Studies of the binding of Escherichia coli RNA polymerase to DNA. V. T7 RNA chain initiation by enzyme-DNA complexes

Initiation of T7 RNA chains by Escherichia coli RNA polymerase-T7 DNA complexes has been followed using incorporation of λ-³²P-labeled ATP and GTP to determine the relation between the enzyme binding sites and RNA chain initiation sites on the T7 genome. If the period of RNA synthesis is limited to less than two minutes, the stoichiometry of RNA chain initiation can be measured in the absence of chain termination and re-initiation. About 70% of the RNA polymerase holoenzyme molecules in current enzyme preparations are able to rapidly initiate a T7 RNA chain. The ratio of ATP- to GTP-initiated T7 RNA chains is not altered by variations in the number of enzyme molecules added per DNA, nor by alterations in the ionic conditions employed for RNA synthesis. This suggests that RNA chain initiation sites are chosen randomly through binding of RNA polymerase to tight (class A) binding sites on T7 DNA.     

DNA of the Streptomyces phage SH10: Binding sites for Escherichia coli RNA polymerase and denaturation map

Summary Escherichia coli RNA polymerase bound to Streptomyces phage SH10 DNA was visualized by electron microscopy. Six specific binding sites were observed at map units 53, 85, 93, 97, 98, and 99 on the physical map of the 48 kb long genome. Electron microscopy of partially denatured SH10 DNA revealed a characteristic melting pattern of A+T-rich regions around map units 1, 3, 48, 52, and 99. A comparison of the denaturation map with the RNA polymerase binding sites indicates that three binding sites are located in the most A+T-rich regions, two in other early melting regions and one in a segment of higher DNA helix stability.     

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.     

Transcription of polyoma virus DNA in vitro. III. Localization of calf thymus RNA polymerase II binding sites.

The binding sites of calf thymus RNA polymerase II on polyoma DNA were monitored by electron microscopy. Six discrete binding sites were located at positions 0.06, 0.25, 0.57, 0.66, 0.85 and 0.98 on the physical map of polyoma DNA. Although most of these sites are located in easily denaturable regions of the DNA, the strongest binding sites do not overlap with the major A + T-rich regions. In addition, the same binding sites were observed on superhelical or linear polyoma DNA. These results suggest that the eucaryotic RNA polymerase II can recognize specific sequences on double-stranded DNA and not only easily denaturable regions. At least five of these sites correspond to the binding and initiation sites mapped previously for the Escherichia coli RNA polymerase (Lescure et al., 1976).Stable initiation complexes can be formed with both E. coli and calf thymus RNA polymerases in the presence of a single dinucleotide (GpU) and a specific ribotriphosphate (CTP). Under these conditions, the binding of both enzymes to the sites in positions 0.06 and 0.57 is stimulated whereas the binding in positions 0.65 and 0.84 is partially suppressed. Both eucaryotic and procaryotic RNA polymerases may recognize similar sequences of the viral DNA in vitro.     

Mapping of Escherichia coli RNA polymerase binding sites on 2-micrometers DNA from Saccharomyces cerevisiae. Heterogeneity within the inverted duplication and evidence for an eukaryotic invertible DNA sequence.

The 2-μm DNA plasmids from Saccharomyces cerevisiae strain H1 and strain HQ/5C were analyzed by electron microscopy for the presence of Escherichia coli RNA polymerase binding sites. On native 2-μm DNA isolated from strain HQ/5C five RNA polymerase binding sites were detected. One further site was mapped on cloned 2-μm DNA type 23 from S. cerevisiae strain H1. This additional site is located at a distance of 2.15 kilobases from EcoRI site B inside one of the inverted duplication (id) sequences. No such binding site could be detected in the other id sequence of the type 23 molecule, thus indicating that the two id sequences of strain H1 differ in at least one short region. The location of the id sequence carrying the RNA polymerase binding site was analyzed in native 2-μm DNA isolated from strain H1 and found to be present on HindIII fragment 2 and absent from HindIII fragment 5. This indicates that at least a part of the id sequences has a fixed position with respect to the unique S segment and further suggests a site specific recombination mechanism for the inversion of one of the unique segments. As a control for the specificity of RNA polymerase binding, we have mapped binding sites on vectors pBR313 and pBR322. The location of the E. coli RNA polymerase binding sites on 2-μm DNA is discussed in relation to the DNA regions expressed in E. coli minicells.     

Transcription of polyoma virus DNA in vitro. II. Transcription of superhelical and linear polyoma DNA by RNA polymerase II.

The protein product of the bacteriophage T4 gene 32 is a single-stranded DNA binding protein which functions during phage DNA repair, replication and recombination. Recently the gene 32 protein was shown to participate in the regulation of its own expression. Although the purified protein is known to interact with DNA, the autoregulation was shown to occur at the translational level. The previous analysis in vivo, although coherent, was indirect. We report here direct cell-free experiments in which purified gene 32 protein specifically represses translation of gene 32 messenger RNA.     

Electron microscopy of a eukaryotic single-stranded DNA binding protein-DNA complex

The single-stranded DNA binding protein of Ustilago maydis decreases the contour length of φX174 DNA. When DNA complexes were prepared with subsaturating amounts of the protein, its distribution on the DNA was markedly non-random, indicating a high degree of co-operativity in its binding to single-stranded DNA. The analagous Escherichia coli, Salmonella typhimurium and bacteriophage T7 binding proteins also reduced DNA contour lengths to a similar extent, whereas the bacteriophage T4 gene 32 protein, as shown previously, increased the contour length. Despite the fact that the U. maydis protein efficiently denatures poly[d(A-T) · d(A-T)], it appears to initiate denaturation of native bacteriophage λ DNA rather inefficiently.     

Pauses at positions of secondary structure during in vitro replication of single-stranded fd bacteriophage DNA by T4 DNA polymerase

Since bacteriophage T4 DNA polymerase is unable to use duplex DNA molecules as templates (B. Alberts, J. Barry, M. Brittner, M. Davies, H. Hama-Inaba, C. C. Liu, D. Mace, L. Moran, C. F. Morris, J. Piperno, and N. Sinha, 1977, in Nucleic Acids and Protein Recognition, Vogel, H. J., ed., pp. 31–63, Academic Press, New York), a technique involving synchronous and uniquely primed synthesis of DNA on the single-stranded fd DNA by the T4 DNA polymerase has been developed to probe regions exhibiting secondary structure on this genome. As the polymerase proceeds, the template secondary structure acts as a kinetic barrier to delay the continuous chain extension catalyzed by this enzyme. These kinetic pause sites can be mapped by denaturing agarose gel electrophoresis of replication intermediates and used to generate a secondary structure map. Using this method, we are able to establish a list including at least seven plausible stable helical regions in fd DNA. Two of the most stable secondary structures have been mapped near fd sequence positions 3350 and 5650, respectively. The latter has been reported to be the region where fd DNA replication begins (C. P. Gray, R. Sommer, C. Polke, E. Beck, and H. Schaller, 1978, Proc. Nat. Acad. Sci. USA, 75, 50–53). However, the biological function associated with the former has yet to be investigated. Following a two-state model, we estimate the first-order rate constant for progression through the duplex regions near position 5650 in fd DNA to be about 0.042 min^?1 for fd DNA synthesis by the T4 DNA polymerase under our reaction conditions. A 7.5-fold increase in this rate constant is obtained upon the addition of the T4 DNA helix destabilizing protein (i.e., gene 32 protein). The general pattern of our secondary structure map agrees well with a computer search for duplex regions on the fd genome. Both the stability and the size of a stable secondary structure at a particular position on the fd template determine the time that the newly made DNA molecules spend at that site. A structure with a stem of less than 8 base pairs does not interrupt significantly the procession of the T4 DNA polymerase during the process of fd DNA synthesis.     

Two ribosome binding sites from the gene 0.3 messenger RNA of bacteriophage T7

Ribosome-protected regions have been isolated and analyzed from the bacteriophage T7 gene 0.3 mRNA labeled in vivo. Two discrete sites which are nearly equally protected by ribosomes are obtained from what was previously assumed to be a monocistronic message. Use of appropriate T7 deletion mutant RNAs has allowed mapping of both ribosome-recognized regions. Site a is positioned very close to the 5′ terminus of the mRNA and is apparently the initiator region for the major gene 0.3 protein, which acts to overcome the host DNA restriction system. Site b is located within several hundred nucleotides of the 3′ end of the RNA and probably initiates synthesis of a small polypeptide of unknown function. Both ribosome binding sites exhibit features common to other initiator regions from Escherichia coli and bacteriophage mRNAs. The proximity of site a to the RNase III cleavage site at the left end of gene 0.3 may explain why processing by RNase III is required for efficient translation of the major gene 0.3 protein.     

Letter: The isolation and properties of the specific binding sites for Escherichia coli RNA polymerase on T4 and T7 bacteriophage DNAs.

RNA polymerase of Escherichia coli was allowed to bind to labeled T4 or T7 bacteriophage DNA. The unbound and “weakly” bound polymerase molecules were removed by adding an excess of poly(I) which has a high affinity for the enzyme (Bautz et al., 1972). After the unbound DNA regions were digested with pancreatic DNAase and snake venom phosphodiesterase, the “protected” DNA-RNA polymerase complexes were isolated by Sephadex G200 column chromatography. The protected DNA sites were then isolated by phenol extraction and hydroxylapatite chromatography. Studies of the DNA recognition regions led to the following conclusions. (1) No binding is observed in the absence of the sigma subunit or at low temperatures. (2) The amount of protection ranges from 0·18% to 0·24% of T4 DNA and from 0·25% to 0·34% of T7 DNA. In the absence of poly(I), higher protections are observed and the protected regions display heterogeneity in size and secondary structure. (3) The protected regions are double-stranded, as shown by hydroxylapatite chromatography, base composition analysis, and thermal chromatography. (4) The length of the protected regions comprise about 50 to 55 nucleotide pairs, as suggested by end-group analysis, sucrose density-gradient centrifugation, and polyacrylamide gel electrophoresis. (5) The results suggest the interaction of dimeric polymerase molecules at these sites. On the basis of DNA sizes, there are 7 to 9 such sites on T4 DNA and 2 to 3 on T7 DNA. (6) The protected regions are high in (A + T): 68% for T4 and 62% for T7 DNA. (7) Thermal chromatograms reflect these base compositions and suggest the homogeneity of these regions with respect to size and base composition.     

Angular Alteration of the DNA Helix by <Emphasis Type="Italic">E. coli</Emphasis> RNA Polymerase

IT has been a source of speculation whether the reading of the genetic code of DNA by RNA polymerase involves the disruption of the DNA helix. While circuitous evidence favouring either affirmative or negative answer has been accumulating, direct experiments have been few^1–11. Kosaganov et al. investigated the possibility of a local unwinding of DNA during RNA synthesis by measuring the kinetics of formaldehyde-induced denaturation of DNA during RNA synthesis¹². They concluded that the binding of RNA polymerase did not cause local unwinding but RNA synthesis produced “defects” in the double helix. Unfortunately, the interpretation of formaldehyde-induced denaturation is not clear, nor is the nature of a “defect”.     

Comparison of the A-T rich regions and the Bacillus subtilis RNA polymerase binding sites in phage phi 29 DNA.

By using a modification of the BAC spreading method for mounting the DNA for electron microscopy, partial denaturation maps of protein-free phi 29 DNA and of phi 29 DNA containing protein p3 were obtained. In phi 29 p3-DNA1 the protein does not seem to influence the melting of the ends of the molecules. The comparison of the partial denaturation map and the B. subtilis RNA polymerase binding sites indicates that five of the seven early promoters (A1, A2, A3, B2 and C2) are located in A-T rich DNA regions whereas the other two early promoters (B1 and C1) are located in less A-T rich sites.     

Repression by the cI protein of phage λ: in vitro inhibition of RNA synthesis

We have studied the in vitro repression of RNA synthesis by the cI protein of phage λ. We find that highly purified cI protein is an effective and specific repressor of RNA synthesis from the early gene region of λ DNA. Under optimal conditions at least 95% of the early gene RNA synthesis is repressed and this repression is eliminated or severely impaired by the use of λ DNA-carrying operator-type mutations which reduce the binding affinity of the cI protein. Highly effective repression can be demonstrated only through the use of the initiation-inhibitor rifampicin, which presumably, selects “properly” initiated RNA chains; thus we can by-pass in vitro but not yet solve the problem of how the host polymerase initiates specifically in vivo from the immediate-early promoter sites.