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.