F.William Studier

Analysis of bacteriophage T7 early RNAs and proteins on slab gels

The RNAs and proteins specified by five early genes of bacteriophage T7 have been identified by electrophoresis on sodium dodecyl sulfate, polyacrylamide gels. Extracts of cells infected by different deletion strains and point mutants of T7 are analyzed on a slab gel system in which 25 samples can be run simultaneously and then dried for autoradiography. The high capacity of this system makes it possible to do many types of experiment that would be extremely tedious by other means. The five early genes are designated 0.3,0.7, 1, 1.1 and 1.3, in order from left to right on the T7 genetic map. The stop signal that prevents host RNA polymerase from transcribing into the late region of T7 DNA is located to the right of gene 1.3 (ligase). Most deletions that affect gene 1.3 also delete the stop signal, and some of them affect at least one late protein, the 1.7 protein. Several small, early RNAs can be resolved that are not affected by any of the deletions. These small RNAs could not come from between the five early genes or from the right end of the early region, and other work (Dunn & Studier, 1973) indicates that at least some of them come from the region to the left of gene 0.3. Deletions have been found that enter either end of the gene 1 RNA or the right ends of the 0.3 or 1.1 RNAs without seeming to affect the proteins specified by these RNAs. Perhaps all of the early messenger RNAs of T7 have untranslated regions at both ends. Some deletions that enter the left end of the gene 1 RNA reduce the amount of gene 1 protein that is synthesized, presumably by interfering with initiation of protein synthesis.

Analysis of restriction fragments of T7 DNA and determination of molecular weights by electrophoresis in neutral and alkaline gels.

The DNA of bacteriophage T7 is cut into seven unique fragments by the restriction endonuclease Dpn II (or the equivalent Mbo I), 19 fragments by Hpa I, and eight additional fragments by the combination of the two enzymes. The relative location of each fragment in the T7 DNA has been determined by a combination of techniques. If it is assumed that the length of any DNA molecule equals the sum of the lengths of the fragments produced from it by cleavage, and that electrophoretic mobility through agarose gels is a smooth function of the length of the DNA, then the known relationships between fragments provide enough conditions to define accurately the relative molecular weight of each fragment in the set. Absolute molecular weights are based on that of full-length T7 DNA. The fragments provide a convenient set of length standards covering the entire range from about 100 to 40,000 base-pairs (the length of T7 DNA). A horizontal slab gel system for electrophoresis on agarose gels is described. In this system, gels of very low concentrations do not distort during electrophoresis and accurate relative mobilities of large DNAs are obtained. Excellent resolution can be obtained for DNAs of molecular weights up to at least 26·5×10 6 , a difference of less than 10% being readily resolved even for molecules of this size. Agarose and polyacrylamide gels can be prepared in alkaline solvents that denature native DNA and completely unfold the single strands. The fragments of T7 DNA have the same relative mobilities whether subjected to electrophoresis as single strands in alkaline gels or as double-stranded DNA in neutral gels, and resolution is comparable in the two states. Thus, electrophoresis in alkaline gels can provide accurate molecular weights for linear, single-stranded DNAs, and should be useful in analyzing DNA for single-strand breaks, depurinations or topological differences such as ring forms. In both neutral and alkaline gels, the relative mobilities of DNAs shorter than about 1000 base-pairs (or bases) are essentially insensitive to changes in voltage gradient, at least over the range of voltage gradients commonly employed. However, relative mobilities become increasingly sensitive to voltage gradient the larger the DNA, with DNAs longer than about 20,000 base-pairs (or bases) being severely affected. This effect is probably due to the ease with which large DNA molecules can be deformed from their equilibrium conformations, thus permitting them to penetrate channels in the gel that would exclude them in their unperturbed conformations. As a practical matter, this means that low voltage gradients must be used for separations of large DNAs by gel electrophoresis.

Vectors for selective expression of cloned DNAs by T7 RNA polymerase

Plasmid vectors are described that allow cloning of target DNAs at sites where they will be minimally transcribed by Escherichia coli RNA polymerase but selectively and actively transcribed by T7 RNA polymerase, in vitro or in E. coli cells. Transcription is controlled by the strong phi 10 promoter for T7 RNA polymerase, and in some cases by the T phi transcription terminator. The RNA produced can have as few as two foreign nucleotides ahead of the target sequence or can be cut by RNase III at the end of the target sequence. Target mRNAs can be translated from their own start signals or can be placed under control of start signals for the major capsid protein of T7, with the target coding sequence fused at the start codon or after the 2nd, 11th or 260th codon for the T7 protein. The controlling elements are contained on small DNA fragments that can easily be removed and used to create new expression vectors.

Complete nucleotide sequence of bacteriophage T7 DNA and the locations of T7 genetic elements

The complete nucleotide sequence of bacteriophage T7 DNA, 39,936 base-pairs, has been determined by the techniques of Maxam & Gilbert. All previously known T7 genes and several unsuspected genes have been identified in the sequence. T7 DNA carries genetic information very efficiently: the coding sequences of 50 genes are close-packed but essentially not overlapping, and occupy almost 92% of the nucleotide sequence. This arrangement strongly suggests that all 50 of these closepacked genes are expressed, although there is as yet evidence for expression of only 38 of them. In addition, five potential overlapping genes have been identified, and there is preliminary evidence that one of them is expressed. Where gaps between coding sequences are found, they usually are less than 100 basepairs long, and usually contain one or more transcription signals, RNAase III cleavage sites, or origins of replication. Transcription signals in the T7 DNA include the three strong early promoters and the early termination site for Escherichia coli RNA polymerase, and 17 promoters and one termination site for T7 RNA polymerase. Ten RNAase III cleavage sites have been located, five in the early region and five in the late region. The primary transcripts are processed at these sites to provide the messenger RNAs observed in vivo . Almost all of the T7 messenger RNAs are polycistronic, but there are few polar effects at the level of transcription or translation, and most T7 proteins seem to be initiated independently, each from its own ribosome-binding and initiation site. The initiation codon for most T7 proteins is AUG, but a few proteins are predicted to begin at GUG. Certain T7 genes specify pairs of overlapping proteins. The two proteins specified by gene 4 are made in about equal amounts, beginning at two different ribosome-binding and initiation sites in the same reading frame and ending at a common termination codon. The two proteins specified by gene 10 are made in very different amounts. They begin at the same initiation site, but the minor gene 10 protein appears to be produced by a shift in translational reading frame just ahead of the normal termination codon, thereby adding 53 amino acids to the COOH-terminal end of the major protein. Gene 10 specifies the major capsid protein of the phage particle, and both the major and minor gene 10 proteins are incorporated into the phage particle. One or two other T7 genes appear to utilize translational frameshifting to produce unequal amounts of proteins that differ at their COOH-terminal ends. The amino acid sequences and compositions predicted for all of the T7 proteins (except the proteins produced by frameshifting) are given. T7 DNA begins and ends with a perfect direct repeat of 160 base-pairs. Immediately adjacent to this terminal repetition, at both ends of the mature DNA, lie very similar, regular arrays of 12 imperfect copies of a seven-base sequence. These arrays occupy about 160 base-pairs, starting about 15 basepairs from the terminal repetition. In the concatemeric form of T7 DNA, a single copy of the terminal repetition is flanked by these two arrays of repeated sequences, and it seems likely that this arrangement is involved somehow in formation of the ends of mature T7 DNA.

Use of bacteriophage T7 lysozyme to improve an inducible T7 expression system

Bacteriophage T7 lysozyme, a natural inhibitor of T7 RNA polymerase, can reduce basal activity from an inducible gene for T7 RNA polymerase and allow relatively toxic genes to be established in the same cell under control of a T7 promoter. Low levels of T7 lysozyme supplied by plasmids pLysS or pLysL, which are compatible with the pET vectors for expressing genes from a T7 promoter, are sufficient to stabilize many target plasmids and yet allow high levels of target protein to be produced upon induction of T7 RNA polymerase. Higher levels of lysozyme supplied by plasmids pLysE or pLysH reduce the fully induced activity of T7 RNA polymerase such that induced cells can continue to grow and produce innocuous target proteins indefinitely. Different configurations of the expression system can maintain several different steady-state levels of target gene expression. The presence of T7 lysozyme has the further advantage of facilitating the lysis of cells in preparing extracts for purification of target gene products.

The genetics and physiology of bacteriophage T7.

Abstract Nineteen genes have been identified in T7 by isolation and characterization of amber mutants. Representative mutants from each gene have been ordered on a linear genetic map by means of two-factor crosses, and the genes are numbered in order from left to right. At present, the map is over 200 map units long. From studies of T7-directed protein synthesis ( Studier and Maizel, 1969 ), it is estimated that not more than another 10 or so genes remain to be discovered. The ability of mutants from each gene to synthesize DNA, lyse the culture, and make DNA-containing particles in the restrictive host, E. coli B, has been determined. Mutants from genes 1–6 (at the left end of the map) are defective in both DNA synthesis and lysis; both functions appear to be essentially normal in mutants from genes 7–19. Mutants in genes 7, 11, 12, 13, and 17 can produce noninfectious, DNA-containing particles when grown on B. It appears that in T7 (as in T4 and lambda) related functions tend to cluster along the genetic map. The development of detailed genetic analysis for T7, together with the simplicity of its physical chemistry, makes this system a very attractive one in which to study a number of biological problems at the molecular level.

Structural genomics: beyond the Human Genome Project

With access to whole genome sequences for various organisms and imminent completion of the Human Genome Project, the entire process of discovery in molecular and cellular biology is poised to change. Massively parallel measurement strategies promise to revolutionize how we study and ultimately understand the complex biochemical circuitry responsible for controlling normal development, physiologic homeostasis and disease processes. This information explosion is also providing the foundation for an important new initiative in structural biology. We are about to embark on a program of high-throughput X-ray crystallography aimed at developing a comprehensive mechanistic understanding of normal and abnormal human and microbial physiology at the molecular level. We present the rationale for creation of a structural genomics initiative, recount the efforts of ongoing structural genomics pilot studies, and detail the lofty goals, technical challenges and pitfalls facing structural biologists.

T7 lysozyme inhibits transcription by T7 RNA polymerase

The selectivity of T7 RNA polymerase for its own promoters is used to direct all transcription and replication to bacteriophage T7 DNA during infection. We now find that T7 lysozyme, which is known to cut a bond in the peptidoglycan layer of the cell wall, forms a specific complex with T7 RNA polymerase and inhibits transcription. Mutations that weaken this interaction have been found in the coding sequence for T7 RNA polymerase; an affinity column containing wildtype polymerase selectively binds T7 lysozyme, but a similar column containing mutant polymerase does not. The lysozyme-polymerase interaction ensures a controlled burst of late transcription during infection, and could possibly have some direct role in replication and/or control of lysis.

Genome Sequences of Escherichia coli B strains REL606 and BL21(DE3)

Escherichia coli K-12 and B have been the subjects of classical experiments from which much of our understanding of molecular genetics has emerged. We present here complete genome sequences of two E. coli B strains, REL606, used in a long-term evolution experiment, and BL21(DE3), widely used to express recombinant proteins. The two genomes differ in length by 72,304 bp and have 426 single base pair differences, a seemingly large difference for laboratory strains having a common ancestor within the last 67 years. Transpositions by IS1 and IS150 have occurred in both lineages. Integration of the DE3 prophage in BL21(DE3) apparently displaced a defective prophage in the lambda attachment site of B. As might have been anticipated from the many genetic and biochemical experiments comparing B and K-12 over the years, the B genomes are similar in size and organization to the genome of E. coli K-12 MG1655 and have >99% sequence identity over approximately 92% of their genomes. E. coli B and K-12 differ considerably in distribution of IS elements and in location and composition of larger mobile elements. An unexpected difference is the absence of a large cluster of flagella genes in B, due to a 41 kbp IS1-mediated deletion. Gene clusters that specify the LPS core, O antigen, and restriction enzymes differ substantially, presumably because of horizontal transfer. Comparative analysis of 32 independently isolated E. coli and Shigella genomes, both commensals and pathogenic strains, identifies a minimal set of genes in common plus many strain-specific genes that constitute a large E. coli pan-genome.

Nucleotide sequence from the genetic left end of bacteriophage T7 DNA to the beginning of gene 4

The nucleotide sequence running from the genetic left end of bacteriophage T7 DNA to within the coding sequence of gene 4 is given, except for the internal coding sequence for the gene 1 protein, which has been determined elsewhere. The sequence presented contains nucleotides 1 to 3342 and 5654 to 12,100 of the approximately 40,000 base-pairs of T7 DNA. This sequence includes: the three strong early promoters and the termination site for Escherichia coli RNA polymerase: eight promoter sites for T7 RNA polymerase; six RNAase III cleavage sites; the primary origin of replication of T7 DNA; the complete coding sequences for 13 previously known T7 proteins, including the anti-restriction protein, protein kinase, DNA ligase, the gene 2 inhibitor of E. coli RNA polymerase, single-strand DNA binding protein, the gene 3 endonuclease, and lysozyme (which is actually an N-acetylmuramyl-l-alanine amidase); the complete coding sequences for eight potential new T7-coded proteins; and two apparently independent initiation sites that produce overlapping polypeptide chains of gene 4 primase. More than 86% of the first 12,100 base-pairs of T7 DNA appear to be devoted to specifying amino acid sequences for T7 proteins, and the arrangement of coding sequences and other genetic elements is very efficient. There is little overlap between coding sequences for different proteins, but junctions between adjacent coding sequences are typically close, the termination codon for one protein often overlapping the initiation codon for the next. For almost half of the potential T7 proteins, the sequence in the messenger RNA that can interact with 16 S ribosomal RNA in initiation of protein synthesis is part of the coding sequence for the preceding protein. The longest non-coding region, about 900 base-pairs, is at the left end of the DNA. The right half of this region contains the strong early promoters for E. coli RNA polymerase and the first RNAase III cleavage site. The left end contains the terminal repetition (nucleotides 1 to 160), followed by a striking array of repeated sequences (nucleotides 175 to 340) that might have some role in packaging the DNA into phage particles, and an A · T-rich region (nucleotides 356 to 492) that contains a promoter for T7 RNA polymerase, and which might function as a replication origin.