The properties of the guanidine hydrochloride induced unfolding transition of iso-2 cytochrome c (iso-2) from Saccharomyces cerevisiae have been investigated by using kinetic and equilibrium techniques and have been compared with previously published studies of horse cytochrome c, which differs from iso-2 by 46% in amino acid sequence. Measurements of absorbance in the ultraviolet and visible spectral regions as a function of guanidine hydrochloride concentration give superimposable equilibrium transition curves with a midpoint of 1.15 M at pH 7.2 and 20 degrees C. A two-state analysis of the equilibrium data gives a Gibbs free energy of unfolding of 3.1 kcal/mol at 20 degrees C in the absence of denaturant. This agrees well with the predicted difference in stability between S. cerevisiae iso-2 and horse cytochrome c estimated from the free energies of transfer of buried hydrophobic groups. Three kinetic phases associated with folding can be detected throughout most of the transition zone. Two of the phases are detected by stopped-flow mixing experiments. The third phase is over within the mixing time of the flow experiments but is detectable by temperature jumps. At 20 degrees C, pH 7.2, the slowest phase (T1) is in the 20-100-s time range, the middle phase (T2) is in the 0.1-3-s range, and the fastest phase (T3) is on the order of 1 ms. For the reactions observed in the stopped flow (T1 and T2), a simplified three-state mechanism can be used to predict quantitatively the relative amplitudes of the phases and the equilibrium unfolding curve from the observed time constant data. Previously this same mechanism has been successful in describing the folding reactions of horse cytochrome c [Hagerman, P. J. (1977) Biopolymers 16, 731]. We suggest that the qualitative features of protein folding reactions may be conserved among homologous proteins.
The enzyme, Qi3 replicase, responsible for the replication of the RNA of Escherichia coli phage Q0, is composed of four nonidentical subunits, three of which, I, III, and IV, are coded for by the bacterial genome, while subunit II is phage-specific. Subunit IV is shown to be identical to the protein synthesis elongation factor EF Ts by the following criteria: coelectrophoresis on polyacrylamide gels in sodium dodecyl sulfate and in urea buffers, identity of the first seven amino acids at the amino-terminus, precipitation of subunit IV by anti-EF T-factor serum, and stimulation of EF Tu-GDP exchange by subunit IV. Subunit III is shown to be identical to the protein synthesis elongation factor EF Tu by the following criteria: coelectrophoresis on sodium dodecyl sulfate gels, precipitation of EF Tu by anti-QB3 replicase serum, binding of guanine nucleotides, and binding of phenylalanyl-tRNA. In addition, QB3 replicase activity can be reconstituted from subunits I and II with EF Tu and EF Ts.The RNA bacteriophage of Escherichia coli, Qu, induces an enzyme, Q0 replicase, that is responsible for replication of the phage RNA. This enzyme has been extensively characterized and purified. It will copy Q0 RNA, but not the RNA of similar E. coli RNA phages. Early in its purification, it is calpable of copying both phage RNA ("plus strands") and the RNA complement of the phage RNA ("minus strands"). The ability to copy "plus strands," however, is lost upoin further purification. The purified core enzyme can be assayed with either "minus strands" or poly(C) as template. "Plus strand" activity can be restored by addition of hostcoded factors (for a review, see Stavis and August, ref. 1).Kamen (2) and Kondo, Gallerani, and Weissmannii (3) found that the purified core enzyme consists of four nonidentical polypeptide chains of approximate molecular weights 70,000, 65,000, 45,000, and 35,000 (designated 1, 11, III, and IV in the nomenclature of Kameni). Subunit II is coded for by the phage genome, while the other three subunits are present in uninfected E. coli. The replicase of the serologically unrelated RNA phage f2 has been purified by Fedoroff and Zinder (4); it contains three host-coded polypeptides of similar, if not identical, molecular weights to those of Q,3 replicase, in addition to the phage-coded subunit.The four polypeptides of Qfl replicase can be separated into complexes of subunits I + II and subunits III + IV by incubation in a buffer of low salt concentration, followed by sedimentation on a low salt-glycerol gradient. Kamen (2) found that neither fraction alone shows activity ill the poly-(C)-dependent assay, but partial activity could be recovered after the two were mixed together.WTe report here that subunits III and IV of QO replicase are identical with EF Tu and EF Ts, respectively, two elongation factors identified as part of the mechanism of protein biosynthesis by Lucas-Lenard and Lipmanni (5,6 Abbreviations: SDS, sodium dodecyl sulfate; Phe-tRNA, phenylalanyl-charged phenylalanine-tRNA.
Treatment of simian virus 40 (SV40) particles at pH 9.8 in the presence of 1 mM dithiothreitol for 5 min at 370C disrupted the virions into a 60S DNA-protein complex and DNA-free 7S protein particles. The DNA-protein complex contained approximately equal amounts of DNA and protein, and appeared by electron microscopy to be relaxed circular structures with an average of 21 beads joined by short, thin bridges. The major protein components in the complex were
Mutants of simian virus 40 (SV40), with deletions ranging in size from fewer than 3 to 750 base pairs located throughout the SV40 genome, were obtained by infecting CV-1P cells with linear SV40 DNA and DNA of an appropriate helper virus. The linear DNA was obtained by complete cleavage of closed circular DNA with Hae II or BamHI endonuclease or partial cleavage with either Hae III endonuclease or nuclease S1, followed, in some cases, by mild digestion with phage A 5'-exonuclease. The following mutants with deletions in the late region of the SV40 genome were obtained and characterized. Ten, containing deletions at the Hae II endonuclease site (map location 0.83), define a new genetic complementation group, E, grow extremely slowly without helper virus, and cause alterations only in VP2. Two mutants with deletions in the region 0.92 to 0.945 affect both VP2 and VP3, demonstrating that VP3 shares sequences with the C-terminal portion of VP2. The mutant with a deletion at 0.93 is the first deletion mutant in the D complementation group and is also temperature sensitive; the mutant with a deletion at 0.94 is viable and grows normally. Three mutants with deletions at the EcoRI endonuclease site (0/1.0) and eleven with deletions at the BamHI endonuclease site (0.15) fall into the B/C complementation group. Six additional mutants with deletions at the BamHI endonuclease site are viable, growing more slowly than wild type. VP1 is the only polypeptide affected by mutants in the B/C group. A mutant with a deletion of the region 0.72 to 0.80 has a polar effect, failing to express the E, D, and B/C genes. Mutants with deletions in the early region (0.67 counterclockwise to 0.17) at 0.66 to 0.59, 0.48, 0.47, 0.33, and 0.285 to 0.205 are all members oftheA complementation group. Thus, the A gene is the only viral gene in the early region whose expression is necessary for productive infection of permissive cells. Since mutants with deletions in the region 0.59 to 0.54 are viable, two separate regions are essential for expression of the gene A function: 0.66 to 0.59 and 0.54 to 0.21. Mutants with deletions at 0.21 and 0.18 are viable. Approximate map locations of SV40 genes and possible models for their regulation are discussed. Deletions in the simian virus 40 (SV40) genome arise during propagation of the virus at high multiplicities of infection (56). Generally, the deletions are extensive, accompanied by compensating duplications, rearrangements (28, 47) and, occasionally, substitutions by cellular DNA sequences (23, 24). Initially, we intended to use such naturally arising deletion mutants to map the genetic organization of the SV40 chromosome, but the extensive alterations and the consequent multiplicity of genetic
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