Advances in Protein Chemistry最新文献

Escherichia coli possesses two members of the newly discovered class of Y DNA polymerases (Ohmori et al., 2001): Pol IV (dinB) and Pol V (umuD'C). Polymerases that belong to this family are often referred to as specialized or error-prone DNA polymerases to distinguish them from the previously discovered DNA polymerases (Pol I, II, and III) that are essentially involved in DNA replication or error-free DNA repair. Y-family DNA polymerases are characterized by their capacity to replicate DNA, through chemically damaged template bases, or to elongate mismatched primer termini. These properties stem from their capacity to accommodate and use distorted primer templates within their active site and from the lack of an associated exonuclease activity. Even though both belong to the Y-family, Pol IV and Pol V appear to perform distinct physiological functions. Although Pol V is clearly the major lesion bypass polymerase involved in damage-induced mutagenesis, the role of Pol IV remains enigmatic. Indeed, compared to a wild-type strain, a dinB mutant exhibits no clear phenotype with respect to survival or mutagenesis following treatment with DNA-damaging agents. Subtler dinB phenotypes will be discussed below. Moreover, despite the fact that both dinB and umuDC loci are controlled by the SOS response, their constitutive and induced levels of expression are dramatically different. In noninduced cells, Pol V is undetectable by Western analysis. In contrast, it is estimated that there are about 250 copies of Pol IV per cell. On SOS induction, it is believed that only about 15 molecules of Pol V are assembled per cell (S. Sommer, personal communication), whereas Pol IV levels reach approximately 2500 molecules. In fact, despite extensive knowledge of the individual enzymatic properties of all five E. coli DNA polymerases, much more work is needed to understand how their activities are orchestrated within a living cell.

The design of photosynthetic systems reflects the length scales of the fundamental physical processes. Energy transfer is rapid at the few angstrom scale and continues to be rapid even at the 50-A scale of the membrane thickness. Electron tunneling is nearly as rapid at the shortest distances, but becomes physiologically too slow well before 20 A. Diffusion, which starts out at a relatively slow nanosecond time scale, has the most modest slowing with distance and is physiologically competent at all biologically relevant distances. Proton transfer always operates on the shortest angstrom scale. The structural consequences of these distance dependencies are that energy transfer networks can extend over large, multisubunit and multicomplex distances and take leaps of 20 A before entering the domain of charge separating centers. Electron transfer systems are effectively limited to individual distances of 15 A or less and span the 50 A dimensions of the bioenergetic membrane by use of redox chains. Diffusion processes are generally used to cover the intercomplex electron transfer distances of 50 A and greater and tend to compensate for the lack of directionality by restricting the diffusional space to the membrane or the membrane surface, and by multiplying the diffusing species through the use of pools. Proton transfer reactions act over distances larger than a few angstroms through the use of clusters or relays, which sometimes rely on water molecules and which may only be dynamically assembled. Proteins appear to place a premium on robustness of design, which is relatively easily achieved in the long-distance physical processes of energy transfer and electron tunneling. By placing cofactors close enough, the physical process is relatively rapid compared to decay processes. Thus suboptimal conditions such as cofactor orientation, energy level, or redox potential level can be tolerated and generally do not have to be finely tuned. The most fragile regions of design tend to come in areas of complex formation and catalysis involving proton management, where relatively small changes in distance or mutations can lead to a dramatic decrease in turnover, which may already be limiting the overall speed of energy conversion in these proteins. Light-activated systems also face a challenge to robust function from the ever-present dangers of high redox potential chemistry. This can turn the protein matrix and wandering oxygen molecules into unintentional redox partners, which in the case of PSII requires the frequent, costly replacement of protein subunits.

Bacteriophage T7 is a double-stranded DNA bacteriophage that has attracted particular interest in studies of gene expression and regulation and of morphogenesis, as well as in biotechnological applications of expression vectors and phage display. We report here studies of T7 capsid assembly by cryoelectron microscopy and image analysis. T7 follows the canonical pathway of first forming a procapsid that converts into the mature capsid, but with some novel variations. The procapsid is a round particle with an icosahedral triangulation number of 7 levo, composed of regular pentamers and elongated hexamers. A singular vertex in the procapsid is occupied by the connector/portal protein, which forms 12-fold and 13-fold rings when overexpressed, of which the 12-mer appears to be the assembly-competent form. This vertex is the site of two symmetry mismatches: between the connector and the surrounding five gp 10 hexamers; and between the connector and the 8-fold cylindrical core mounted on its inner surface. The scaffolding protein, gp9, which is required for assembly, forms nubbin-like protrusions underlying the hexamers but not the pentamers, with no contacts between neighboring gp9 monomers. We propose that gp9 facilitates assembly by binding to gp10 hexamers, locking them into a morphogenically correct conformation. gp9 is expelled as the procapsid matures into the larger, thinner walled, polyhedral capsid. Several lines of evidence implicate the connector vertex as the site at which the maturation transformation is initiated: in vivo, maturation appears to be triggered by DNA packaging whereby the signal may involve interaction of the connector with DNA. In the mature T7 head, the DNA is organized as a tightly wound coaxial spool, with the DNA coiled around the core in at least four and perhaps as many as six concentric shells.