Dual translational initiation sites control function of the lambda S gene.

Lysis gene S of phage lambda has a 107 codon reading frame beginning with the codons Met1-Lys2-Met3. Genetic data have suggested that translational initiation occurs at both Met1 and Met3, generating two polypeptides, S107 and S105 respectively. We have proposed a model in which the proper scheduling of lysis depends on the partition of translational initiations between the two start codons. Here, using in vitro methods, we show that two stem-loop structures, one immediately upstream of the reading frame and a second approximately 10 codons within the gene, control the partitioning event. Utilizing primer-extension inhibition or 'toeprinting', we show that the two S start codons are served by two adjacent Shine-Dalgarno sequences. Moreover, the timing of lysis supported by the wild-type and a number of mutant alleles in vivo can be correlated with the ratio of ternary complex formation over Met1 and Met3 in vitro. Thus the regulation of the S gene is unique in that the products of two adjacent in-frame initiation events have opposing function.     

Relationship between utilization of dual translational initiation signals and protein processing in Streptomyces

Summary Two sets of the Shine-Dalgarno sequence and the initiation codon (ATG) for translation of a gene encoding the protein SSI (Streptomyces subtilisin inhibitor) were studied in vivo by site-directed mutagenesis. The result shows that each ATG can function as an initiator of translation in either Streptomyces lividans 66 or Escherichia coli. The choice of initiation codon seems dependent on the host strain and is closely related to the processing mechanism of pre-SSI protein. The upstream ATG is presumed to be utilized preferentially giving two cleavage sites in pre-SSI in S. albogriseolus S-3253, the original SSI producer strain.Abbreviations SD Shine-Dalgarno - SSI Streptomyces subtilisin inhibitor     

Bacteriophage lambda preconnectors. Purification and structure

The morphogenesis of bacteriophage lambda proheads is under the control of the four phage genes B, C, Nu3 and E, and the two Escherichia coli genes groEL and groES . It has been shown previously that extracts prepared from cells infected with a lambda C-E- mutant accumulate a gpB polymer, which behaves as a biologically active intermediate in prohead assembly. This gpB activity has been called a preconnector , as it is probably a precursor to the head-tail connector. We now report the partial purification of biologically active preconnectors and the characterization of its structure. In the electron microscope, preconnectors appear as donut -like structures composed of several subunits displaying radial symmetry. Optical filtration of periodic arrays of preconnectors showed that the structure has 12-fold rotational symmetry. Side views of the preconnector reveal that it resembles an asymmetrical dumbell . This information has been used to construct a three-dimensional model of the preconnector . The implications of this structure for prohead shape and function, and for DNA packaging are discussed.     

Bacteriophage lambda: the untold story.

The study of bacteriophage lambda has provided key insights into fundamental biological processes. This review recalls some highlights in the history of lambda research, and relates how simple (but elegant) experiments yielded major scientific breakthroughs. What we know about recombination, gene regulation, and protein folding, for example, derives in large part from bacteriophage lambda genetics. Lambda not only represents a model system of scientific logic in a technology-driven age, but continues to reveal new principles of molecular biology.     

Bacteriophage lambda and plasmid vectors, allowing fusion of cloned genes in each of the three translational phases.

We have constructed vectors from bacteriophage lambda and from plasmid pBR322 having a single EcoRI restriction site which is immediately downstream from the lac UV5 promotor. Each vector allows the fusion of a cloned gene to the lac Z gene in a different phase relative to the translation initiation codon of the lac Z gene. These vectors were constructed through modification of the initial EcoRI restriction site by S1 endonuclease treatment and then addition of octadeoxyribonucleotides (EcoRI linkers), which shifted the restriction site by 2 or 4 nucleotides. Used in combination these vectors should allow translation of a cloned gene in any one of the three coding phases. The bacteriophages vectors are certified as B2 (EK2) safety level vectors by the French "recombinaison génétique in vitro" committee (D.G.R.S.T.).     

Bacteriophage lambda FII gene protein: role in head assembly

The in vitro completion of bacteriophage lambda F_II^? heads to form phage can be used as an assay for the λ F_II gene protein. F_II protein activity is released from highly purified phage particles or phage heads by treatment with heat or denaturing agents. F_II protein was purified from isolated phage particles and from an extract of E^? infected cells in which it is not bound to any large structures. No differences in molecular weight (11,500), isoelectric point (4.75), electrophoretic mobility, or purification properties could be demonstrated between the F_II proteins from the two sources. Thus the polypeptide does not seem to be modified during assembly.Phage φ80 is closely related to λ. φ80 heads will join to φ80 tails in vitro but will not join to λ tails, though λ heads will join to either type of tail. Mixing experiments between F_II^? heads, tails, and F_II protein from λ or φ80 show that the specificity of head-tail joining is correlated with the source of the F_II protein and not with the source of the other head proteins. Thus, F_II protein is apparently responsible for this specificity of head-tail joining.     

Cis-acting signals controlling translational initiation in the thermophilic archaeon Sulfolobus solfataricus

In this work, we have studied the in vitro translational features of a bicistronic mRNA of the extremely thermophilic Archaeon Sulfolobus solfataricus, with the aim of determining the nature of the cis-acting signals controlling the recognition of the translation initiation sites in the Archaea. We found that the most important feature for efficient initiation was the presence of a Shine-Dalgarno (SD)-like ribosome-binding motif, whose disruption entirely abolished the translation of the corresponding cistron. The influence of other features, such as the type of initiation codon, was variable and depended upon the gene and its position in the mRNA. However, the translational block caused by the disruption of the SD sequences could be removed by deleting the 5' untranslated region altogether, thereby creating a 'leaderless' mRNA. This suggests that 'leaderless' initiation operates by a default mechanism that does not require a specific mRNA-rRNA interaction and may be common to all three primary domains of life.     

Recognition of messenger RNA during translational initiation in Escherichia coli

The structural aspects of recognition by E. coli ribosomes of translational initiation regions on homologous messenger RNAs have been reviewed. Also discussed is the location of initiation region on mRNA, its confines, typical nucleotide sequences responsible for initiation signal, and the influence of RNA macrostructure on protein synthesis initiation. Most of the published DNA nucleotide sequences surrounding the start of various E. coli genes and those of its phages have been collected.     

Exclusion of bacteriophage T1 by bacteriophage lambda. I. Early exclusion requires lambda N gene product and host factors involved in N gene expression.

Two modes of exclusion of T1 by lambda are distinguished. "Early" exclusion depends on gene N, but not on gene Q. It is at least partially ineffective against T1am23. "Late" exclusion depends on gene Q and effects T1am23 as well as T1+. Early exclusion is a direct effect of N gene product, rather than N gene being required for the expression of some other lambda gene. Three host mutations, groN, nusA, and nusB, known to interfere with lambda replication by affecting N gene expression, also interfere with the ability of lambda to exclude T1.     

Bacteriophage P2-lambda interference. III. Essential role of an early step in the initiation of lambda replication.

Induction of bacteriophage λ in the presence of a P2 prophage results in inactivation of cellular transfer RNA, inhibition of amino acid and uridine incorporation in the host, as well as inhibition of phage replication. A red gam double mutation allows λ to escape from interference, and a mutation in gene O or P abolishes the effects on the host.It is shown here that phage and plasmid DNA extracted from cells undergoing P2-λ interference are still active in a transfection assay. Mutations in bacterial gene dna B or in phage site ori suppress the inhibition of amino acid incorporation, whereas genes dnaE and dna G have no such effect. Derepression of bacterial exonuclease VIII totally suppresses the interference, and mutations in genes recA and lexA, which control the SOS functions, suppress it partially if the λ phage is red⁺. Our results suggest that P2-λ interference is due to the action of old at an early step of the initiation of λ replication.     

The first and third uORFs in RSV leader RNA are efficiently translated: implications for translational regulation and viral RNA packaging.

Rous sarcoma virus (RSV) RNA leader contains three short upstream open reading frames. We have shown recently that both uORFs 1 and 3 influence in vivo translation of the downstream gag gene and are involved in the virus RNA packaging process. In this report, we have studied the translational events occurring at the upstream AUGs in vivo. We show that (i) the first and third AUGs are efficient translational initiation sites; (ii) ribosomes reinitiate efficiently at AUG3; and (iii) deletions in the intercistronic distance between uORF1 and 3 (which is well conserved among avian strains) prevent ribosome initiation at AUG3, thus increasing translation efficiency at the downstream AUGgag. The roles of the uORFs in translation and packaging are discussed.