Construction and characterization of a plasmid containing a nearly full-size DNA copy of bacteriophage MS2 RNA.

An apparently full-length complementary DNA copy of in vitro polyadenylated MS2 RNA was synthesized with avian myeloblastosis virus RNA-dependent DNA polymerase. After the MS2 RNA template was removed from the complementary DNA strand with T₁ and pancreatic RNase digestion, the complementary DNA became a good template for the synthesis of double-stranded MS2 DNA with Escherichia coli DNA polymerase I. We then constructed molecular chimeras by inserting the double-stranded MS2 DNA into the PstI restriction endonuclease cleavage site of the E. coli plasmid pBR322 by means of the poly(dA)· poly(dT) tailing procedure. An E. coli transformant carrying a plasmid with a nearly full-length MS2 DNA insertion, called pMS2-7, was chosen for further study. Correlation between the restriction cleavage site map of pMS2-7 DNA and the cleavage map predicted from the primary structure of MS2 RNA, and nucleotide sequence analysis of the 5′ and 3′ end regions of the MS2 DNA insertion, showed that the entire MS2 RNA had been faithfully copied, and that, except for 14 nucleotides corresponding to the 5′-terminal sequence of MS2 RNA, the fulllength DNA copy of the viral genetic information had been inserted into the plasmid. Restriction endonuclease analysis of the chimera plasmid DNA also revealed the presence of an extra DNA insertion which was identified as the translocatable element IS1³ (see following paper).     

Nucleic acid binding properties of Escherichia coli ribosomal protein S1. I. Structure and interactions of binding site I.

Escherichia coli ribosomal protein S1 plays a central role in initiation of protein synthesis, perhaps via participation in the binding of messenger RNA to the ribosome. S1 protein has two nucleic acid binding sites with very different properties: site I binds either single-stranded DNA or RNA, while site II binds single-stranded RNA only (Draper et al., 1977). The nucleic acid binding properties of these sites have been explored using the quenching of intrinsic protein fluorescence which results from binding of oligo- and polynucleotides, and are reported in this and the accompanying paper (Draper &; von Hippel, 1978).Site I has been studied primarily using DNA oligomers and polymers, and has been found to have the following properties. (1) The intrinsic binding constant (K) of site I for poly(dA) and poly(dC) is ~3 × 10⁶m^?1 at 0.12 m-Na⁺, and the site size (n, the number of nucleotide residues covered per S1 bound) is 5.1 ± 1.0 residues. (2) Binding of site I to polynucleotides is non-co-operative. (3) The K value for binding of S1 to single-stranded polynucleotides is ~10³ larger than K for binding to double-stranded polynucleotides, meaning that S1 (via site I) is a potential “melting” or “double-helix destabilizing” protein. (4) The dependence of log K on log [Na⁺] is linear, and analysis of the data according to Record et al. (1976) shows that two basic residues in site I form charge-charge interactions with two DNA phosphates. In addition, a major part of the binding free energy of site I with the nucleic acid chain appears to involve non-electrostatic interactions. (5) Oligonucleotides bound in site II somewhat weaken the binding affinity of site I. (6) Binding affin is virtually independent of base and sugar composition of the nucleic acid ligand; in fact, the total absence of the base appears to have little effect on the binding, since the association constant for 2′-deoxyribose 5′-phosphate is approximately the same as that for dAMP or dCMP. (7) Two molecules of d(ApA) can bind to site I, suggesting the presence of two “subsites” within site I. (8) Iodide quenching experiments with S1-oligonucleotide complexes show differential exposure of tryptophans in and near the subsites of site I, depending upon whether neither, one, or both subsites are complexed with an oligonucleotide.     

Physical mapping of the transfer RNA genes on lambda-h80dglytsu+36

The three Escherichia coli transfer RNA genes of the DNA of the transducing phage λ80cI857S^?t68dglyTsu⁺₃₆tyrTthrT (abbreviated λh80T), which specify the structures of tRNA^Gly₂(su⁺₃₆), tRNA^Tyr₂ and tRNA^Thr₃, have been mapped by hybridizing ferritin-labeled E. coli tRNA to heteroduplexes of λh80T DNA with the DNA of the parental phage (λh80cI857S^?t68) and examining the product in the electron microscope. The DNA of λh80T contains a piece of bacterial DNA of length 0·43 λ unit³ that replaces a piece of phage DNA of length 0·46 λ unit, proceeding left from B · P′ (the junction of bacterial DNA and phage DNA) (i.e. att⁸⁰). A cluster of three ferritin binding sites, and thus of tRNA genes, is seen at a position of 0·24 λ unit (1·1 × 10⁴ nucleotides) to the left of B· P′. The three tRNA genes of the cluster are separated by the unequal spacings of 260 (±30) and 140 (± 30) nucleotides, proceeding left from B·P′. The specific map positions have been identified by hybridization competition between ferritin-labeled whole E. coli tRNA with unlabeled purified tRNA^Tyr₂ and with unlabeled partially purified tRNA^Gly₂. The central gene of the cluster is tRNA^Tyr₂. The tRNA^Gly₂gene is probably the one furthest from B·P′. Thus, the gene order and spacings, proceeding left from B·P′, are: tRNA^Thr₃, 260 nucleotides, tRNA^Try₂, 140 nucleotides, tRNA^Gly₂.     

Nucleic acid binding and unfolding properties of ribosomal protein S1 and the derivatives S1-F1 and m1-S1.

The nucleic acid binding and unwinding properties of wild-type Escherichia coli ribosomal protein S1 have been compared to those of a mutant form and a large trypsin-resistant fragment, both reported recently [J. Mol. Biol. 127, 41-45 (1979) and J. Biol. Chem. 254, 4309-4312 (1979). The mutant (m1-S1) contains 77% and the fragment (S1-F1) 66% of the polypeptide chain length (approximately 600 amino acid residues) of protein S1. The mutant is active in protein synthesis in vitro; the fragment, although retaining one or more of the functional domains of S1, is inactive in protein synthesis. We find that m1-S1 is is almost as effective as S1 in binding to poly(rU), phage MS2 RNA and simian virus 40 (SV40) DNA, and in unfolding poly(rU) and the helical structures present in MS2 RNA and phi X174 viral DNA. S1-F1, however, binds to poly(rU) and denatured SV40 DNA, but not to MS2 RNA. It unfolds neither poly(rU), nor the residual secondary structure of MS2 RNA or phi X174 viral DNA. Thus, there appears to be a correlation between the loss in ability of S1 to unwind RNA and the loss in its ability to function in protein synthesis.     

Electron microscopic determination of the preferential binding sites of Escherichia coli RNA polymerase to phi X174 replicative form DNA

Binding of Escherichia coli RNA polymerase to φX174 DNA replicative form (RF) has been studied by electron microscopy. Samples of the binary complexes were spread for observation upon polylysine-coated carbon films. Binding was obtained with both RFI and RFIII forms of the DNA; complexes formed with the former were treated with restriction enzyme PstI before spreading. A histogram constructed from the positions of 558 polymerase molecules bound to 181 DNA strands exhibited three prominent, sharp peaks at 3.3 × 10² nucleotides, 39.7 × 10² nucleotides and 49.0 × 10² nucleotides from the PstI cleavage point. These positions correspond closely to those of the D, A and B promoter sequences, as derived from φX174 DNA sequence data by Sanger et al. (1977).     

Additional Nucleotide Sequences in Precursor 16S Ribosomal RNA from <Emphasis Type="Italic">Escherichia coli</Emphasis>

MATURE 5S, 16S and 23S ribosomal RNA species present in E. coli ribosomes are the end products of complex biosyn-thetic pathways. They are formed by reduction in length, and methylation of longer RNA chains transcribed on the ribosomal RNA cistrons of E. coli DNA. While these modifications take place the ribosome structure is formed by progressive addition of ribosomal proteins and conformational changes in the resulting ribonucleoprotein precursor particles¹.     

Molecular Basis for Repressor Activity of Qβ Replicase

WITH the purification and characterization of viral replicases, a novel feature of nucleic acid polymerases—stringent template specificity—was recognized^1,2. Qβ replicase, the most extensively studied viral RNA polymerase^2–8, is now known to replicate Qβ RNA², the complementary Qβ minus strand⁹, RNA molecules described as “variants” of Qβ RNA^10,11 and a set of small RNAs of unknown origin which accumulate in Qβ-infected Escherichia coli, collectively designated as “6S RNA”¹². On the other hand, the RNA from phages related distantly, if at all, to Qβ^13,14, such as MS2 or R17 and of other viruses such as TMV² or AMV (Diggelmann and Weissmann, unpublished results) are completely inert as templates, as are ribosomal and tRNA from E. coli². Poly C and C-rich synthetic copolymers at high concentrations elicit synthesis which, however, remains restricted to the formation of a strand complementary to the template^15,16.     

Nucleic acid binding properties of Escherichia coli ribosomal protein S1. II. Co-operativity and specificity of binding site II.

The following properties characterize the interaction of nucleic acid binding site II of Escherichia coli ribosomal protein S1 with oligo- and polyribonucleotides; all have been determined with site I complexed with oligo- or polydeoxyribonucleotides. (1) The intrinsic binding constant (K) of site II to single-stranded polyribonucleotides is fairly independent of base composition, though cytidinecontaining polymers bind with approximately threefold higher intrinsic affinities than do the comparable adenine-containing species. (2) Poly(rC) is bound to site II co-operatively; the co-operativity parameter (ω) ? 31. Poly(rA) shows no binding co-operativity. The site size (n) for both polyribonucleotides binding at site II is about ten nucleotide residues. (3) The K value for site II is ? 4 × 10⁵m^?1 for poly(rA), and ? 1 × 10⁶m^?1 for poly(rC), in 0.12 m-Na⁺. Unlike site I, the binding affinity of site II increases somewhat with increasing salt concentration, suggesting that phosphate—basic protein residue contacts are not involved. (4) Varying Mg^{2 +} concentration has no effect on K, and changes in the concentration of either Mg²⁺ or Na⁺ do not affect the magnitude of site II co-operativity. (5) Reaction of the exocyclic amino groups of poly (rC) with formaldehyde drastically reduces the affinity of site II for this polynucleotide, while the affinity of poly (rC) for site I is not altered by this treatment. (6) No major sequence specificity of K for site II is found with either homogeneous polynucleotides or the 3′ terminal dodecanucleotide of 16 S ribosomal RNA; we conclude that selectivity of S1 binding via site II depends largely on the presence or absence of base compositiondependent binding co-operativity.The binding properties of site II probably account for the ability of S1 to inhibit translation at high S1 to ribosome ratios (“factor i” activity). Possible mechanisms for the role of S1 protein as a part of the phage Qβ replicase complex and in protein synthesis are discussed in relation to the binding properties of site I and site II.     

Destabilization of the secondary structure of RNA by ribosomal protein S1 from escherichia coli

S1 is an acidic protein associated with the 3′ end of 16S RNA; it is indispensable for ribosomal binding of natural mRNA. We find that S1 unfolds single stranded stacked or helical polynucleotides (poly rA, poly rC, poly rU). It prevents the formation of poly (rA + rU) and poly (rI + rC) duplexes at 10–25 mM NaCl but not at 50–100 mM NaCl. Partial, salt reversible denaturation is also seen with coliphage MS2 RNA, $\text{E. coli}$ rRNA and tRNA. Generally, only duplex structures with a Tm greater than about 55° are formed in the presence of S1. The protein unfolds single stranded DNA but not poly d(A·T).     

Subunit interactions of rabbit muscle aldolase. Conformational change of aldolase in magnesium chloride and its relationship to the dissociation of subunits.

The effect of Escherichia coli ribosomal protein S1 on translation has been studied in S1-depleted systems programmed with poly(U), poly(A) and MS2 RNA³. The translation of the phage RNA depends strictly on the presence of S1. Optimum poly(U)-directed polyphenylalanine synthesis and poly(A)-programmed polylysine synthesis also require S1. Excess S1 relative to ribosomes and messenger RNA results in inhibition of translation of MS2 RNA and poly(U), but not of poly (A). In the case of phage RNA translation, this inhibition can be counteracted by increasing the amount of messenger RNA. Three other 30 S ribosomal proteins (S3, S14 and S21) are also shown to inhibit MS2 RNA translation. The effects of S1 on poly(U) translation were studied in detail and shown to be very complex. The concentration of Mg²⁺ in the assay mixtures and the ratio of S1 relative to ribosomes and poly(U) are crucial factors determining the response of this translational system towards the addition of S1. The results of this study are discussed in relation to recent developments concerning the function of this protein.     

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.     

Inhibition of nucleic acid synthesis in Ehrlich tumor cells by periodate-oxidized adenosine and adenylic acid

Periodate-oxidized adenosine and AMP were inhibitory to both RNA and DNA synthesis in Ehrlich tumor cells in culture. With periodate-oxidized adenosine, the inhibition of RNA synthesis paralleled the inhibition of DNA synthesis. Periodate-oxidized AMP, however, was more inhibitory to DNA synthesis than to RNA synthesis. With both compounds, there was a decrease in the conversion of [¹⁴C]cytidine nucleotides to [¹⁴C]deoxycytidine nucleotides in the acid-soluble pool. The borohy-dride-reduced trialcohol derivative of the periodate-oxidized adenosine compound was not inhibitory to DNA or RNA synthesis in the tumor cells. The incorporation of [³H]uridine into 28S and 18S ribosomal RNA was inhibited by both periodate-oxidized adenosine and AMP, but the incorporation of [³H]uridine in 45S, 5S, and 4S RNA was essentially unaffected by these compounds. Periodate-oxidized adenosine inhibited Ehrlich tumor cell growth in vivo.     

A technique for mapping transfer RNA genes by electron microscopy of hybrids of ferritin-labeled transfer RNA and DNA: the phi-80hpsu+3-system

A method for mapping transfer RNA genes on single strands of DNA is described. tRNA is covalently coupled to the electron-opaque label, ferritin. The ferritinlabeled tRNA, Fer-tRNA, is hybridized to a single strand of DNA, or to a single- strand region of a DNA in a heteroduplex. The sites where the Fer-RNA binds to the complementary sequence on the DNA are then mapped by electron microscopy. Several alternative coupling procedures are described (see Fig. 1). In HzI a — COCH₂Br group is attached to ferritin by acylation. 3'-Oxidized tRNA is joined to HSRCONHNH₂ by hydrazone formation. Ferritin is then coupled to tRNA by reaction of the CBr and SH bonds. In the BI procedure a lysine amino group of ferritin is coupled by Schiff base formation with 3'-oxidized RNA. The conjugate is stabilized by borohydride reduction. In the BII procedure, a —COCH₂Br group is attached to ferritin. (H₂NCH₂CH₂S—)₂ is coupled to oxidized tRNA by Schiff base formation and borohydride reduction. An SH group is exposed by reduction. This HS-tRNA is coupled to a —COCH₂Br group attached to ferritin. All the procedures work but BII is recommended. Methods for purifying the Fer-tRNA and the Fer-tRNA-DNA hybrid are described. For the transducing phages, φ80hpsu^+,?_III and φ80hpsu^?_III, the DNA molecules each carry a piece of bacterial DNA of length 0·066±0·007 λ unit (3100 nucleotide pairs; we find the length of λ is 8·99 φX174 units) replacing a piece of phage DNA of φ80h of length 0·045±0·005 λ unit. The left junction of this bacterial DNA with phage DNA (referred to as P-B′) is at or close to the att site. The two tandem tyrosine genes of φ80hpsu^+,?_III and the single tRNA gene of φ80hpsu^?_III have been mapped at a position 1100 nucleotides to the right of the left (P·B′) junction of phage DNA and bacterial DNA, by hybridizing Escherichia coli Fer-tRNA to φ80hpsu_III/φ80h heteroduplexes. The separation of the two ferritin labels in φ80hpsu^+,?_III hybrids gives 140±20 nucleotides as the size of a single tyrosine tRNA gene.     

A comparison of the structural organization of amplified ribosomal DNA from Xenopus mulleri and Xenopus laevis.

Secondary structure maps of long single strands of amplified ribosomal DNA from two closely related species of frogs, Xenopus laevis and X. mulleri, have been compared. The secondary structure pattern of the gene region is identical in both ribosomal DNAs while the patterns in the non-transcribed spacers² differ. In X. mulleri, the spacer shows an extended region without any secondary structure adjacent to the 28 S ribosomal RNA sequence. In contrast, the same region in the X. laevis spacer has extensive secondary structure. A comparison of secondary structure maps and denaturation maps of these two ribosomal DNAs (Brown et al., 1972) reveals that the portion without secondary structure in the X. mulleri spacer corresponds to an early melting A + T-rich region. As in X. laevis ribosomal DNA, Escherichia coli restriction endonuclease (EcoRI) makes two cuts in each repeating unit of amplified ribosomal DNA from X. mulleri. The position of the cleavage sites is identical in the two species as judged from secondary structure mapping of the two classes of EcoRI fragments generated. The small fragments of X. mulleri ribosomal DNA are homogeneous in size with a duplex molecular weight of 3.0 × 10⁶, and contain about 85% of the 28 S ribosomal RNA gene and about 17% of the 18 S ribosomal RNA gene. The large fragments are heterogeneous in size with molecular weights ranging from 4.2 to 4.9 × 10⁶, and contain the remaining portions of the gene regions and the nontranscribed spacer. Heteroduplexes made between large fragments of different lengths show only deletion loops. The position of these loops indicates that the length heterogeneity resides in the non-transcribed spacer region. Electrophoretic analysis of EcoRI digests of chromosomal ribosomal DNA from X. mulleri demonstrates that this DNA is heterogeneous in length as well.