Defective lysis of streptomycin-resistant escherichia coli cells infected with bacteriophage f2

A lysis defect was found to account for the failure of a streptomycin-resistant strain of Escherichia coli to form plaques when infected with the male-specific bacteriophage f2. The lysis defect was associated with the mutation to streptomycin resistance. Large amounts of apparently normal bacteriophage accumulated in these cells. Cell-free extracts from both the parental and mutant strains synthesized a potential lysis protein in considerable amounts in response to formaldehyde-treated f2 RNA but not in response to untreated RNA. As predicted from the nucleotide sequence of the analogous MS2 phage, the protein synthesized in vitro had the expected molecular weight and lacked glycine. The cistron for the lysis protein overlapped portions of the coat and replicase cistrons and was translated in the +1 reading frame. Initiation at the lysis protein cistron may be favored by translation errors that expose the normally masked initiation site, and streptomycin-resistant ribosomes, known to have more faithful translation properties, may be unable to efficiently synthesize the lysis protein.     

Transfection: enhancement by assembly protein of bacteriophage R17.

Assembly protein was isolated by DEAE cellulose chromatography from disrupted R17 bacteriophage and reconstituted with purified R17 phage RNA. Following reconstitution, ¹²⁵I labeled assembly protein co-sediments with 27S R17 phage RNA in a sucrose gradient. SDS-polyacrylamide gel analysis of the 27S ¹²⁵I labeled protein-RNA complex confirmed that assembly protein was the only phage protein associated with the RNA. The specific infectivity (PFU/μg RNA) of the R17 phage RNA-assembly protein complex was 35-fold greater than that of R17 phage RNA when assayed on $\text{Escherichia coli}$ spheroplasts. Infectivity of both preparations was destroyed by treatment with pancreatic ribonuclease A. Furthermore, the assembly protein-RNA complex was infectious for intact cells whereas phage RNA was not infectious. Infectivity of this 27S complex for intact cells was totally eliminated by pretreatment with ribonuclease.     

Study of the assembly of vesicular stomatitis virus N protein: role of the P protein

To derive structural information about the vesicular stomatitis virus (VSV) nucleocapsid (N) protein, the N protein and the VSV phosphoprotein (P protein) were expressed together in Escherichia coli. The N and P proteins formed soluble protein complexes of various molar ratios when coexpressed. The major N/P protein complex was composed of 10 molecules of the N protein, 5 molecules of the P protein, and an RNA. A soluble N protein-RNA oligomer free of the P protein was isolated from the N/P protein-RNA complex using conditions of lowered pH. The molecular weight of the N protein-RNA oligomer, 513,879, as determined by analytical ultracentrifugation, showed that it was composed of 10 molecules of the N protein and an RNA of approximately 90 nucleotides. The N protein-RNA oligomer had the appearance of a disk with outer diameter, inner diameter, and thickness of 148 +/- 10 A, 78 +/- 9 A, and 83 +/- 8 A, respectively, as determined by electron microscopy. RNA in the complexes was protected from RNase digestion and was stable at pH 11. This verified that N/P protein complexes expressed in E. coli were competent for encapsidation. In addition to coexpression with the full-length P protein, the N protein was expressed with the C-terminal 72 amino acids of the P protein. This portion of the P protein was sufficient for binding to the N protein, maintaining it in a soluble state, and for assembly of N protein-RNA oligomers. With the results provided in this report, we propose a model for the assembly of an N/P protein-RNA oligomer.     

Membrane damage in abortive infections of colicin Ib-containing Escherichia coli by bacteriophage T5.

Strains of Escherichia coli K-12 containing the colicin Ib (Col Ib) factor did not produce progeny phage when infected by T5 bacteriophage. The cells were killed but did not lyse. If sodium dodecyl sulfate (SDS) was added to T5-infected E. coli (Col Ib), lysis occurred prematurely, but no phage were produced. SDS had no effect on infected cells that did not contain the Col Ib factor or on uninfected cells with or without the Col Ib factor. Cells that contained a mutant Col Ib factor that allowed phage production were not prematurely lysed after infection in the presence of SDS. When the Col Ib-containing cells were infected, protein and RNA synthesis stopped at about 10 min postinfection, and the cells released abnormal amounts of 32P-containing material, ATP, and beta-galactosidase into the medium. They also became inhibited in their ability to accumulate thiomethyl-beta-D-galactopyranoside and to utilize glycerol. Two alternative hypotheses are presented to explain these results.     

Molecular architecture of protein-RNA recognition sites

The molecular architecture of protein-RNA interfaces are analyzed using a non-redundant dataset of 152 protein-RNA complexes. We find that an average protein-RNA interface is smaller than an average protein-DNA interface but larger than an average protein–protein interface. Among the different classes of protein-RNA complexes, interfaces with tRNA are the largest, while the interfaces with the single-stranded RNA are the smallest. Significantly, RNA contributes more to the interface area than its partner protein. Moreover, unlike protein–protein interfaces where the side chain contributes less to the interface area compared to the main chain, the main chain and side chain contributions flipped in protein-RNA interfaces. We find that the protein surface in contact with the RNA in protein-RNA complexes is better packed than that in contact with the DNA in protein-DNA complexes, but loosely packed than that in contact with the protein in protein–protein complexes. Shape complementarity and electrostatic potential are the two major factors that determine the specificity of the protein-RNA interaction. We find that the H-bond density at the protein-RNA interfaces is similar with that of protein-DNA interfaces but higher than the protein–protein interfaces. Unlike protein-DNA interfaces where the deoxyribose has little role in intermolecular H-bonds, due to the presence of an oxygen atom at the 2′ position, the ribose in RNA plays significant role in protein-RNA H-bonds. We find that besides H-bonds, salt bridges and stacking interactions also play significant role in stabilizing protein-nucleic acids interfaces; however, their contribution at the protein–protein interfaces is insignificant.     

Computational analysis of hydrogen bonds in protein-RNA complexes for interaction patterns

Structural analysis of protein-RNA complexes is labor-intensive, yet provides insight into the interaction patterns between a protein and RNA. As the number of protein-RNA complex structures reported has increased substantially in the last few years, a systematic method is required for automatically identifying interaction patterns. This paper presents a computational analysis of the hydrogen bonds in the most representative set of protein-RNA complexes. The analysis revealed several interesting interaction patterns. (1) While residues in the beta-sheets favored unpaired nucleotides, residues in the helices showed no preference and residues in turns favored paired nucleotides. (2) The backbone hydrogen bonds were more dominant than the base hydrogen bonds in the paired nucleotides, but the reverse was observed in the unpaired nucleotides. (3) The protein-RNA complexes contained more paired nucleotides than unpaired nucleotides, but the unpaired nucleotides were observed more frequently interacting with the proteins. And (4) Arg-U, Thr-A, Lys-A, and Asn-U were the most frequently observed pairs. The interaction patterns discovered from the analysis will provide us with useful information in predicting the structure of the RNA binding protein and the structure of the protein binding RNA.     

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.     

Phospholipase activity in bacteriophage-infected Escherichia coli. III. Phopholipase A involvement in lysis of T4-infected cells.

Bacteriophage studies with Escherichia coli K-12 (gamma)DR-DS-, a mutant lacking the major known fatty acyl hydrolases (phospholipases), and its wild-type parent showed equivalent phage infection with regard to phage production and time of phage release. Further examination of the DR-DS- mutant, however, revealed that the progeny bacteriophage were released without complete dissolution of the host cell. Prolonged cell integrity of the infected mutant was noted by spectrophotometry and supported by direct microscope examination. The phage release occurred at normal "lysis" time with phage yields comparable to that of the wild-type bacteria. Inner membrane degradation was indicated by the release of beta-galactosidase, a cytoplasmic enzyme, and of trichloracetic acid-precipitable RNA. Thus, outer membrane degradation is required for dissolution of phage-infected cells, and this degradation is at least partly dependent on activation of host phospholipases.     

Discovering the interaction propensities of amino acids and nucleotides from protein-RNA complexes

With the availability of many genome sequences, the mining of biological data is attracting much attention, most of it limited to the sequences of macromolecules. Sequence data are easy to analyze as they can be treated as strings of characters, whereas the structure of a macromolecule is much more complex. We developed a set of algorithms to analyze the structures of protein-RNA complexes at the atomic level and used them to analyze protein-RNA interactions using structural data on 51 protein-RNA complexes. The analysis revealed, among other things, that: (1) polar and charged amino acids have a strong tendency to interact with nucleotides, (2) arginine and asparagine tend to hydrogen bond with uracil, and (3) histidine favors uracil in water-mediated bonding with RNA. We analyzed a large set of structural data of protein-RNA complexes involving water-mediated hydrogen bonds as well as direct hydrogen bonds. The interaction patterns discovered from the analysis provide useful information for predicting the structure of RNA that binds proteins, and of proteins that bind RNA.     

Replication of bacteriophage f1: A complex containing gene II protein in gene V mutant-infected bacteria

A dense complex has been isolated from bacteria infected with gene V amber mutant f 1 bacteriophage. The major protein in this complex is the f 1 bacteriophage-specific gene II protein. Other proteins in the complex include the f 1 bacteriophage coat protein and proteins which migrate on sodium dodecyl sulfate/polyacrylamide gel electrophoresis with the f1 bacteriophage-specific gene III, gene IV and X protein. A protein of approximately 20,000 M_r is also present in the complex. Examination of bacteria infected with gene V mutant f1 bacteriophage revealed the complex as a densely staining amorphous body which appears to be associated with the cytoplasmic membrane. Bacteria infected with f1 bacteriophage that contain amber mutations in genes other than gene V do not contain this complex.     

The lysis function of RNA bacteriophage Qbeta is mediated by the maturation (A2) protein

Complete or partial cDNA sequences of the RNA bacteriophage Qbeta were cloned in plasmids under the control of the lambdaP(L) promoter to allow regulated expression in Escherichia coli harbouring the gene for the temperature-sensitive lambdaCI857 repressor. Induction of the complete Qbeta sequence leads to a 100-fold increase in phage production, accompanied by cell lysis. Induction of the 5'-terminal sequence containing the intact maturation protein (A2) cistron also causes cell lysis. Alterations of the A2 cistron, leading to proteins either devoid of approximately 20% of the C-terminal region or of six internal amino acids, abolish the lysis function. Expression of other cistrons in addition to the A2 cistron does not enhance host lysis. Thus, in Qbeta, the A2 protein, in addition to its functions as maturation protein, appears to trigger cell lysis. This contrasts with the situation in the distantly related group I RNA phages such as f2 and MS2 where a small lysis polypeptide is coded for by a region overlapping the end of the coat gene and the beginning of the replicase gene.     

A basic region neighboring the lysine-rich C-terminus of protein SRP19 is required for binding to signal recognition particle RNA.

To identify some of the determinants in the 19-kilodalton protein of signal recognition particle (SRP19) for binding to signal recognition particle RNA, two mutant derivatives of the SRP19 were constructed, lacking 14 and 24 C-terminal amino acids. Polypeptides were transcribed and translated in vitro and tested for their ability to bind to signal recognition particle RNA by retention of protein-RNA complexes on DEAE-Sepharose. Both mutant polypeptides form complexes with the RNA, demonstrating that the 24 C-terminal amino acids, which include a lysine-rich sequence at positions 136-144, are dispensable. A third mutant polypeptide, in which eight additional amino acids were removed by oligonucleotide-directed digestion of the mRNA, was unable to bind. The amino acids in the sequence PKLKTRTQ correspond to positions 113-120; they are suggested to be involved in interaction with signal recognition particle RNA.     

PRI-Modeler: extracting RNA structural elements from PDB files of protein-RNA complexes

A complete understanding of protein and RNA structures and their interactions is important for determining the binding sites in protein-RNA complexes. Computational approaches exist for identifying secondary structural elements in proteins from atomic coordinates. However, similar methods have not been developed for RNA, due in part to the very limited structural data so far available. We have developed a set of algorithms for extracting and visualizing secondary and tertiary structures of RNA and for analyzing protein-RNA complexes. These algorithms have been implemented in a web-based program called PRI-Modeler (protein-RNA interaction modeler). Given one or more protein data bank files of protein-RNA complexes, PRI-Modeler analyzes the conformation of the RNA, calculates the hydrogen bond (H bond) and van der Waals interactions between amino acids and nucleotides, extracts secondary and tertiary RNA structure elements, and identifies the patterns of interactions between the proteins and RNAs. This paper presents PRI-Modeler and its application to the hydrogen bond and van der Waals interactions in the most representative set of protein-RNA complexes. The analysis reveals several interesting interaction patterns at various levels. The information provided by PRI-Modeler should prove useful for determining the binding sites in protein-RNA complexes. PRI-Modeler is accessible at http://wilab.inha.ac.kr/primodeler/, and supplementary materials are available in the analysis results section at http://wilab.inha.ac.kr/primodeler/.     

Template activity of complexes formed between bacteriophage f2 RNA and coat protein.

Formation of complexes between f2 RNA polymerase cistron was partially inhibited, some RNA and coat protein was studied using salt conditions which are optimum for phage protein synthesis. In this ionic environment, coat protein precipitation can be prevented by sulfhydryl group-protecting agents. Complexes formed at different protein-RNA input molar ratios were isolated and tested for template activity in an in vitro protein synthesizing system. Simultaneously, the number of protein molecules bound per RNA strand in such complexes was measured by the membrane (Millipore) filtration technique. Under conditions in which translation of the RNA strands were complexed with six molecules of coat protein, whereas some remained unbound. Strong inhibition of the translation of the RNA polymerase cistron was observed when each of the RNA strands present in the mixture was associated with six molecules of coat protein.     

Dissecting the protein-RNA interface: the role of protein surface shapes and RNA secondary structures in protein-RNA recognition

Protein-RNA interactions are essential for many biological processes. However, the structural mechanisms underlying these interactions are not fully understood. Here, we analyzed the protein surface shape (dented, intermediate or protruded) and the RNA base pairing properties (paired or unpaired nucleotides) at the interfaces of 91 protein-RNA complexes derived from the Protein Data Bank. Dented protein surfaces prefer unpaired nucleotides to paired ones at the interface, and hydrogen bonds frequently occur between the protein backbone and RNA bases. In contrast, protruded protein surfaces do not show such a preference, rather, electrostatic interactions initiate the formation of hydrogen bonds between positively charged amino acids and RNA phosphate groups. Interestingly, in many protein-RNA complexes that interact via an RNA loop, an aspartic acid is favored at the interface. Moreover, in most of these complexes, nucleotide bases in the RNA loop are flipped out and form hydrogen bonds with the protein, which suggests that aspartic acid is important for RNA loop recognition through a base-flipping process. This study provides fundamental insights into the role of the shape of the protein surface and RNA secondary structures in mediating protein-RNA interactions.     

A chemical interference study on the interaction of ribosomal protein L11 from Escherichia coli with RNA molecules containing its binding site from 23S rRNA.

The interaction between ribosomal protein L11 from Escherichia coli and in vitro synthesized RNA containing its binding site from 23S rRNA was characterized by identifying nucleotides that interfered with complex formation when chemically modified by diethylpyrocarbonate or hydrazine. Chemically modified RNA was incubated with L11 under conditions appropriate for specific binding of L11 and the resulting protein-RNA complex was separated from unbound RNA on Mg(2+)-containing polyacrylamide gels. The ability to isolate L11 complexes on such gels was affected by the extent of modification by either reagent. Protein-bound and free RNAs were recovered and treated with aniline to identify their content of modified bases. Exclusion of RNA containing chemically altered bases from L11-associated material occurred for 29 modified nucleotides, located throughout the region corresponding to residues 1055-1105 in 23S rRNA. Ten bases within this region did not reproducibly inhibit binding when modified. Multiple bands of RNA were consistently observed on the nondenaturing gels, suggesting that significant intermolecular RNA-RNA interactions had occurred.