F1 Coat Protein Research Articles

Synthesis of Nonadeca- and Octadecaribonucleotides Using the Solid-Phase Phosphotriester with Tetrahydropyranyl Groups as the 2′-Hydroxyl-Protecting Group

Abstract Nonadeca- and octadecaribonucleotides corresponding to the D-loop of tRNAPhe from yeast and the leader sequence of phage f1 coat protein mRNA were synthesized by the activated phosphotriester method. Coupling yield in the synthesis of oligoribonucleotides depended on the extent of nucleosides loaded on controlled pore glass beads (CPG). N-Acyl-5′-O-dimethoxytrityl-2′-O-tetrahydropyranyl derivatives were used as fully protected ribonucleotide monomer units. The 18mer and 19mer corresponding to the D-loop did not serve as substrates for tRNA (guanosine-2′-)methyltransferase from T. thermophilus, but inhibited methylation of the 5′-half fragment of tRNAPhe. This indicates that both fragments possess some affinity with the enzyme.

Partial Synthesis of Leader Sequence of Phage f1 Coat Protein mRNA

Abstract Tetra to undecaribonucleotides of the leader sequence in messenger RNA(mRNA) of f1 coat protein were synthesized on a polystyrene solid support through simple phosphotriester approach.

Effects of bacteriophage f1 gene III protein on the host cell membrane.

Plasmids which encode bacteriophage f1 coat protein genes VIII and III are responsible for a number of unusual properties suggesting that they have a drastic effect on the bacterial outer membrane. Analysis of several such recombinant plasmids and selection of mutant plasmids unable to cause this effect established that the properties were caused by gene III protein or its amino-terminal fragment.

The orientation of the major coat protein of bacteriophage f1 in the cytoplasmic membrane of Escherichia coli.

The orientation of the major coat (B) protein of the bacteriophage f1, an integral membrane protein in the cytoplasmic membrane of infected Escherichia coli, was examined. Pyridoxal 5'-phosphate and [3H]NaBH4 were used to label the cytoplasmic membrane proteins in spheroplasts and membrane vesicles of E. coli infected with bacteriophage f1. Under the conditions described, tritium incorporation was almost completely dependent on the presence of pyridoxal 5'-phosphate and little if any of the cytoplasmic proteins were labeled when the reaction was applied to intact spheroplasts. The major coat protein was isolated from the cytoplasmic membranes labeled in this manner and the chymotryptic peptides were analyzed for the presence of tritium in the pyridoxamine 5'-phosphate conjugate. When the proteins were labeled in the intact spheroplast, only the NH2-terminal chymotryptic peptide of the coat protein was labeled. If the proteins were labeled during osmotic lysis of the spheroplasts or in isolated vesicles, the chymotryptic peptide containing the COOH terminus of the coat protein as well as the NH2-terminal peptide was labeled. The NH2-terminal peptide was labeled to approximately the same extent as occurred in the intact spheroplast. These results are consistent with the hypothesis that the mature f1 coat protein asymmetrically spans the cytoplasmic membrane of the infected host with its NH2 terminus exposed on the outside and COOH terminus exposed on the cytoplasmic surface.

Processing of alkaline phosphatase precursor to the mature enzyme by an Escherichia coli inner membrane preparation.

An inner membrane preparation co-translationally cleaved both the alkaline phosphatase and bacteriophage f1 coat protein precursors to the mature proteins. Post-translational outer membrane proteolysis of pre-alkaline phosphatase generated a protein smaller than the authentic monomer.

Deuterium nuclear magnetic resonance investigation of the effects of proteins and polypeptides on hydrocarbon chain order in model membrane systems

Deuterium Fourier-transform nuclear magnetic resonance spectra have been obtained of 1-myristoyl 2-(14,14,14-trideutero)myristoyl phosphatidylcholine bilayers at 34.1 MHz by using the quadrupole echo pulse technique. Thereby, we have investigated the effects upon the deuterated dimyristoyl phosphatidylcholine bilayers of the following proteins and polypeptides: gramicidin A, bacteriophage f1 coat protein, beef brain myelin proteolipid apoprotein, cytochrome b(5), and cytochrome c oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1). Above T(c), the transition temperature between the gel and liquid crystal phases, the quadrupole splitting of the deuterium-labeled methyl group is reduced or collapsed in the presence of protein or polypeptide. No evidence has been found for ordered "boundary lipid." Below T(c), the spectra show that the hydrocarbon chains are prevented from crystallizing by the protein (or polypeptide) incorporated in the membrane. Similar disordering effects above T(c) are also seen when an unsaturated lipid, 1-(16,16,16-trideutero)palmitoyl 2-palmitoleyl phosphatidylcholine is complexed with cytochrome oxidase.

Studies on bacteriophage fd DNA: IV. The sequence of messenger RNA for the major coat protein gene

One of the RNA species transcribed in vitro on phage fd replicative form DNA is initiated at a site preceding the major coat protein gene and terminated immediately after this gene. The total sequence of this RNA seecies was determined. The transcript was 369 bases long, and contained the sequence identical to the ribosome-binding site for phage f1 coat protein gene (Pieczenik et al., 1974) at positions 88 to 119 and the sequence for coat protein at positions 175 to 324. The coat protein sequence was immediately followed by two termination codons UGA and UAA. The AUG codon appeared at the fifth and 23rd tripletframe upstream from the codon for the first amino acid (Ala) of coat protein. The latter AUG codon was located in the middle of the ribosome-binding site. The result strongly suggests that coat protein is formed from a precursor containing 23 extra amino acid residues at the N-terminus. The transcript was usually terminated with a sequence of eight U residues. It was also noted that there is a region of strong secondary structure near the 3′-end.

Hexamer of bacteriophage f2 coat protein as a repressor of bacteriophage RNA polymerase synthesis.

Formation of complex I between phage f2 RNA and coat protein, leading to repression of phage RNA polymerase synthesis, depends nonlinearly upon the concentration of the coat protein. Maximum formation of complex I was observed when six molecules of coat protein were bound to one molecule of RNA. RNase digestion of a glutaraldehyde-fixed complex left, as the products, coat protein oligomers. The heaviest, hexamers, predominated in the mixture. It was also shown that, in an ionic environment required for phage protein synthesis, coat protein at a concentration optimum for complex I formation exists in solution as a dimer. The results indicate that the translational repression of the RNA polymerase cistron is due to a cooperative attachment to phage template of three dimers of coat protein, forming a hexameric cluster on an RNA strand.

Proteolytic digestion of the micellar complex of f1 coat protein and deoxycholate

The major coat protein of bacteriophage f1 radioactively labeled with specific amino acids was solubilized with deoxycholate and digested with trypsin or alpha-chymotrypsin. The degree of proteolysis of the coat protein was assayed by gel filtration chromatography of the digest in the presence of deoxycholate. Hydrolysis occurred at residues in the hydrophilic termini of the coat, releasing peptides containing proline, lysine, and phenylalanine. No cleavage occurred at the tyrosine or methionine residues in the hydrophobic core. However, chymotrypsin could cleave somewhat at these residues in the absence of deoxycholate. A model for the topography of the micellar complex of coat protein and deoxycholate is presented in which the hydrophobic sequence of the coat is bound to deoxycholate within a micelle, while the hydrophilic termini of the coat project from the micelle.

Negamycin inhibits termination of protein synthesis directed by phage f2 RNA in vitro

In the presence of negamycin, the amount of ribosome-bound peptides during protein synthesis increased to 2 or 3 times that of controls and the release of completed proteins from ribosomes was severely inhibited. This unusual accumulation of ribosome-bound peptides did not occur if protein synthesis was completely inhibited by simultaneous addition of chloramphenicol. Negamycin counteracted kasugamycin, an inhibitor of initiation, but did not inhibit the puromycin reaction. The major fraction of ribosome-bound peptides accumulated in the presence of negamycin consisted of peptides which were as large or nearly as large as phage f2 coat protein.

F1 Coat protein synthesis and altered phospholipid metabolism in f1 infected Escherichia coli

The phospholipid composition of cytoplasmic membranes prepared from bacteria grown in the presence of [ 32P]phosphate and infected with f1 wild type and f1 amber mutant bacteriophages was determined. Ninety minutes after infection with f1 amber mutants in genes 1, 3, 4, 5, 6, and 7 the percentage of cardiolipin was increased from the level in uninfected bacteria of 5% to about 20–35%, and the percentage of phosphatidylethanolamine was decreased from 70% to about 50–60%. The phospholipid composition of cytoplasmic membranes from bacteria infected with a phage containing an amber mutation in the coat cistron (gene 8) did not differ from that of uninfected bacteria. Although late in infection there were no detectable alterations in phospholipid metabolism in wild type infected bacteria, transient alterations in phospholipid metabolism occurred in these bacteria 10 to 20 min after infection. During this time period, the f1 coat protein was found to be rapidly synthesized but was not being packaged into mature phage and released from bacteria. Both the long-term alterations of phospholipid metabolism found in the amber mutant infected bacteria and the transient alterations found in wild-type infected bacteria resulted from an increase in the rate of synthesis of phosphatidylglycerol and cardiolipin and a decrease in the rate of synthesis of phosphatidylethanolamine. These results are discussed in terms of the relationship between the accumulation of f1 coat protein in infected bacteria and the observed alterations in phospholipid metabolism.

Abortive infection of Escherichia coli with the bacteriophage f1: cytoplasmic membrane proteins and the f1 DNA-gene 5 protein complex.

A partially purified cytoplasmic membrane fraction has been isolated from non-permissive Escherichia coli infected with various amber mutants of the bacteriophage f1. The bacteria were labeled with [14C]lysine 20–40 min after infection and the proteins extracted and analyzed by SDS polyacrylamide gel electrophoresis and radio-autography. The f1 coat protein was present in the membrane of bacteria infected with all f1 amber mutants except gene 8. A complex composed of f1 single-stranded DNA and the f1 gene 5 protein was isolated with the inner membrane fraction. Approximately 300 molecules of this complex were found in bacteria infected with f1 wild type and all f1 amber mutants except those in gene 2 and gene 5. In bacteria infected with f1 wild type, the f1 single-stranded DNA was chased from the complex into phage and the displaced gene 5 protein appeared able to associate with another f1 single-stranded DNA molecule. In contrast, the f1 single stranded DNA-gene 5 protein complex was stable in the amber mutant infected bacteria. Two other proteins associated with the membrane of the amber mutant infected bacteria were tentatively identified as the products of gene 2 and gene 4.

Extent of variation in three related bacteriophage RNA molecules

The isolation and sequence analysis of a series of oligonucleotides from complete ribonuclease T 1 digests of bacteriophage R17 RNA have been reported (Jeppesen, 1971). We have obtained the homologous sets of large T 1-resistant oligonucleotides from the related phages f2 and MS2. As expected from earlier findings (Nichols & Robertson, 1971), each RNA can be uniquely characterized by its ribonuclease T 1 fingerprint. Comparison of nucleotide sequences from the sets of RNA fragments from R17, f2 and MS2 allows an estimate of the over-all degree of variation among the three RNA's (2.8 to 3.4%), and yields additional evidence that non-coding regions show significantly lower variation. R17, f2 and MS2 are apparently not linear descendants, but could have diverged from a common ancestor. Base substitutions involving U and C transitions occur at a higher frequency than would be expected on a random basis. It appears probable that a total of 15 to 20 base replacements in the phage coat protein cistron may accompany the single amino-acid difference between f2 coat protein and that of either R17 or MS2. We conclude that analysis of this sort should prove useful in determining the extent of variation among members of any set of closely related nucleic acid molecules.

Inhibition of replication of ribonucleic acid bacteriophage f2 by superinfection with bacteriophage T4.

Superinfection by phage T4 of cells infected by the ribonucleic acid (RNA) phage f2 results in inhibition of further f2 production. Experiments using rifampin show that the exclusion of f2 requires T4 gene function soon after T4 infection. By using a sensitive new peptide-mapping procedure to identify f2 coat protein in infected cells, we show that synthesis of the f2 coat occurs at a reduced level until 4 min after T4 superinfection and then ceases abruptly. Within 4 min after T4 superinfection, there are also several changes in f2 RNA metabolism, all of which require T4 gene function: preexisting f2 replicative intermediate RNA and f2 single-stranded RNA are degraded to small but still acid-precipitable fragments, and most f2-specific RNA is released from polyribosomes. We favor the hypothesis that T4 induces the synthesis of a specific endoribonuclease which degrades f2 RNA and that the inhibition of f2 protein synthesis may be a consequence of this degradation, rather than a direct effect of T4 upon translation.

Synthesis of the major bacteriophage fl coat protein.

A method which allows one to follow the synthesis of the major f1 coat protein in normal, unirradiated f1-infected cells is reported. The N-terminal tryptic peptide of this protein, labeled with (14)C-lysine, has a negative charge at pH 4.5 and is readily separated from the contaminating peptides of host cell proteins. This technique was used to study several aspects of the synthesis of the major f1 coat protein in infected cells.

Biosynthesis of Phage-specific Initiation Dipeptides: A PURIFIED BIOSYNTHETIC SYSTEM DERIVED FROM ESCHERICHIA COLI

Abstract This report describes the properties of a purified Escherichia coli protein synthetic system which accurately translates the initial codons of the bacteriophage f2 maturation, coat, and RNA synthetase cistrons. In order to assay cistron recognition easily and to accumulate specific intermediates in the translation of a natural message, we have restricted in vitro synthesis to the formation of the initial formyldipeptides corresponding to the three f2 cistrons. This is accomplished by preventing the initial, G factor-requiring translocation reaction through the use of small amounts of monospecific anti-G factor antibody or the antibiotic fusidic acid. The reaction requires initiation-competent ribosomes which are depleted of elongation factors by repeated washings in low ionic strength buffer. Initiation in this system is quite specific. The initial f2 coat dipeptide, N-formylmethionylalanine, is readily converted into the expected coat tripeptide, N-formylmethionylalanylserine, if translocation is permitted. In addition, synthesis of the initial f2 RNA synthetase dipeptide, N-formylmethionylserine, is specifically repressed in the presence of f2 coat protein, indicating that this purified system is sensitive to translational control.

Sequences of RNA fragments from the bacteriophage f2 coat protein cistron which differ from their R17 counterparts

We have isolated two RNA fragments from a partial T 1 ribonuclease digest of bacteriophage f2 RNA and determined their sequences. Both fragments are located in the coat protein cistron of the virus. Comparison of their primary structures with the corresponding sequences previously determined for the closely related bacteriophage R17 reveals four differences out of a total of 100 nucleotides. Three of these occur within the coat protein cistron, including one which accounts for the only amino acid difference between the coat proteins of the two phages; the other is located immediately after the termination triplets of the coat cistron.

Release factor translation of RNA phage terminator codons.

Release factors R1 or R2 stimulate equally well the release of f2 coat protein, suggesting that UAA is the first coat terminator codon. The suppression of a nonsense codon can be regulated in vitro by the concentration of R factor of appropriate codon specificity.

Amino Acid Sequence of the f2 Coat Protein

Abstract The complete amino acid sequence of the f2 coat protein has been determined by enzymatic fragmentation of the tryptic peptides. The sequences within these smaller units were established by a combination of methods such as subtractive Edman degradation, leucine aminopeptidase, carboxypeptidases A and B digestion, and hydrazinolysis. Since the tryptic peptides have already been ordered, the data presented in this paper provide sufficient evidence for the proposed structure of the coat protein.

The Arrangement of the Tryptic Peptides in the Coat Protein of the f2 Bacteriophage

Abstract In separate experiments, involving chymotryptic and peptic digestion of the f2 coat protein, a sufficient number of overlap peptides were isolated to permit us to propose an unambiguous arrangement of the tryptic peptides in the intact coat protein. The assignment of linkages between tryptic peptides was made by comparing the amino acid compositions of chymotryptic and peptic peptides containing lysine and arginine with the compositions of the tryptic peptides. In some cases these assignments were confirmed by digesting the overlap peptides with trypsin and by end group determinations. The order proposed for the tryptic peptides is T11-T8-T4-T3-T7-T9-T5-T2-T10-T6-T1. Two chymotryptic peptides were isolated which were derived from the NH2-terminal region of the protein. The order and amino acid sequence of these peptides were determined, making it possible to establish the sequence of the first 7 residues from the NH2-terminal end of the protein. This sequence is: Ala-Ser-Asn-Phe-Thr-Gln-Phe.