The action of N prevents polymerase from terminating at t L1 and t R1 as well as at other more distant terminators in the p L and p R operon (see review by Friedman, 1988). In each of these operons, between the promoter and the first terminator, there is a target sequence which is also required for the N- antitermination process (Friedman et al., 1973; Rosenberg et al, 1978). These targets, nutL and nutR, have been defined genetically by mutations to block antitermination in the respective operons (Salstrom and Szybalski, 1978; Olson, et al., 1984). These mutations have defined two regions of nut, boxA and boxB (Friedman & Gottesman, 1983). The boxA site is conserved in sequence among other phages and E. coli whereas the box B site is unique for each phage type and is thought to be the N recognition site (Lazinski, et al., 1989) (See Figure 1).