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Presents forthcoming papers on biochemistry published in the February 1982 issue of the "European Journal of Biochemistry." "The Receptor of Bacteriophage Lambda: Evidence for a Biosynthesis Dependent on Lipid Synthesis," by C. Pages, C. Lazdunski and A. Lazdunski; "Citrate Synthesis in Intact Rat-Liver Mitochondria Is Irreversible," by M. Greksák, M. Lopez-Cardozo and S. G. van den Bergh; "Kinetic Equivalence of the Subunits of Liver Alcohol Dehydrogenase," by P. Anderson and G. Pettersson.

The paper presents various articles, which are to be published in the future issues of the European Journal of Biochemistry. Some of the articles to be published are — "Mechanisms for Albumin-Mediated Membrane Damage," by D. Dramas, E. Harvey, Al Lawrence and A. Thomas, "Transfer RNA Precursors Are Accumulated in Escherichia Coli in the Absence of RNase E," by B.K. Ray and D. Apirion, "A New Procedure for the Simultaneous Large-Scale Purification of Bacteriophage-T4-Induced Polynucleotide Kinase, DNA Ligase, RNA Ligase and DNA Polymerase," by G.M. Dolganov, A. V. Chestukhin and M.F. Shemyakin.

In this paper we have studied the effect of raising the intracellular EF-Tu concentration on the expression of tufB. To this aim cells were transformed with multicopy plasmids carrying either tufA or tufB. The intracellular EF-Tu concentrations were determined by the specific immunoelectrophoresis assay described in the preceding paper in this journal. We have cloned the tufA gene in a plasmid, containing the powerful major leftward promoter (PL of phage λ. Transcription from PL can be repressed at low temperature by a temperature-sensitive repressor and activated by heat induction. Cloning occurred in two orientations in a single EcoRI site about 150 base pairs downstream of PL. Cells carrying either plasmid were shown to contain an almost doubled amount of EF-Tu at temperatures from 25°C to 37°C. This indicates that transcription of tufA can proceed from a possible binding site for RNA polymerase on these cloned fragments. The EF-Tu level was further increased to about 30% of toal cellular protein after a temperature shift from 37°C to 43°C. The multicopy plasmid pTuB1 described by Miyajima et al. [FEBS Lett. 102, 207-210 (1979)] and a derivative (pTuBO, compare preceding paper in this journal) were used to study the expression of both chromosomal and plasmid-borne tufB. Transformation with either plasmid raised the intracellular EF-Tu concentration by 30-60% depending on the nutritional conditions. Suppressions of tufB expression was observed when the intracellular level of EF-Tu increased after transformation with all plasmids mentioned above. The results are in accord with the concept that EF-Tu acts as an autogenous feedback inhibitor involved in the regulation of tufB. [ABSTRACT FROM AUTHOR]

The physical map of 2C DNA (cf. following paper in this journal) was compared to the maps of SP0t, SP82 and Φe (three other Bacillus subtilis phages containing hydroxymethyluracil in place of thymine in their DNA). The overall organization of the four genomes was remarkably similar, as indicated by the topology of HaeIII and Sa/J cleavage segments. The proof was gathered for the presence in the four phage DNAs of large redundant ends carrying a single HaeIII recognition site. The location of the latter proved identical for 2C and SP01, but was shifted in the DNAs of SP 82 and Φe. Since the redundant end components of these hydroxymetbyluracil genomes are colinear, as shown by crossahybridization studies, the shifting of the HaeIII cleavage site is presumably due to two base substitutions, suppressing an endonuclease recognition site and establishing a new site elsewhere. Relatedness between the genomes of this family of viruses was evaluated from the fraction of conserved restriction fragments. According to these calculations. 6% base substitutions have occurred within the four viral DNAs, in the course of evolution. However, specific segments of 2 C DNA were not present in SP01 and Φe DNA, as shown by cross- hybridization with restriction fragments. These data indicate the occurrence of deletions, in addition to base substitutions, as evolutionary mechanisms prevailing in the genomes of this family of phages. [ABSTRACT FROM AUTHOR]

Lysozyme from bacteriophage T4 was found to digest a soluble, uncrosslinked peptidoglycan which is secreted by cells of Micrococcus luteus when incubated in the presence of penicillin G. Analysis of the enzymatic degradation products shows that T4 acts as an endo-acetylmuramidase capable of cleaving glycosidic bonds only at muramic acid residues that are substituted with peptide side-chains. The results indicate that the secreted peptidoglycan may consist of a mixture of chains, approximately half of which are substituted by peptide side chains on most of their muramic acid residues, while the other half is made up of chains in which the muramic acid moieties are unsubstituted. [ABSTRACT FROM AUTHOR]

Hybridization studies have indicated that on the M13 genome there are located regions which are transcribed in vivo with a high as well as with a low frequency. The region with the highest transcriptional activity coincides with the region coding for the polypeptides which during the infection cycle are needed most abundantly (gene V and gene VIII protein). The hybridization studies furthermore have indicated that at least a number of the promoters previously detected in vitro are operative in the infected cell. In addition the experimental data are consistent with the presence of three transcription termination signals on the M13 genome. With the aid of analytical and preparative hybridization studies we have been able to establish that during the infection cycle at least eleven M13-specific RNA species are made which range in size from 280 to 2000 nucleotides. The six major RNA species (i.e. an 8-S RNA with 370 nucleotides, a 9-S with 420, three 11-S species with 760, 810 and 860 nucleotides and a 14- species with 1140 nucleotides) are encoded by the region with the highest transcriptional activity (located between the N-terminal end of gene X and the C-terminal end of gene VIII). Characterization of these RNA species with respect to their genetic origin, codogenic properties and nucleotide sequences has indicated that their synthesis is terminated at the previously detected rho-independent transcription termination signal T0.25, which is located immediately after gene VIII. From the data obtained it furthermore could be deduced that the 5ʹ termini of these RNA .species are located as follows: for the 8-S RNA immediately in front of gene IX (promoter G0.18), the 9-S RNA within or immediately distal to the 5ʹ end of gene VII, the three 11-S RNAs within the C-terminal end of gene X and the 14-S RNA immediately in front of gene X. Analyses of the 5ʹ ends of the major RNA chains synthesized in vivo have indicated that four (i.e. the 9-S and three 11-S species) out of the six RNA species are the result of processing of precursor molecules. For this processing the RNA processing enzymes RNase III, RNase E, and RNase P are not required. The meaning of these results, in terms of the mechanisms by which the expression of the M13 genome is regulated, is discussed. [ABSTRACT FROM AUTHOR]

The cell wall peptidoglycan of Escherichia coli or Salmonella typhi were, hydrolysed to dialysable non-reducing fragments by partially purified preparations of the bacteriolytic activity present in lysates of phage λ or Vi II, The structure of the two main fragments resulting from this digestion was determined. In each case the lack of reducing power is explained by the presence of a 1,6-anhydro-N-acetylmuramic acid residue at what should normally be the reducing end of the peptidoglycan fragment. Furthermore, no D-alanyl-(D)-meso-diaminopimelyl peptide crosslinkages were detected in these fragments. These results indicate that the partially purified preparations of phage λ or phage Vi II endolysin contain at least two different types of hydrolytic activities: a phage endopeptidase responsible for the cleavage of the peptide crosslinkages present in peptidoglycan and a new type of N-acetylmuramidase activity which not only cleaves β-1 ... 4 linkages between N-acetylmuramic acid and. N-acetylglucosamine residues in the glycan moiety of peptidoglycan, but also catalyses a dehydration with the formation of 1,6-anhydro-N-acetylmuramic acid residues. [ABSTRACT FROM AUTHOR]

The physical map of 2C DNA (cf. following paper in this journal) was compared to the maps of SP01, SP82 and phi e (three other Bacillus subtilis phages containing hydroxymethyluracil in place of thymine in their DNA). The overall organization of the four genomes was remarkably similar, as indicated by the topology of HaeIII and SalI cleavage segments. The proof was gathered for the presence in the four phage DNAs of large redundant ends carrying a single HaeIII recognition site. The location of the latter proved identical for 2C and SP01, but was shifted in the DNAs of SP82 and phi e. Since the redundant end components of these hydroxymethyluracil genomes are colinear, as shown by cross-hybridization studies, the shifting of the HaeIII cleavage site is presumably due to two base substitutions, suppressing an endonuclease recognition site and establishing a new site elsewhere. Relatedness between the genomes of this family of viruses was evaluated from the fraction of conserved restriction fragments. According to these calculations, 6% base substitutions have occurred within the four viral DNAs, in the course of evolution. However, specific segments of 2C DNA were not present in SP01 and phi e DNA, as shown by cross-hybridization with restriction fragments. These data indicate the occurrence of deletions, in addition to base substitutions, as evolutionary mechanisms prevailing in the genomes of this family of phages.

Temperate bacteriophages can display in a stable way two essentially different behaviours. In the immune state, a gene (cI) produces a repressor which prevents expression of all the other viral genes; in the non-immune state the typically viral functions are expressed. The choice between the two pathways and the establishment of one of them have much in common with cell determination and differentiation. This choice depends on a complex control system, in fact one of the most intricate nets of regulation known in some detail. Our paper provides a formal description and partial analysis of this regulatory net. It is shown that even for relatively simple known models, this kind of analysis uncovers predictions which had previously remained hidden. Some of these predictions were checked experimentally. The experimental part chiefly deals with the efficiency of lysogenization by thermoinducible lambda phage carrying mutations in one or more of the regulatory genes, N, cro and cII. Although N- mutations are widely known for preventing efficient integration, and both N- and cII mutations for preventing efficient establishment of immunity, it is shown that, as predicted by a simple model, both N- and cII- phage efficiently lysogenize at low temperature if they are in addition cro-. In contrast with lambda N- cro+, lambda N- cro- is not propagated as a plasmid at low temperature, precisely because it establishes immunity too efficiently. Genetic control circuits are described in terms of sets of logic equations, which relate the state of expression of genes or of chemical reactions (functions) to input (genetic and environmental) variables and to the presence of gene and reaction products (internal, or memorization varibles). From the set of equations, one derives a matrix which shows the stable stationary states (if any) of the system, and from which one can derive the pathways (temporal sequences of states) consistent with the model. This kind of analysis is complementary to the more widely used analysis based on differential equations; it allows one to analyze in less detail more complex systems. The language might be used as well, mutatis mutandis, in fields very different from genetics. The last part of the discussion deals with the role of positive feedback loops in our specific problem (establishment and maintenance of immunity in temperate bacteriophages) and in developmental genetics in general. As a generalization of an old idea, it is suggested that cell determination (for a given character) depends on a set of genes whose interaction constitutes a positive feedback loop. Such a system has two stable stationary states: which one is chosen will usually depend on additional controls grafted on the loop.

The substrate specificity of 49[SUP+]-enzyme was investigated in vitro. The enzyme showed a marked preference for rapidly sedimenting T4 DNA (> 1000 S) when helix-destabilizing proteins from Escherichia coli or phage T4 were added to the reaction. Regular replicative T4 DNA (200-S DNA) or denatured T4 DNA was not cleaved by the enzyme in the presence of these proteins but if they were omitted from the reaction both DNAs become good substrates for the enzyme. 200-S DNA was cleaved at its natural sites of single strandedness which occur at one-genome intervals. Gaps in T4 DNA which were constructed by treatment of a nicked DNA with exonuclease III were also cleaved by 49[SUP+]-enzyme in the absence of helix-destabilizing proteins. Single-stranded T4 DNA was extensively degraded and up to 50% of the material was found to be acid-soluble in a limit digest. The degradation products were predominantly oligonucleotides of random size. No preference for a 5'-terminal nucleotide was observed in material from a limit digest with M13 DNA. Double-stranded DNA was nicked upon exposure to 49[SUP+]-enzyme and double-strand breakage finally occurred by an accumulation of single-strand interruptions. No acid-soluble material was produced from native T4 DNA. The introduction of nicks in native DNA did not improve its properties as a substrate for the enzyme. Double-stranded DNA was about 100-fold less sensitive to the enzyme than single-stranded DNA. [ABSTRACT FROM AUTHOR]

The lipid-containing bacteriophage PM2 was reconstituted stepwise from its purified denatured subunits. In the first step the nucleocapsid was reconstituted from the DNA and the two nucleocapsid proteins. Slight biochemical differences between reconstituted nucleocapsids and those isolated from native virus were seen. Combination of reconstituted nucleocapsid or nucleocapsid from virions with the coat and spike proteins in the presence of the viral lipids resulted in the formation of infectious virus in both cases. The reconstituted particles contained amounts of viral components similar to those in native virus, except for the lipid content. The amount of lipids present in the reconstituted particles was twice as high as the lipid content of native bacteriophage. [ABSTRACT FROM AUTHOR]

Tiamulin, a water-soluble and highly effective semisynthetic derivative of pleuromutilin leads to the formation of physiologically inactive polypeptide chain initiation complexes which readily decompose and do not enter the phase of peptide chain elongation. Once elongation has begun it continues even in the presence of tiamulin as has been shown by measuring the formation of N-acetylphenylalanine-poly(phenylalanine). The formation of abortive initiation complexes was observed regardless of whether AcPhe-tRNA of fMet-tRNA was used as an initiator or whether artificial messengers or a natural messenger, like RI7 bacteriophage RNA, was used. When this drug was acting on whole cells, it led to the disappearance of polysomes. The only structures which could be detected were of the monosome size. Therefore, polysomes seem to elongate the polypeptide chains in whole cells in the presence of this antibiotic, but since effective reinitiation is blocked, the polysome pool of the cell soon becomes depleted. [ABSTRACT FROM AUTHOR]

The lipid-containing bacteriophage PM2 has four distinct structural proteins, as shown by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate or electrophoresis on cellulose acetate strips in the presence of 6-8 M urea. The molecular weights of these four polypeptides determined in several polyacrylamide gel systems are: I = 43000, II = 26000-27000, III = 12500, IV = 4700. The isoelectric points of the four viral proteins were determined on disrupted virus particles by electrophoresis on cellulose acetate strips in the presence of 6 M urea at various pH values. The mobilities were extrapolated to zero mobility, a method preferable to isoelectric focusing where the denatured viral proteins aggregate and precipitate near the isoelectric point. The method is well suited for the determination of the isoelectric point of proteins in thc native or only partially denatured state. Whereas proteins I, III and IV have isoelectric points slightly under neutrality (pH 6.2, 5.8 and 5.5, respcctively), thc isoelectric point of protein II is around 12.3. Calcium ions are tightly bound to protein II. After removal of the calcium ions in the presence of 6 M guanidine hydrochloride and EDTA, the pi of protein II is 9. The isoelectric point of thc intact virus, determined on cellulose strips, is around 7.3. Protein I, the spike protein, is the only structural polypeptide which is soluble in aqueous solutions around pH 7. All of the proteins are soluble in 6-8 M urea or 6 M guanidine hydrochloride at pH 7-8.5. Proteins III and IV. the nucleocapsid core proteins, have the solubility properties of proteolipids or highly lipophilic polypeptides as shown by their solubility in nonionic detergents and by their distribution in a Bligh and Dyer fractionation. Neither glycolipids nor glycoproteins are present in the virus. The distribution of phospholipids has been determined by chemical modification of the outer lamella or outer and inner lamellae of the bacteriophage lipid bilayer. The viral lipid bilayer is asymmetric; most of the phosphatidylglycerol residues form the outer lipid layer, most of the phosphatidylethanolamine residues the inner lipid layer. A complete model of the virus, including the assembly process, is discussed at the end of this report. [ABSTRACT FROM AUTHOR]

Highly purified RNA polymerase from Escherichia coli terminates RNA synthesis in vitro at defined sites on the DNA of coliphage TT. One such site maps at the end of the major ‘early’ transcription region of the genome. A second, distal site is most readily studied in certain mutant strains from which the first site has been deleted. At both sites, termination in vitro is simple (independent of rho or other factors) and both appear to be functional in vivo: We have applied to these sites a technique designed to study base sequences at and beyond RNA termini. The RNA terminated at the first site has cytosine at its 3′ end, and the first base coded by the DNA template beyond the RNA terminus is guanine. The specificity of this sequence is at least 92%. At the second terminator, which is found to be less efficient in its action, the corresponding bases are cytosine and adenine. [ABSTRACT FROM AUTHOR]

Some techniques, facilitating the determination of the amino-acid sequence of proteins or protein fragments available in 10 nanomole amounts are presented. Fluorescamine was used to follow the purification of proteins and peptides without ineterfering in the determination of the amino-acid composition and sequence. These techniques were applied on the coat protein of coliphage MS[SUB2] and on some peptides obtained from a chymotryptie digest of the the assembly protein of MS2. [ABSTRACT FROM AUTHOR]

Undegraded, uniformly 32P-labelled replicative form and replicative intermediate were prepared and fractionated according to sedimentation rate. 1. For each fraction the amount of 5'-terminal residues, which after alkaline hydrolysis are released as 5'-triphosphoryl, 3'(2')-phosphoryl nucleosides (nucleoside tetraphosphates), was quantitatively determined. The replicative form contained O, and the replicative intermediate 2.5 nascent chains, if the assumption is made that the latter are on the average half completed. The number of nascent chains apparently increases with increasing sedimentation constant of the replicative intermediate molecules. 2. The nucleoside tetraphosphate, released from each fraction, was identified as exclusively pppGp. This is interpreted as evidence for a 5' to 3' polarity in the synthesis of viral RNA, i. e. a growing 3'OH end. 3. The replicative form and replicative intermediate were also prepared from cells, in which the 32P-Iabel was chased for 5 mm before harvest. The amount of nucleoside tetraphosphate released agreed with a single end group per complete chain. Presumably 50 to 100% of the residual labelled terminal was derived from the complementary strand, and again only pppGp was found. It follows that the 3'-terminal adenosine residue of the viral RXA is presumably not part of the genetic information transferred during replication. [ABSTRACT FROM AUTHOR]

The chromosome of the Bacillus subtilis phage 2C, a linear molecule of double-stranded DNA of about 108 Da, in which thymine is completely replaced by hydroxymethyluracil, was cleaved by different endonucleases. In some cases restriction segments were much fewer than expected, suggesting a possible interference of the unusual base with the recognition mechanism of endonucleases. The physical map of 2C DNA was established by use of Sail and Hae III restriction endonucleases, which yielded a limited number of fragments. The expected number of fragments was 240 for Hae III and 23 for Sa/I; in reality, five segments were observed upon cleavage with Hae III and four with Sa/I The terminal fragments of the genome were first identified; the other fragments were ordered by hybridization and molecular weight determination of restriction fragments obtained by cleavage with the two endonucleases. In addition hybridization of restriction fragments showed the presence of homologous regions at the ends of the 2 C genome. The structure of these direct repetitive sequences was analyzed by cleavage with Rae III and hybridization with EcoRl restriction fragments. Their size (9.2 MDa) was found to be about 1/11 of that of the whole chromosome. [ABSTRACT FROM AUTHOR]

We have studied the regulation of the expression of tufA and tufB, the two genes encoding EF-Tu in Escherichia coli. To this aim we have determined the intracellular concentrations of EF-TuA and EF-TuB under varying growth conditions by an immunological assay in mutant of E. coli constructed for this purpose. The data show that in wild-type cells the expression of tufA and tufB is regulated coordinately. This coordination is not restricted to steady-state growth conditions but is maintained throughout the life cycle of the cells up till the stationary phase. The ratio in which the two genes are expressed, however, may vary among cells with different genetic constitutions. Neither complete elimination of EF-TuB from the cell (by insertion of bacteriophage Mu DNA into tufB) nor elevation of the intracellular EF-TuB concentration (by transformation with plasmids harbouring tufB) has any effect on the expression of tufA. A specific single-site mutation of tufA and tufB, enhancing tufB expression exclusively. These results have been interpreted by assuming that in wild-type cells the EF-Tu protein itself is involved in the regulation of the expression of tufB and that the mutant species of EF-Tu has lost this capacity either partially or completely. In agreement with this hypothesis are experiments performed in vitro with a coupled transcription/translation system programmed with DNA from a plasmid harbouring the entire tRNA-tufB transcriptional unit as a template. They show that addition to this system of EF-Tu in concentrations 2-5% of the endogenous amount results in strong inhibition of EF-Tu synthesis. We hypothesize that EF-Tu acts as an autogenous repressor, inhibiting tufB expression post-transcriptionally. [ABSTRACT FROM AUTHOR]

The FI gene product of bacteriophage lambda has been purified extensively using a biochemical assay that measures assembly of lambda phage particles in vitro. The molecular weight of the native protein was estimated to be 21 700 with an S20,w of 2.1 S and a Stokes radius of 2.5 nm. The molecular weight in dodecylsulfate was estimated to be 19000. The protein is highly acidic with an isoelectric point less than 4.1. [ABSTRACT FROM AUTHOR]

DNA-binding protein I from Escherichia coli was purified from cells carrying the ssh gene on a multicopy plasmid. In comparison to the strain without the recombinant plasmid the DNA-binding protein was overproduced more than 20-fold. The amount of the protein was measured after the purification steps by gel electrophoresis and radial immunodiffusion. The protein was purified to homogeneity and was active in replication assays like the wild-type DNA-binding protein. The assays were enzymatic replication of single-stranded and double-stranded fd DNA. E. coli DNA-binding protein I was further subjected to amino acid sequence analysis. A monomer of the protein consists of 187 residues which correspond to a molecular weight of 19715 with 5% error in analysis. The sequence of the amino-terminal 40 residues was determined and includes several basic residues of the protein. Sequence comparison between the DNA-binding protein I from E. coli and that coded by bacteriophage fd reveals similarities suggesting that DNA-binding protein I may use amino-terminal residues for binding to DNA like the phage protein. [ABSTRACT FROM AUTHOR]

The rate of formation of dinucleoside tetraphosphate, pppApU, from ATP and UTP by RNA polymerase on the A1 promoter of the mutant D111 of bacteriophage T7 is distinctly and specifically reduced not only by the third template-directed nucleotide, CTP, but also by CMP. The inhibitory effect of CMP is not changed when the enzyme contains prebound rifampicin. The synthesis of pppApU is also strongly reduced after preincubation of the enzyme with RNA. This inhibitory effect of RNA is, however, distinctly diminished by rifampicin bound to the enzyme prior to the addition of RNA. On the other hand RNA can suppress the specific binding of the antibiotic to the RNA polymerase subassembly α2β. [ABSTRACT FROM AUTHOR]

The binding of gene-5 protein to oligo(deoxyadenylic acid)s varying in length from 2 to 16 nucleotides has been studied by titrating the protein with the oligonucleotides and recording the 1H NMR spectra at 360 MHz. To obtain information about the mode of binding of the protein the aromatic parts of the spectra have been analysed by performing spectral simulations, starting from the assignments obtained from nuclear Overhauser enhancements at 500 MHz [Alma. N. C. M., Harmsen, B. J. M., Hull. W. F., Van der Marel, G., Van Boom, J. H,, and Hilbers, C. W. (1981) Biochemistry, 20, 4419-4428]. The 1H NMR spectra of the complexes of gene-5 protein with (dA)8, (dA),2 and (dA)11, appear to he identical except for differences in linewidth. The 1H NMR spectra ot' the complexes with the smaller oligonucleotides (dA)2, (dA)3 and (dA)4 differ from each other and from the spectra obtained from the complexes with longer oligonucleotides. However, binding of all oligo- nucleotides basically influences the same aromatic residues, namely two tyrosines and one phenylalaminc. In the protein-oligonucleotide complexes, one protein monomer covers three nucleotide residues, in contrast to the stoichiometry of 1: 4 found for protein-polynucleotide complexes. It was found that the binding to oligo- nucleotides is cooperative and ionic-strength-dependent hut far less so than found for the binding to polynucleotides. [ABSTRACT FROM AUTHOR]

The purification of the neck appendage protein of φ29, p12*, which is involved in the adsorption of the phage to Bacillus subtilis, is described. The purified native protein is in a dimeric form and can be assembled, in vitro, onto purified 12- particles that lack the neck appendages, suggesting that the incorporation of p12* to the rest of the phage structure is a self-assembly process. The assembly of protein p12* in vitro follows cooperative kinetics and it occurs with an efficiency of about 4 %. [ABSTRACT FROM AUTHOR]

An endonuclease has been purified more than 300-fold from Escherichia coli infected with bacteriophage T4. The enzyme degrades rapidly sedimenting (≫ 1000 S) DNA in vitro by introducing a limited number of breaks. The substrate is the replicative DNA isolated from cells infected with gene-49-defective phage [Kemper, B. and Janz, E. (1976) J. Virol. 18, 992-999]. Molecules of approximately a third the size of unit-length T4 DNA are exclusively found in a limit digest. The enzyme also reacts with single-stranded DNA from various sources. Heat-denatured T4 DNA is converted into acid-soluble oligonucleotides. Circular single-stranded M13 DNA is linearized by endonucleolytic cleavage causing a reduction of infectivity during transfection. The enzyme behaves like a typical late-gene product. Its activity is 100-fold reduced in cells infected with gene55-defective phage (defect in expression of late functions). A 30-fold reduction in its specific activity was found in cells infected with gene-49-defective phage suggesting that gene 49 codes for the enzyme or controls its expression. The purified enzyme binds to native or denatured DNA from various sources. The protein has a molecular weight of 42000 as determined by gel filtration and sedimentation analysis. Optimal activity on rapidly sedimenting DNA is obtained at pH 8.6 in Tris/HC1 buffer in the presence of 10 mM MgCl[SUB2]. Some 75% of the activity can be obtained with 7 mM MnC1[SUB2]. 5 mM CaC1[SUB2] has a stimulatory effect on the reaction with MgCl[SUB2] or MnCl[SUB2] each present at its individual optimal concentration. The enzyme does not require the addition of sulfhydryl reagent for full activity. The reaction can be inhibited by compounds like KCI, spermidine, p-hydroxymercuribenzoate or tRNA. [ABSTRACT FROM AUTHOR]

Interactions between lipids and the DNA-binding protein (protein IV) purified from bacteriophage PM2 were studied in vitro. The efficiency of incorporation of protein IV into single-walled liposomes was more than 90 %. Protein IV embedded in liposomes interacted more strongly with PM2 DNA than protein IV alone. The DNA - protein-IV--liposome complex was relatively stable as observed by sedimentation behavior on a sucrose gradient. The interaction between DNA and the protein-IV - liposome was abolished by tryptic digestion, even though 40 % of the protein remained in the vesicle. More than 70 % of the amino acids of this embedded peptide segment were hydrophobic. Carboxypeptidase digestion of the protein-IV-liposome caused a release of 20% of the radioactivity of the vesicle without changing the DNA-binding ability of the complexes. Modification of the protein-IV - liposome with the chemical probe, 2,4-dinitrofluorobenzene, and analysis of the tryptic peptides released from the protein- IV - liposome demonstrated that the N-terminal basic amino acid cluster segment responsible for the DNA binding was located on the outer surface of the bilayer. These results support an earlier model in which protein IV anchors itself in the inner leaflet of the PM2 bilayer membrane, interacting with the DNA in the virion. [ABSTRACT FROM AUTHOR]

The A and A* proteins of the bacteriophage 04 have been purified. The proteins have been analyzed for their enzymatic activities on single-stranded and double-stranded DNA. The A protein introduces a single-stranded break at a specific place in the G4 replicative form I DNA. This cleavage site has been localized between nucleotides 506 and 507 in the viral (+) strand. The A protein binds covalently to the 5' end of the cleavage site. The A protein initiates the replication of the viral (+) DNA [Borrias, et al. (1979) Virology, 31, 288-298]: the cleavage site therefore identifies the origin of replication. The A* protein cleaves viral (+) strand DNA at many different sites and also binds covalently to the 5' ends of the nick sites. The properties of both proteins strongly resemble the properties of the A and A* proteins of the related and much butter analyzed phage ∅X 174. These results indicate that the G4 and ∅XI 74 A and A* proteins have comparable functions and also that both phages initiate the replication of their replicative form DNA in a similar way. [ABSTRACT FROM AUTHOR]

A protein-synthesizing system in vitro, programmed with bacteriophage T7 DNA as template, changed the specificity of gene expression in the course of incubation as a result of newly synthesized T7 early proteins. The system mimics largely the situation in vivo on both the transcriptional and the translational levels, i.e. early gene expression is turned off shortly after late synthesis has been started. These results suggest that the switch from early to late expression does not necessarily require changes in the cellular environment nor is it dependent on the presence of membranes. The main part in this process is played by the phage-dependent RNA polymerase (gene 1 product), whose activity appears 8 -10 mm after start of incubation. When its activity is reduced by inhibitors or creation of non-optimal conditions, the system is not able to manage the early - late switch. [ABSTRACT FROM AUTHOR]

30-S ribosomal subunits of Escherichia coli form a stable complex with MS2 RNA or Qβ RNA at 37βC in the absence of initiator tRNA. The complex functions as a precursor of initiation since it can enter the ribosome cycle in the presence of inhibitors of de novo initiation. [ABSTRACT FROM AUTHOR]

The nucleocapsid of bacteriophage PM2, prepared according to Schäller et al. [Eur. J. Biochem. 92, 579–588 (1978)], was studied by biochemical and biophysical methods. It was not possible to isolate the lipid-free nucleocapsid. More than 95% of the lipids were associated with the nucleocapsid. The asymmetric distribution of phospholipids across the viral membrane was retained in the nucleocapsid since less than 10% of the phosphatidylethanolamine was accessible to the non-penetrable membrane probe, 2,4,6-trinitrobenzenesulfonate. Micro-dissection of the nucleocapsid with thermolysin demonstrated the asymmetric orientation of core proteins across the nucleocapsid membrane. Protein III was embedded deeply in the lipid bilayer and about 20% of the molecule extended to the exterior. Protein IV interacts with PM2 DNA and is partially in the inner leaflet of the bilayer. Small-angle X-ray scattering studies on the nucleocapsid enabled us to localize the lipid bilayer structure at radii from 20.5 nm to 24.0 nm. [ABSTRACT FROM AUTHOR]

P protein of phage λ and dnaB protein of Escherichia coli were isolated from (a) bacteria containing an inducible λ P gene on a plasmid, and (b) phage-λ-infected bacteria. P protein from both sources copurifies with part of the dnaB protein during four purification steps. A highly purified preparation contains the multimeric dnaB and the P protein in a complex as revealed by glycerol gradient centrifugation. The complex is composed of two major polypeptides. Their molec- ular weights of 52000 and 26000 are identical to those previously determined for the dnaB and P polypeptides, respectively. The complex contains a DNA-dependent ribonucleoside triphosphatase activity which can be inactivated by anti-dnaB globulin. Both the dnaB complementing and the ribonucleoside triphosphatase activities are partially masked by the P protein as shown by their stimulation following a treatment with sodium chloride and N-ethylmaleimide. [ABSTRACT FROM AUTHOR]

The adsorption constant of the irreversible adsorption of the bacteriophage K3 to Escherichia coli K12 bacteria is strongly dependent on the incubation temperature. Two inflection points are observed in an Arrhenius plot. For cells grown at 37°C the inflection points are found at 20°C and 28°C whereas these inflection points shift to 10°C and 19°C for cells grown at 12°C. To study the lipid environment of the receptor the temperature dependence of the inactivation of bacteriophage K3 was measured in vitro in the presence of various lipids. The Arrhenius plots of the rate of inactivation of phage K3 by complexes of protein d and lipopolysaccharide are very similar to those observed for whole cells. With lipopolysaccharide isolated from cells grown at 37°C inflection points are observed at 20°C and 28°C. With lipopolysaccharide from cells grown at 12°C the inflection points are found at 10°C and 21°C. These results show that the environment of protein d in vivo can be mimicked perfectly in vitro by protein d/lipopolysaccharide complexes. The fatty acid composition of lipopolysaccharide isolated from cells grown at 37°C and at 12°C differs in that in the latter case the amounts of mono-unsaturated fatty acids (mainly palmitoleic acid) are increased at the expense of lauric acid. This difference in fatty acid composition probably explains the difference in the phase transition temperatures caused by the two lipopolysaccharide preparations. A transition at the inflection point of the highest temperature is also found for lipopolysaccharide using light-scattering measurements and appears to be a thermal transition, since it is also observed in differential scanning calorimetry. [ABSTRACT FROM AUTHOR]

The properties of Dp-4 phage and its DNA have been studied. The phage has a polyhedral head of 60 nm diameter, a tail 155 nm long and a buoyant density in CsCl of 1.48 g/cm3. Analysis by acrylamide gel electrophoresis indicates the presence of five polypeptides. Dp-4 DNA has a molecular weight of 37×106, a melting point of 83.5°C (when dissolved in 0.15 M NaCl/0.015 M sodium citrate, pH 7.0) and a G+C content of about 33%. Denaturation of DNA yields two strands of different buoyant density in a neutral CsCl gradient. The interaction of poly(U.G) with the heavy strand strongly enhances its density and allows the preparative separation of both strands. Native Dp-4 DNA has unusual physical properties: abnormally low buoyant densities both in CsCl (1.666 g/cm3) and in Cs2SO4, (1.410g/cm3) (which do not correspond to the value predictable from the G+C content of the DNA), and a high thermal stability at low ionic strength. [ABSTRACT FROM AUTHOR]

An enzyme which specifically cleaves very-fast-sedimenting DNA of bacteriophage T4 is synthesized after infection of T4, and its synthesis is controlled by gene 49 [1,2]. This enzyme has been proved to be a DNase [2]. We have purified this DNase 3000-fold from extracts of E. coli infected with T4. The purified preparation was practically free from other DNases, and the DNase activity was not detectable in cells infected with a mutant defective in gene 49. The enzyme activity from cells infected with a temperature-sensitive mutant of gene 49 was also temperature-sensitive, suggesting strongly that gene 49 is a structural gene of the DNase. The molecular weight of the wild-type enzyme was estimated to be 50×103 by gel filtration chromatography. The purified DNase did not cleave native and denatured DNAs of T3 and T4, but cleaved renatured T4 DNA and DNA which was constructed by hybridization of denatured T3 DNA with enzymatically fragmented T3 DNA, indicating that gaps in the DNA duplex are structures susceptible to the DNase. Cleavage of the hybridized T3 DNA occurred when the fragmented DNA was phosphorylated at either the 3′ or 5′-strand termini. [ABSTRACT FROM AUTHOR]

Purified DNA-dependent RNA polymerase from Sulfolobus acidocaldarius is composed of 10 different subunits, one of which is present as four copies. Their molecular weights are 122000, 101000, 44000, 32000, 24000, 17500, 13800, 11800 (four copies), 11200, 10800, summing up to a total Mr of 423500. The sedimentation velocity is 13.5 S, indicating that at 0.5 M NH4Cl the enzyme exists in the monomeric form. At pH 9.2 in cellogel electrophoresis two of the subunits migrate towards the cathode. The composition is quite different from that of a typical eubacterial RNA polymerase. Its complexity reminds one of eucaryotic RNA polymerase. Maximal transcription of DNA from a Halobacterium halobium phage ΦH (ΦH DNA) proceeds at ph 8.5 and 75 °C. The enzyme is stable up to 75 °C and strictly requires a DNA template. ΦH DNA and poly[d(A-T)] are the most efficient. The temperature dependence of the transcription rate is characteristic for the template. Actinomycin D and heparin prevent transcription. While rifampicin, streptolydigin and α-amanitin have not effect. During storage, even at −70 °C, the enzyme loses its activity to transcribe ΦH DNA, whereas transcription of poly[d(A-T) · d(A-T)] remains unaffected. [ABSTRACT FROM AUTHOR]

Growth conditions and a purification procedure for mycobacteriophage ATCC 11759, a lyric phage for Mycobacterium smegmatis, are described. The phage is a large DNA phage with a very long tail (240 nm). A study of its DNA revealed three interesting features. 1. After denaturation the DNA molecule yields two strands of different buoyant densities. 2. The native DNA has unusual physical properties: its buoyant density in CsCl is very low (1.654 g/cm³), its sedimentation rate is lower than expected for the molecular weight, its thermal stability at low ionic strength is high. 3. The DNA (in its native form or after reannealing) is resistant to various restriction endonucleases. [ABSTRACT FROM AUTHOR]

The contractile sheaths of five defective, PBS X-like bacteriophages from Bacillus subtilis and B. licheniformis were investigated by electron microscopy, dodecylsulphate gel electrophoresis and immunodiffusion. Electron microscope images of the extended and contracted sheaths were of similar appearance, although their lengths were different. The surface lattices of both the extended and the contracted sheaths were determined by optical diffraction. This showed that the quaternary structure of the sheaths of all five defective phages originated from identical surface lattices, which could be approximately expressed by the selection rules L = -2n' + 3m and L = 9n' + 17m for the extended and contracted sheaths respectively, in which 6n'= n with n = 0 or an integer multiple of 6. These results indicated that the packing of the protein subunits in these sheaths differed from those of other bacteriophages, for example T4 and Mu [Amos and Klug, J. Mol. Biol. 99, 51-73 (1975); Admiraal and Mellema, J. Ultrastruct. Res. 56, 48-64 (1976)]. The molecular weight of the main sheath protein of the defective phages, as determined by dodecylsulphate gel electrophoresis, was approximately 50 000. This value differed from that for T4, but was similar to that of Mu [Admiraal and Mellema, J. Ultrastruct. Res. 56, 48-64 (1976); King and Laemmli, J. Mol. Biol. 75, 315-337 (1973)]. The results of immunodiffusion experiments, however, pointed to a chemical difference between the sheath proteins of the defective phages and Mu, in addition to T4. [ABSTRACT FROM AUTHOR]

The transcription products of phage T4 DNA in vitro are separated on polyacrylamide gels. The influence of salt, polymerase, triphosphate concentration and glucosylation on the RNA synthesis are shown. Individual transcripts are initiated selectively with dinucleotides and a single triphosphate. This technique allows the prediction of the initiation sequences of several T4 transcripts. [ABSTRACT FROM AUTHOR]

The competition of the DNA-binding proteins I and II of Escherichia coli and ol the phage fd DNA-binding protein for single-stranded DNA was investigated. Their roles in cells might be judged from their binding affinities to DNA and their mutual exchange in the DNA. protein complexes. Strongest binding on single strands was found for the phage protein. DNA-binding protein II displaced half of the protein I in the complex with single-stranded DNA when no double- stranded DNA was present. Protein-complexed single strands were protected against degradation. The protection is less pronounced for protein II which can increase the stability of the fd DNA complex with DNA-binding protein I against nucleolytic cleavage. [ABSTRACT FROM AUTHOR]

Three methods, chromatographic, spectrophotometric and tritium-release assay, were used and compared for the assay of deoxycytidylate methyltransferase. All three methods can be used for assay of this enzyme but the tritium-release assay appears to be the most simple and convenient. With the help of this assay the deoxycytidylate methyltransferase has been isolated and purified from sonically disrupted cells of Xp12-infected Xanthomonas oryzae. Using a procedure that involves fractionation with streptomycin sulfate and ammonium sulfate, filtration through Sephadex G-100 and chromatography on DEAE-cellulose, a 214-fold increase in specific activity was obtained. The enzyme displays a narrow pH optimum at 6.0. Among the buffers tested, 6-morphotino-ethane sulfonate with the addition of Mg2+ is the best. The enzyme can utilize dCMP and CMP as a substrate. The enzyme can also convert tetrahydrofolic acid into dihydrofolic acid. The Km value for dCMP is 31.3 μM and the Km value for tetrahydrofolic acid is 71.4 μM. There is no absolute requirement of ions for the activity of the enzyme; however, the presence of ions causes stimulating or inhibiting effects on enzyme activity that are dependent on the variety and concentration of ions used. [ABSTRACT FROM AUTHOR]

After polyadenylation in vitro, several viral RNAs become excellent templates for the synthesis of complementary DNA using RNA-dependent DNA polymerase from avian myeloblastosis virus. in the presence of oligo(dT) as a primer. If dTTP was the only deoxynucleoside triphosphate present, the reverse transcriptase catalyzed a 'slippage' reaction on the poly(A) tail, especially if the dTTP concentration exceeds 1 µM. In the case of MS2 RNA-poly(A), this poly(dT) synthesis could be specifically inhibited, but not completely abolished, by addition of dGTP. Lowering the dTTP concentration also decreased the average size of the poly(dT) products formed. In the presence of all four dNTPs, but using a low dTTP concentration to avoid slippage of the primer, discrete partial transcripts could be synthesized, complementary to the 3′-terminal region of the viral RNA. Although the average size of the products increases at higher temperatures of incubation, the pattern of the labeled transcripts, as revealed by gel electrophoresis, was mainly dependent on the nature of the deoxynucleoside triphosphate present in limiting concentration. These results indicate that it is not the secondary structure of the template but its primary sequence that is the major determinant of the discrete partial transcripts. Restricted synthesis, using a combination of only three or less dNTPs, results in very small cDNA products. The successive incorporation of the different dNTPs added to the primer, using MS2 RNA-poly(A) or Qβ RNA-poly(A) as a template, was in complete agreement with the known 3′-terminal sequence. Under the conditions used and with MS2 RNA-poly(A) or poly(A) as template, a ribosyl primer could not be substituted for the deoxyribosyl primer. Similarly, with MS2 RNA-oligo(C) only (dG)10 was an efficient primer for reverse transcription. Ribonucleoside triphosphates were very poor substrates. [ABSTRACT FROM AUTHOR]

Endolysin was induced in Pseudomonas BAL-31 infected with bacteriophage PM2 and was also associated with the purified virion. This enzyme required divalent cations for its activity, Ca2+ being the most effective cation. Endolysin activity in the virion increased up to three-fold upon disruption and the activity could be localized in the viral nucleocapsid. Thus the enzyme is localized within the virion. After purification of the structural proteins of bacteriophage PM2, only the nucleocapsid protein (III) had endolysin activity. [ABSTRACT FROM AUTHOR]

RNA bacteriophage f2, treated in vitro with rifampicin, loses infectivity dramatically. Rifampicin interacts with phage RNA, binding to a few specific sites. Inhibition of phage RNA infectivity occurs at 10–100 times lower molar excess of rifampicin than inhibition of infectivity of intact phage particles. Thus the phage capsid acts as a barrier, diminishing interaction of the drug with phage RNA. [ABSTRACT FROM AUTHOR]

With the aid of transcription studies on restriction fragments of bacteriophage M13 DNA it has been demonstrated that at least eight promoter sites are located on the M13 genome. Five of these promoters initiate the synthesis of RNA chains which contain at their 5′-terminal end pppG (G promoters), while the other three promoters initiate RNA chains which start exclusively with pppA (A promoters). The positions of these promoter sites on the physical map are: 0.82 (G0.82), 0.88 (G0.88), 0.94 (G0.94), 0.01 (G0.01). 0.08 (G0.08), 0.36 (A0.36), 0.51 (A0.51) and 0.56 (A0.56). The G promoters were found to be clustered within a distance of one-third of the genome length from the central termination site for transcription (map position 0.77). The A promoters, however, were found at greater distances from this termination signal. Based upon the incorporation of [γ-32P]ATP or [γ-32P]GTP, the capacity of these promoters to initiate the synthesis of RNA chains varies. The strongest G promoters are G0.82, G0.94 and G0.08 and the strongest A promoter is A0.36. As judged from their position on the genetic map, it is postulated that two promoters, namely G0.94 and G0.01, are located within gene II. The other promoters are most probably located immediately in front of the gene VIII/VII boundary (G0.82), and immediately in front of gene V (G0.88), gene II (G0.08), gene IV (A0.36), gene I (A0.51) and gene VI (A0.56). No evidence has been obtained so far for the existence of a promoter immediately in front of gene III. [ABSTRACT FROM AUTHOR]

The four structural proteins of the lipid-containing bacteriophage PM2 have been purified by dissociation of the virus in the presence of acetic acid followed by a combination of gel filtration and ion-exchange chromatography in the presence of sodium dodecylsulfate and guanidine hydrochloride. Amino acid analyses of each of the proteins were performed and correlated with the properties and functions of the proteins. Protein I has the highest polarity and is the only water-soluble protein. Protein II has a rather high polarity and hydrophobicity index and probably interacts electrostatically and hydrophobically with the bilayer. Proteins III and IV have low polarities and possess the solubility properties of proteolipids. At least protein Ill and perhaps also protein IV may interact with the bilayer. No fatty acids are covalently linked to these proteins. Tryptic fingerprints showed that proteins I and II contain a high proportion of hydrophobic peptides, but especially protein I also contains a large number of hydrophilic peptides. Proteins III and IV have relatively few hydrophobic peptides despite their relatively high hydrophobicity. Protein IV has two distinct regions, as shown by partial sequence studies. Basic amino acids at the N-terminus would serve for interaction with the viral DNA, the following hydrophobic sequence might interact with protein III or with the bilayer. [ABSTRACT FROM AUTHOR]

An unsaturated fatty acid auxotroph, strain UFA, isolated from the marine pseudomonad Pseudomonas BAL-31, host cell of the lipid-containing bacteriophage PM2, was grown in media supplemented with different unsaturated fatty acids. Under these conditions the fatty acid composition of the cell could be altered drastically. The phase transition in the native membrane and in the extracted lipids was analyzed by electron spin resonance using a nitroxide spin probe. Membranes prepared from strain UFA grown in cis16:1 or trans16:1 showed one transition at 9.4 °C and 12.4 °C respectively. Extracted lipids in both cases had almost the same transition temperature as that of the intact membrane. Membranes prepared from Pseudomonas BAL-31 had one transition at ≈ 12 °C, on the other hand there was no clear cut phase transition using extracted lipids. Replication of bacteriophage PM 2 took place below the transition temperature of the membrane lipids in the case where strain UFA was grown in trans 16 : 1. Other cases were not studied. [ABSTRACT FROM AUTHOR]

An immunoassay for tyrosinase, using the modified bacteriophage technique, was developed: Tyrosinase of Neurospora was conjugated to bacteriophage T4 using glutaraldehyde as a cross-linking agent. The conjugated phage that survived the coupling process could be inactivated by antiserum raised in rabbits against pure tyrosinase, but not by normal serum. This inactivation was specifically inhibited by pure Neurospora tyrosinase, and the degree of inhibition was proportional to the concentration of tyrosinase within the range of 30-150 ng/ml. Crude mycelial extracts possessing tyrosinase activity could similarly inhibit the inactivation of the conjugated phage by the antiserum. To evaluate the tyrosinase content of crude extracts, their inhibitory capacity was compared to that of known amounts of pure tyrosinase, and the amounts thus calculated agreed with those predicted from an enzymatic assay. The tyrosinase-bacteriophage immunoassay was used for the quantitation of tyrosinase-antigen in crude extracts of Neurospora cultures that had been induced to form tyrosinase by the addition of ethionine. Enzymatic activity appeared after a lag of several hours, increased for 2-3 days and then declined. Immunological assays of these cultures showed: (a) serologically reactive protein started to accumulate upon culture starvation and was evident during the lag period; (b) specific activity (units per mg antigen) was constant throughout induction: (c) at the phase of decrease in mycelial enzyme content, increasing amounts of serologically reactive protein were detected in the medium, indicating that some enzyme was eventually excreted. These results show that the lag is not a qualitatively distinct period, and support the previously forwarded notion that tyrosinase is synthesized de novo upon induction. [ABSTRACT FROM AUTHOR]

Cell-free translation of Qβ RNA and subsequent partial purification of the enzyme resulted in replicase activity. From 0.5 to 1.5% of all R chains synthesized were found in the 7-S replicase complex. The presence in the 7-S complex of the host subunits of authentic replicase, I(= S1) and EF-Ts, was shown by the effect of antisera directed against ribosomal protein S1 and EF-Ts, respectively. Furthermore, the presence of EF-Ts was demonstrated by thermal denaturation of in vitro replicase made by a cell extract from a Escherichia coli mutant with a thermolabile EF-Ts. In vitro replicase did not assemble spontaneously during protein synthesis but was formed upon subsequent purification. Assembly could be induced by ammonium sulphate precipitation (60% saturation) alone. It is concluded that the functional phage-coded subunit synthesized in vitro recognizes 1 and the EF-Tu · EF-Ts complex among a mixture of host proteins. [ABSTRACT FROM AUTHOR]

Protein kinase, which was isolated from cells infected with T7, is indeed a viral gene product. This is shown by DNA-dependent synthesis in vitro. The protein kinase transfers phosphate from ATP to seryl or threonyl residues in protein. The enzyme has only a relative requirement for magnesium ions, but is only active at low ionic strength. The best substrate is lysozyme. T7 protein kinase activity is not stimulated by cyclic 3′:5′-AMP and/or cyclic 3′:5′-GMP. The T7 protein kinase carries — SH groups essential for activity. There is indication that the enzyme phosphorylates itself and causes self inactivation, which may explain the fast disappearance of enzyme activity in vivo. Bacteriophage T3 also induces a protein kinase which is similar to the T7-induced enzyme in all respects tested. [ABSTRACT FROM AUTHOR]

1. The 5′-terminal sequence of the RNA transcribed from bacteriophage fd replicative form DNA under the control of promoter region I has been detennined to be ppp(Gp)nUpApApApGp- ApCpCpUpGpApUpUp… 2. This sequence is complementary to the 5′-terminal sequence of the minus strand of the corresponding RNA polymerase binding site I, the starting point for RNA synthesis lying approximately in the middle of the binding site. 3. This initial sequence is also transcribed faithfully from isolated complexes of RNA polymerase and binding site I, obtained by DNase digestion of complexes between RNA polymerase and fd replicative form DNA. These highly stable complexes can not be reconstituted from binding site and enzyme. 4. It is concluded that RNA polymerase binding site and initiation site are identical parts of a promoter region, and that no ‘drift’ between these sites is required as a step in RNA chain initiation. An additional non-transcribed outside region is implicated as essential for full promoter function. [ABSTRACT FROM AUTHOR]