Replication initiation in bacteriophage lambda appears to require wrapping of origin DNA on an approximately 50 angstrom radius in or around the complex with the initiator protein O. Since short lengths of DNA are not that flexible, it may be that runs of coherently spaced deoxyadenylate residues constitute bend sites in the ori sequence that facilitate the process. Earlier data showed that ori DNA has electrophoretic anomalies characteristic of bend sites and that these are augmented by initiator protein binding. Here origin bending is examined by direct measurement of the ability of polymerized ori sequences to form small circles. The smallest circles observed (84 residues) are compatible with the required radius of curvature. Bend sites within the O protein binding sites, bend sites in the spacers between them, plus the inherent flexibility of non-bent DNA in the origin may all contribute to origin bending. The data also show that a bend site is required for O protein binding to DNA.

No abstract

DNA replication in bacteriophage lambda begins at a unique origin between residues 39,000 and 39,200 of the lambda genome. This segment of DNA serves a dual function since it also lies within the coding sequence of the lambda replication initiator protein O which binds origin DNA. The lambda origin sequence contains four 19-base-pair (bp) segments (iterons) which have dyad symmetry, followed by a 40-bp A + T-rich zone of highly asymmetrical base composition. It was noted earlier that lambda origin DNA exhibits an anomalous electrophoretic mobility on gels; that is, the length of DNA as determined by DNA sequencing is approximately 20% less than is predicted from electrophoretic mobility. Recent studies of kinetoplast minicircle DNA (K-DNA) from the protozoan Leishmania tarentolae have led to the proposal that sequence-induced DNA curvature could account for such electrophoretic anomalies by alteration of the shape of the DNA molecule. We now present evidence that the lambda origin contains a static curve.

's int gene contains an unusually high frequency of the rare arginine codons AGA and AGG, as well as dual rare Arg codons at three positions. Related work has demonstrated that Int protein expression depends on the rare AGA tRNA. Strong transcription of the int mRNA with a highly efficient ribosome-binding site leads to inhibition of Int protein synthesis, alteration of the overall pattern of cellular protein synthesis, and cell death. Synthesis or stability of int and ampicillin resistance mRNAs is not affected, although a portion of the untranslated int mRNA appears to be modified in a site-specific fashion. These phenotypes are not due to a toxic effect of the int gene product and can be largely reversed by supplementation of the AGA tRNA in cells which bear plasmids expressing the T4 AGA tRNA gene. This indicates that depletion of the rare Arg tRNA due to ribosome stalling at multiple AGA and AGG codons on the overexpressed int mRNA underlies all of these phenomena. It is hypothesized that int mRNA's effects on protein synthesis and cell viability relate to phenomena involved in lambda phage induction and excision.The arginine codons AGA and AGG are the rarest codons in genes of Escherichia coli (1), and limitation of the respective tRNAs has been proposed to be a regulatory mechanism in genes enriched in these codons (6). Direct measurements of the level of the Arg-4 or argU (AGA) and Arg-5 (AGG) tRNAs have shown that they are present in extremely low concentrations during all phases of cell growth (11,17,32). Indirect experiments suggest that rare Arg tRNA may become limiting in mid-log phase (5, 6). Mutant forms of the Arg-4 tRNA gene, known as argU (or dnaY) (12, 16), strongly affect replication and cell growth.The lambda integrase (int) mRNA is highly enriched in the rare arginine codons AGA and AGG, which represent 59% (20 of 34) of the total Arg codons and occur three times in tandem (9, 38). Translation of these codons has been demonstrated to be slower than that of the major arginine codons (2, 35). Previous studies have shown that efficient translation of int mRNA depends on the level of rare Arg tRNA (38).The Int protein plays a critical catalytic role in integration of lambda phage DNA during lysogenization and also during the excision process accompanying phage induction (37). Transcriptional and posttranscriptional regulation of the bacteriophage lambda int gene is complex and has been the subject of intensive study (23). int is expressed from the N-antiterminated p L transcript during excision and from the cII-activated p I promoter during integration (27, 34). The two int transcripts show different patterns of termination and mRNA processing (23).The major new result presented here is that high-level expression of int mRNA made from a plasmid-borne tac promoter (p tac ) and containing the T7 gene 10 translational enhancer (ε) and ribosome-binding site (RBS) produces inhibitory phenotypes which depend on the supply of rare Arg tRNA. In view of the other established inhibitory functions of lamb...

We have characterized the binding of lambda phage replication initiation protein O to the phage origin of replication. The minimal DNA segment required for O binding is the single iteron, a 19‐bp sequence of hyphenated dyad symmetry that is repeated with variations four times in the origin. The isolated amino terminus of O protein is also sufficient to bind DNA. Electrophoretic studies show that the amino terminus of O protein induces bending of a single iteron. The DNA‐protein interaction was characterized by ethylation interference, dimethyl sulfate protection and neocarzinostatin footprinting. Points of DNA‐protein contact are largely concentrated in two areas symmetrically disposed with respect to the dyad symmetry of the iteron. This suggests the protein interacts as a dimer with half sites in the DNA. However, a few non‐symmetrical contacts are found, indicating that O protein may distort the helix. This may correlate with the bending effects demonstrated electrophoretically. Cylindrical DNA projections were used to model O protein binding to the lambda origin and compare it with the lambda repressor‐operator interaction. Whereas bound repressor nearly encircles the DNA in the major groove, O protein leaves the major groove on the opposite side exposed.

A 15-kb cryptic plasmid was obtained from a natural isolate of Rhodopseudomonas palustris. The plasmid, designated pMG101, was able to replicate in R. palustris and in closely related strains of Bradyrhizobium japonicum and phototrophic Bradyrhizobium species. However, it was unable to replicate in the purple nonsulfur bacterium Rhodobacter sphaeroides and in Rhizobium species. The replication region of pMG101 was localized to a 3.0-kb SalI-XhoI fragment, and this fragment was stably maintained in R. palustris for over 100 generations in the absence of selection. The complete nucleotide sequence of this fragment revealed two open reading frames (ORFs), ORF1 and ORF2. The deduced amino acid sequence of ORF1 is similar to sequences of Par proteins, which mediate plasmid stability from certain plasmids, while ORF2 was identified as a putative rep gene, coding for an initiator of plasmid replication, based on homology with the Rep proteins of several other plasmids. The function of these sequences was studied by deletion mapping and gene disruptions of ORF1 and ORF2. pMG101-based Escherichia coli-R. palustris shuttle cloning vectors pMG103 and pMG105 were constructed and were stably maintained in R. palustris growing under nonselective conditions. The ability of plasmid pMG101 to replicate in R. palustris and its close phylogenetic relatives should enable broad application of these vectors within this group of ␣-proteobacteria.Purple nonsulfur bacteria (PNSB) are an assemblage of phenotypically diverse species. Under anaerobic conditions in the light, all species grow photoheterotrophically when supplied with various organic substrates or photoautotrophically with CO 2 as a sole carbon source. Under microaerobic to aerobic conditions in the dark, many representatives can grow chemoheterotrophically, and some grow chemoautotrophically (40).To develop a new CO 2 -fixing bioprocess, we have been performing biochemical and genetic analyses of intermediary metabolism, including CO 2 fixation, underlying the complex modes of growth in the PNSB, using Rhodopseudomonas palustris as a model microorganism (4,23,24). For this purpose, development of a versatile host-vector system would be helpful.In R. palustris and other PNSB, broad-host-range vectors have been used to provide the tools for gene transfer. The most widely used vectors are derivatives of RK2 such as pRK415 (25) and pLAFR1 (14). Cloning vector pRK415 has been utilized for genetic analyses of several R. palustris genes (8, 17), and cosmid vector pLAFR1 has been used to make a library of R. palustris DNA (8). However, these plasmids were unstable in R. palustris under nonselective conditions (M. Inui, unpublished data), as also observed in R. sphaeroides (9) and in Rhodospirillum rubrum (42).Other vectors derived from the broad-host-range plasmid RSF1010 such as pDSK519 (25) can also replicate in PNSB, including R. palustris (Inui, unpublished data). But this vector was also unstable under nonselective conditions in R. palustris and R. sphaeroides (Inui, unpublish...

Lambda's int gene contains an anomalously high frequency of the rare arginine codons AGA and AGG when compared to genes of Escherichia coli or to the rest of phage lambda. These are the least frequent codons in genes of E. coli and are recognized by the rarest tRNAs. The presence of these codons reduces the translation rate and, depending on the context, this can strongly modulate translational efficiency by a variety of mechanisms. In this study, we show that expression of the natural int gene may also be modulated by rare arginine codon usage, and we explore this mechanism.

Widespread occurrence of a separate small RNA derived from the 5'-end of 23S rRNA and of an intervening sequence (IVS) which separates this domain from the main segment of 23S rRNA in the alpha-proteobacteria implies that processing reactions which act to excise the IVS are also maintained in this group. We previously characterized the first example of processing of this IVS in Rhodopseudomonas palustris, which is classified with the Bradyrhizobia In this case, IVS excision occurs by a multistep process and RNase III appears to act at an early step. Here, we characterize in vivo and in vitro IVS processing in two other related, but phenotypically distinct, Bradyrhizobia We also examine in vivo and in vitro processing of rRNA precursors from a more distantly related alpha-proteobacterium, Rhodobacter sphaeroides which produces a separate 5' 23S rRNA domain but has different sequences in the 5' 23S rRNA IVS. The details of the in vivo processing of all of the Bradyrhizobial rRNAs closely resemble the R. palustris example and in vitro studies suggest that all of the Bradyrhizobia utilize RNase III in the first step of IVS cleavage. Remarkably, in vivo and in vitro studies with R.sphaeroides indicate that initial IVS cleavage uses a different mechanism. While the mechanism of IVS cleavage differs among these alpha-proteobacteria, in all of these cases the limits of the internal segments processed in vivo are almost identical and occur far beyond the initial cleavage sites within the IVSs. We propose that these bacteria possess common secondary maturation pathways which enable them to generate similarly processed 23S rRNA 5'- and 3'-ends.