Binding of the structural protein soc to the head shell of bacteriophage T4

Qβ plus strands with a 70 S ribosome bound to the coat cistron initiation site were used as template for Qβ replicase. Minus strand synthesis proceeded until the replicase reached the ribosome. The ribosome was removed and elongation was continued in a substrate-controlled, stepwise fashion. The nucleotide analog N⁴-hydroxyCMP was introduced into the positions complementary to the third and fourth nucleotides of the coat cistron. The minus strands were elongated to completion, purified and used as template for Qβ replicase. The final plus strand preparation consisted of four species, with the sequences -A-U-G-G- (wild type), -A-U-A-G- (mutant C₃), -A-U-G-A- (mutant C₄) and -A-U-A-A- (mutant $\text{C}{}_3\text{C}{}_4$) at the coat initiation site. The ribosome binding capacity of the mutant RNAs relative to wild type was <0.1 (C₃), 3.2 (C₄) and 0.3 ($\text{C}{}_3\text{C}{}_4$). The finding that mutant C₃ no longer formed an initiation complex suggests that the interaction of the ribosome binding site with fMet-tRNA plays an essential role in the formation of the 70 S initiation complex. The fact that mutant C₄ RNA bound more efficiently than wild type, and that mutant $\text{C}{}_3\text{C}{}_4$ RNA showed substantial ribosome binding capacity whereas the single mutant C₃ did not, can be explained by assuming that an A residue following the A-U-G triplet interacts with a complementary U residue in the anticodon loop sequence. In the case of $\text{C}{}_3\text{C}{}_4$ this additional base-pair may offset the reduced codon-anticodon interaction resulting from the modification of the A-U-G codon.     

The regulatory region of MS2 phage RNA replicase cistron. Functional activity of individual MS2 RNA fragments

The present work deals with the structural-functional organization of regulatory regions of messenger RNAs. Some principles of the action of a translational repressor (coat protein) and the formation of the ribosomal initiation complex at the replicase cistron have been studied with MS2 phage RNA. When the complex of MS2 RNA with the coat protein is treated with T₁ ribonuclease, the coat protein selectively protects mainly two fragments (59 and 103 nucleotides in length) from digestion; these fragments contain the intercistronic regulatory region and the beginning of the MS2 replicase cistron. These polynucleotides have been isolated in a pure state and their primary structure has been established.It has been established that both MS2 RNA fragments contain all the necessary information for specific interaction with MS2 coat protein and form a complex with it with an efficiency close to that observed in the case of native MS2 RNA. They also provide the normal polypeptide chain initiation at the replicase cistron. Enzymatic binding of the second aminoacyl-tRNA and electrophoretic analysis of N-terminal dipeptides prove that only the true initiator codon of the replicase cistron is recognized by a ribosome despite the presence of a few additional AUG triplets within the polynucleotides. Under conditions of limited hydrolysis by T₁ ribonuclease, the beginning of the replicase cistron has been removed from the shortest polynucleotide leading to a complete loss of its ability to bind both the coat protein and a ribosome.Some principles of the functioning of the regulatory region in MS2 RNA as well as the nature of the initiator signal of protein biosynthesis are discussed.     

Interactions of Q beta replicase with Q beta RNA

The interactions of Qβ replicase with Qβ RNA were investigated by treating replicase-Qβ RNA complexes under various conditions with ribonuclease T₁, and by characterizing enzyme-bound RNA fragments recovered by a filter binding technique. Evidence for replicase binding at two internal regions of Qβ RNA was obtained. One region (at about 1250 to 1350 nucleotides from the 5′ end) overlaps with the initiation site for coat protein synthesis; this interaction is thought to be inessential for template activity but rather to be involved in the regulation of protein synthesis. Binding to this site (called the S-site) requires moderate concentrations of salt but no magnesium ions. The other region (at about 2550 to 2870 nucleotides from the 5′ end) is probably essential for template activity; binding to this site (called the M-site) is dependent on the presence of magnesium ions. The nucleotide sequences of the RNA fragments from the two sites were determined and found to have no common features. Under the conditions tested, replicase binding at the 3′ end of Qβ RNA could not be demonstrated, except when initiation of RNA synthesis was allowed to occur in the presence of GTP and host factor. If instead of intact Qβ RNA, a complete RNAase T₁ digest of Qβ RNA was allowed to bind to replicase, oligonucleotides from the S-site and the M-site, and oligonucleotides from a region close to the 3′ end, were found to have the highest affinity to the enzyme.The RNA fragments recovered in highest yield, M-2 and S-3 from the M and S-site, respectively, were isolated on a preparative scale and their enzyme binding properties were studied. In competition assays with random RNA fragments of the same size, selective binding was observed both for the M and the S-site fragment. Partial competition for replicase binding was found if M-2 and S-3 were presented simultaneously to the enzyme. Either fragment, if preincubated with replicase, caused a specific inhibition of initiation of Qβ RNA-directed RNA synthesis, without inhibiting the poly(rC)-directed reaction.The results are discussed in terms of a model of replicase-Qβ RNA recognition. Template specificity is attributed to binding of internal RNA regions to replicase, resulting in a specific spatial orientation of the RNA by which the inherently weak, but essential, interaction at the 3′ end is allowed to occur and to lead to the initiation of RNA synthesis.     

Mechanism of translational coupling between coat protein and replicase genes of RNA bacteriophage MS2.

We have analyzed the molecular mechanism that makes translation of the MS2 replicase cistron dependent on the translation of the upstream coat cistron. Deletion mapping on cloned cDNA of the phage shows that the ribosomal binding site of the replicase cistron is masked by a long distance basepairing to an internal coat cistron region. Removal of the internal coat cistron region leads to uncoupled replicase synthesis. Our results confirm the model as originally proposed by Min Jou et al. (1). Activation of the replicase start is sensitive to the frequency of upstream translation, but never reaches the level of uncoupled replicase synthesis.     

Escherichia coli ribosomes bind to non-initiator sites of Q beta RNA in the absence of formylmethionyl-tRNA.

Escherichia coli ribosomes and Qβ [³²P]RNA were incubated with or without fMet-tRNA under protein initiation conditions, treated with RNase A, and centrifuged through a sucrose density gradient. The sample incubated with fMet-tRNA gave a main radioactivity peak in the 70 S region, which consisted predominantly of coat cistron initiator fragments. After incubation without fMet-tRNA, equal amounts of radioactivity were found in the 70 S and the 30 S regions, but in both peaks almost all of the radioactivity was duo to three RNase A-resistant oligonucleotides, A-G-A-G-G-A-G-G-Up (P-2a), A-G-G-G-G-G-Up (P-15) and G-G-A-A-G-G-A-G-Cp (P-4). These three oligonucleotides are derived from three different RNA regions, none of which is close to a protein initiation site. All three fragments show striking complementarity to the 3′-terminal region of E. coli 16 S RNA. Ribosomes incubated with an RNase A digest of Qβ [³²P]RNA bound almost exclusively oligonucleotide P-2a; treatment with cloacin DF13 cleaved off a complex consisting of a 49-nucleotide long segment of 16 S rRNA and oligonucleotide P-2a. These experiments show that the interaction of 30 S ribosomes with the “Shine-Dalgarno” region preceding the initiator codon of the Qβ coat cistron is insufficient to direct correct placement of the ribosome on the viral RNA, and that an additional contribution from the interaction of fMet-tRNA with the initiator triplet is required for ribosome binding to the initiator region.     

Oligonucleotide Sequence of Replicase Initiation Site in Qβ RNA

THE single stranded RNA genome of bacteriophage Qβ has been variously estimated to consist of from 3,500¹ to 4,500² nucleotides. It contains three known cistrons³, which correspond to three of the four Qβ-specific proteins synthesized in vivo and in vitro^4–6. These are: (1) the gene for the maturation or A protein (molecular weight 41,000 (refs. 4, 5)), (2) that for the major coat protein of the virus (molecular weight 14,000 (ref. 9)) and (3) the gene for the phage-specific subunit of the Qβ replicase (molecular weight 64,000 (ref. 10) or 69,000 (ref. 24)), listed in the probable order^7,8 that they occur on the Qβ RNA. The fourth Qβ-specific protein, A₁ or IIb (molecular weight 36,000 (refs. 4–6, 10)), has recently been shown by Weiner and Weber to have an N-terminal sequence which is identical (for eight amino-acids) to that of the coat protein⁷. Because increased amounts of A₁ appear in virus particles grown in cells containing a UGA suppressor, Weiner and Weber postulate⁷ that this protein is the product of natural read-through at the UGA termination signal of the Qβ coat cistron. Such read-through (involving about 600 nucleotides) could occur entirely within a large “intercistronic” region between the coat and replicase genes, or could involve translation, either in or out of phase, of the replicase cistron. In hopes of distinguishing between these alternatives, I have isolated and examined the nucleotide sequence of the region surrounding the initiator codon of the Qβ replicase gene.     

The binding site for coat protein on bacteriophage Qbeta RNA.

The site of interaction of phage Qbeta coat protein with Qbeta RNA was determined by ribonuclease T1 degradation of complexes of coat protein and [32P]-RNA obtained by codialysis of the components from urea into buffer solutions. The degraded complexes were recovered by filtration through nitrocellulose filters, and bound [32P]RNA fragments were extracted and separated by polyacrylamide gel electrophoresis. Fingerprinting and further sequence analysis established that the three main fragments obtained (chain lengths 88, 71 and 27 nucleotides) all consist of sequences extending from the intercistronic region to the beginning of the replicase cistron. These results suggest that in the replication of Qbeta, as in the case of R17, coat protein acts as a translational repressor by binding to the ribosomal initiation site of the replicase cistron.     

A single UGA codon functions as a natural termination signal in the coliphage q beta coat protein cistron

The coat protein cistron of coliphage Qβ has been shown previously to code for two proteins, both of which are structural components of the mature virion (Weiner and Weber, 1971). The predominant translational product is normally terminated Qβ coat protein, but a second product is also made resulting from inefficient translational termination at the end of the coat protein cistron and subsequent read-through into the intercistronic region. Because the molar fraction of this read-through (or IIb) protein relative to normal coat protein in the viral capsid increases from 2.2% to 7.2% when a UGA suppressor strain is used as host for Q/gb infection, the inefficient termination signal in the Qβ coat cistron must be either a single UGA codon or two UGA codons in tandem.A partial amino acid sequence, which includes the suppressed termination signal, has now been obtained for the IIb protein. This sequence proves that a single UGA codon is used alone as the natural translational termination signal of the coliphage Qβ coat cistron. Evidence is also presented that in both the su^- and su⁺_uga host, the ratio of read-through protein to normally terminated coat protein is 1.5 to threefold higher in vivo than in the purified virus. Thus, in the process of self-assembly, the viral capsid prefers to incorporate normally terminated coat protein rather than the read-through product.     

Function of bacteriophage Qbeta replicase containing an altered subunit IV

In order to elucidate the function of elongation factor Ts in Qβ replicase, enzyme was obtained from a Qβ-infected Escherichia coli mutant HAK88, which carries an altered EFTs² with a thermolabile catalytic activity. HAK88 Qβ replicase was found to be quite unstable at 42 °C. Further studies indicated that the mutant enzyme exhibits temperature sensitivity with regard to GTP binding ability but not with Qβ RNA and poly(C) binding. These results suggest that the function of EFTs in Qβ replicase is closely related to the binding of GTP to the enzyme.A defect in Qβ replicase also appears when it is reconstituted from the Qβ replicase subunit complex I–II and the HAK88 EFTu-EFTs complex. Several lines of evidence obtained by using the reconstituted enzyme suggest strongly that the EFTs function is involved specifically in initiation of RNA synthesis, but not in the elongation reaction.     

The inhibition of nucleic acid-binding proteins by aurintricarboxylic acid

Qβ replicase, $\text{Escherichia coli}$ RNA polymerase, and T7 RNA polymerase are inhibited by low concentrations of the dye aurintricarboxylic acid (ATA). In each case initiation by the enzyme was preferentially inhibited. The elongation of initiated polynucleotide chains by Qβ replicase was insensitive to ATA in the range of concentrations required to inhibit initiation. Treatment of Qβ replicase, RNA polymerase and $\text{lac}$ repressor with ATA prevented enzymemediated binding of the templates to nitrocellulose filters. We propose that the inhibitor combines with the template binding site of these proteins to prevent initiation.     

Binding of mammalian ribosomes to MS2 phage RNA reveals an overlapping gene encoding a lysis function.

The main binding site for mammalian ribosomes on the single-stranded RNA of bacteriophage MS2 is located nine tenths of the way through the coat protein gene. Translation initiated at an AUG triplet in the +1 frame yields a 75 amino acid polypeptide which terminates within the synthetase gene at a UAA codon, also in the +1 frame. Partial amino acid sequence analysis of the product synthesized in relatively large amounts by mammalian ribosomes confirms this assignment of the overlapping cistron. The same protein is made in an E. coli cell-free system, but only in very small amounts. Analysis of the translation products directed by RNA from op3, a UGA nonsense mutant of phage f2, identifies the overlapping cistron as a lysis gene. In this paper we show that the op3 mutation is a C yield U transition occurring in the second codon of the synthetase cistron, which explains the lowered production of phage replicase (as well as lack of lysis) upon op3 infection of nonpermissive cells. We discuss the properties of the overlapping gene in relation to its lysis function, recognition of the lysis initiator region by E. coli versus eucaryotic ribosomes and op3 as a ribosome binding site mutant for the f2 synthetase cistron.     

Regulation of RNA replication in RNA-containing bacteriphages. RNA synthesis in coat protein polar mutants]

The synthesis of RNA by polar coat protein mutants f2sus3 and Qbetaam12 under suppressor (Escherichia coli S26R1E, Su+-1; H12R8a Su+-3) and non-suppressor (E. coli AB259; S26) conditions was examined. It was demonstrated that the synthesis of viral RNA under non-suppressor conditions in the presence of rifamycin produced the same gaussian pattern of rates as the synthesis of RNA by wild type phage or non-polar coat protein mutants. However, the total amount of RNA was decreased approximately 10-fold and the peak of RNA synthesis was displaced 7--10 min later. The number of infective centers was reduced also 10-fold indicating that a certain time-lapse was required to overcome the polarity of the parental RNA, this process being of single occurrence, exclusively on the parental RNA, but not on the progeny strains. As a consequence, it was concluded that the initiation of translation at the replicase cistron starts on the nascent RNA chains within the replicative complexes and not on the fully-synthesized templates with their complete secondary structure. The data obtained are not in contradiction with the hypothesis concerning the role of the repressor complex II (replicase-RNA) to slow down the synthesis of replicase and RNA in the coat protein mutants. The polarity can not be responsible probably for the blocking of the replicase cistron on the nascent chain following the block of coat protein cistron. Therefore, it appears appropriate to assume the existence of two binding sites for the replicase as repressor which is in keeping with the conclusions of Weissmann and co-workers.     

The regulatory region of MS2 phage RNA replicase cistron. III. Characterization of fragments resulting from S1 nuclease digestion.

The functionally active fragments MS2 R(-53 leads to 6) and MS2 R(-53 leads to 3) comprising the regulatory region for the replicase cistron have been isolated from MS2 RNA-coat protein complex following T1 RNase digestion. In order to obtain shorter fragments, active in coat protein binding and initiation of translation, MS2 R(-53 leads to 6) was cleaved with S1 nuclease. The results indicate that S1 nuclease attacks the most susceptible loop regions of the two hairpin helices of MSZ R(-53) leads to 6). Among the three fragments which have been isolated, only MS2 R(-35/33 leads to 6) containing the intact hairpin (b) region with initiation codon AUG is active in the coat protein binding. Functional activity exerted by another polynucleotide MS R(-17 leads to 6) supports the assumption that specific binding with the coat protein is determined by the hairpin (b) region prior to the replicase cistron.     

Translational control by a long range RNA-RNA interaction; a basepair substitution analysis.

One of the two mechanisms that regulate expression of the replicase cistron in the single stranded RNA coliphages is translational coupling. This mechanism prevents ribosomes from binding at the start of the replicase cistron unless the upstream coat cistron is being translated. Genetic analysis had identified a maximal region of 132 nucleotides in the coat gene over which ribosomes should pass to activate the replicase start. Subsequent deletion studies in our laboratory had further narrowed down the regulatory region to 12 nucleotides. Here, we identify a long-distance RNA-RNA interaction of 6 base pairs as the basis of the translational polarity. The 3' side of the complementarity region is located in the coat-replicase intercistronic region, some 20 nucleotides before the start codon of the replicase. The 5' side encodes amino acids 31 and 32 of the coat protein. Mutations that disrupt the long-range interaction abolish the translational coupling. Repair of basepairing by second site base substitutions restores translational coupling.     

A polarity gradient in the expression of the replicase gene of RNA bacteriophage Q beta

Nine mutants of bacteriophage Qβ were studied, each having an amber mutation in the coat protein gene. The N-terminal coat protein fragments synthesized in vitro by a non-suppressing Escherichia coli cell extract directed by the mutant RNA's were characterized by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, agarose column gel filtration, and their relative content of certain amino acids. These methods permitted the mutant codon in the coat protein gene to be identified unambiguously; in three cases the amber mutation was at position 17; in five cases, at position 37, and in one case at position 86.Phage-specific uracil incorporation and Qβ replicase activities were measured in infected, non-suppressing cells. Their amounts for each mutant were related to the position of the amber mutation, indicating that across the coat protein gene of Qβ there exists a gradient of polarity for the expression of the replicase gene.     

Crystal Structure of the Bacteriophage Qβ Coat Protein in Complex with the RNA Operator of the Replicase Gene

The coat proteins of single-stranded RNA bacteriophages specifically recognize and bind to a hairpin structure in their genome at the beginning of the replicase gene. The interaction serves to repress the synthesis of the replicase enzyme late in infection and contributes to the specific encapsidation of phage RNA. While this mechanism is conserved throughout the Leviviridae family, the coat protein and operator sequences from different phages show remarkable variation, serving as prime examples for the co-evolution of protein and RNA structure. To better understand the protein–RNA interactions in this virus family, we have determined the three-dimensional structure of the coat protein from bacteriophage Qβ bound to its cognate translational operator. The RNA binding mode of Qβ coat protein shares several features with that of the widely studied phage MS2, but only one nucleotide base in the hairpin loop makes sequence-specific contacts with the protein. Unlike in other RNA phages, the Qβ coat protein does not utilize an adenine-recognition pocket for binding a bulged adenine base in the hairpin stem but instead uses a stacking interaction with a tyrosine side chain to accommodate the base. The extended loop between β strands E and F of Qβ coat protein makes contacts with the lower part of the RNA stem, explaining the greater length dependence of the RNA helix for optimal binding to the protein. Consequently, the complex structure allows the proposal of a mechanism by which the Qβ coat protein recognizes and discriminates in favor of its cognate RNA.     

Analysis of the complete nucleotide sequence of the group IV RNA coliphage SP.

We report the nucleotide sequence of the Group IV RNA bacteriophage SP. The entire sequence is 4276 nucleotides long. Four cistrons have been identified by comparison with the related Group III phage Q beta. The maturation protein contains 449 amino acids, the coat protein contains 131 amino acids, the read-through protein contains 330 amino acids and the replicase beta-subunit contains 575 amino acids. SP is 59 nucleotides longer than Q beta. We have analyzed both sequence and structural conservation between SP and Q beta and shown that the sequences for the coat and central region of the replicase are strongly conserved between the two genomes. We also show that the S and M replicase binding sites of Q beta are strongly conserved in SP. Interestingly, the base composition of SP and Q beta differ significantly from one another, and most of the differences can be accounted for by a strong preponderance of U in the third position of each codon of Q beta relative to SP. We also compare conserved hairpins associated with potential coat protein and replicase binding sites.     

Translation of bacteriophage R17 and Qbeta RNA in a mammalian cell-free system

The polycistronic RNAs from both bacteriophage R17 and Qβ are translated in a mammalian cell-free system of purified and partially purified components. The requirement of one of the partially purified initiation factors (IF-E₃ from rabbit reticulocytes) for the phage RNA translation is strikingly different from that for rabbit globin messenger RNA translation. The phage RNA-directed products are characterized by acrylamide gel electrophoresis and compared with those synthesized in an Escherichia coli cell-free system. There is good agreement between the respective coat proteins and the presumptive synthetase proteins. R17 RNA directs the synthesis of two additional defined polypeptides. However, their possible relationship with the A-protein cistron has not yet been investigated. The RNA from the amB2 mutant of R17, which carries an amber triplet at position 6 in the coat protein cistron, directs the synthesis of the same polypeptides as the wild-type RNA with the exception of the coat protein which is completely abolished. This identifies the product made with wild-type RNA as coat protein and provides a direct in vitro assay for the suppression of nonsense mutations in eukaryotic cells.     

The regulatory region of MS2 phage RNA replicase cistron. IV. Functional activity of specific MS2 RNA fragments in formation of the 70 S initiation complex of protein biosynthesis.

The initiation region of the MS2 replicase cistron can be isolated as a fragment 59 bases in length protected from RNAase by the binding of the coat protein which serves as a translational repressor. This fragment MS2 R(-53 leads to 6) starts 53 bases before the initiation codon and retains full activity in binding ribosomes. We have investigated the functional activity in initiation of a series of fragments from this region variously shortened from the 5'-end. Ribosome protected fragments starting 17 or 21 bases before the AUG are unable to rebind to ribosomes. The shortest fragment which has this activity was produced by partial S1 nuclease digestion and starts 33 to 35 bases before the AUG. The initiation signal comprises some nucleotides between 21 and 33 bases before the initiation codon and the regulatory region responsible for initiation is longer than that protected by the ribosome in the final initiation complex.