No abstract

The complete coding sequence of the gene for bacteriophage T7 RNA polymerase (T7 gene 1) has been cloned in the plasmid pBR322. Large amounts of active enzyme can be accumulated in Escherichia coli when the cloned gene is transcribed from the lac UV5 promoter. A protease activity that apparently can nick the protein without causing it to fall apart can be a problem during purification, but a procedure is described that gives good yields of essentially homogeneous, highly active enzyme suitable for biochemical and physical studies. T7 RNA polymerase has a stringent specificity for its own promoters and will selectively transcribe DNA that has been linked to such a promoter. This specificity makes the enzyme useful both for producing specific RNAs in vitro and for directing the expression of selected genes inside the cell. Having the cloned gene also makes possible a detailed mutational analysis of the functioning of T7 RNA polymerase.Bacteriophage T7 RNA polymerase (EC 2.7.7.6) is produced early in T7 infection and plays a central role in regulating gene expression (for a review, see ref. 1). A single-chain enzyme with a molecular weight close to 100,000, T7 RNA polymerase has a stringent specificity for its own promoters, which contain a highly conserved sequence of 23 continuous base pairs including the start site for the RNA. Seventeen such promoters are found in T7 DNA, but few if any such promoters are found in host DNA or in any other DNAs unrelated to T7. Once T7 RNA polymerase has been produced during infection, other T7 gene products inactivate the host RNA polymerase, leaving all transcription in the cell directed to T7 DNA.The great specificity of T7 RNA polymerase for its own promoters is not only central to the strategy of T7 infection but is also interesting and potentially useful to biochemists. Understanding the basis for this specificity is a challenge in its own right. Furthermore, the purified enzyme can be used to produce large amounts of specific RNAs simply by transcribing DNA that has been joined to a promoter for T7 RNA polymerase. Such RNAs could be useful as hybridization probes, mRNAs for in vitro translation, substrates for analyzing processing reactions or RNA splicing, or for any purpose requiring a specific RNA. T7 RNA polymerase, together with a suitably positioned promoter, can also be used to direct the transcription of selected genes inside the cell. Target genes potentially could be expressed at very high levels, using the same strategy employed by T7 itself: once T7 RNA polymerase had been made, the host RNA polymerase could be inactivated, thereby eliminating the synthesis of competing mRNAs. The T7 RNA polymerase made during T7 infection has already been shown to be capable of directing the transcription of genes cloned in plasmids (2-4).The yield of purified T7 RNA polymerase from infected cells is not particularly good, because the enzyme is synthesized for only a few minutes during infection and does not accumulate to high levels. Nor does T7 infection provide an ...

The structural organization of encapsidated T7 DNA was investigated by cryo-electron microscopy and image processing. A tail-deletion mutant was found to present two preferred views of phage heads: views along the axis through the capsid vertex where the connector protein resides and via which DNA is packaged; and side views perpendicular to this axis. The resulting images reveal striking patterns of concentric rings in axial views, and punctate arrays in side views. As corroborated by computer modeling, these data establish that the T7 chromosome is spooled around this axis in approximately six coaxial shells in a quasi-crystalline packing, possibly guided by the core complex on the inner surface of the connector.

No abstract

A system for testing the effects of specific codons on gene expression is described. Tandem test and control genes are contained in a transcription unit for bacteriophage T7 RNA polymerase in a multicopy plasmid, and nearly identical test and control mRNAs are generated from the primary transcript by RNase III cleavages.Their coding sequences, derived from T7 gene 9, are translated efficiently and have few low-usage codons of Escherichia coli. The upstream test gene contains a site for insertion of test codons, and the downstream control gene has a 45-codon deletion that allows test and control mRNAs and proteins to be separated by gel electrophoresis. Codons can be inserted among identical flanking codons after codon 13, 223, or 307 in codon test vectors pCT1, pCT2, and pCT3, respectively, the third site being six codons from the termination codon. The insertion of two to five consecutive AGG (low-usage) arginine codons selectively reduced the production of full-length test protein to extents that depended on the number of AGG codons, the site of insertion, and the amount of test mRNA. Production of aberrant proteins was also stimulated at high levels of mRNA. The effects occurred primarily at the translational level and were not produced by CGU (high-usage) arginine codons. Our results are consistent with the idea that sufficiently high levels of the AGG mRNA can cause essentially all of the tRNAAGG in the cell to become sequestered in translating peptidyl_tRNAAGG_mRNA-ribosome complexes stalled at the first of two consecutive AGG codons and that the approach of an upstream translating ribosome stimulates a stalled ribosome to frameshift, hop, or terminate translation.Most amino acids are encoded by more than one codon, and frequencies of use of synonymous codons tend to reflect the relative abundance of the tRNAs that recognize them (7,29). Synonymous codons may be translated at different rates (16,17) MATERUILS AND METHODSCodon test plasmids. Plasmids pCT1, pCT2, and pCT3 ( Fig. 1) were assembled from elements of pET vectors (13,24) and of T7 DNA (6). Elements were assembled by using a natural XbaI site (T'CTAGA, where the primer indicates the position of cleavage) between the 410 promoter and the slO translation initiation region for the gene 10 major capsid protein of T7, a natural NdeI site (CA'TATG) at the slO initiation codon, and newly introduced XbaI or NheI (G'CTAGC) sites at the ends of elements. Plasmids were assembled in modular fashion by fusions of compatible ends of DNA fragments from plasmids carrying individual elements or combinations of elements. Most of the fusions were between fragments that had one end at the PstI site in the on May 7, 2018 by guest