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The ring-shaped helicase of bacteriophage T7 (gp4), the product of gene 4, has basic beta-hairpin loops lining its central core where they are postulated to be the major sites of DNA interaction. We have altered multiple residues within the beta-hairpin loop to determine their role during dTTPase-driven DNA unwinding. Residues His-465, Leu-466, and Asn-468 are essential for both DNA unwinding and DNA synthesis mediated by T7 DNA polymerase during leading-strand DNA synthesis. Gp4-K467A, gp4-K471A, and gp4-K473A form fewer hexamers than heptamers compared to wild-type helicase and alone are deficient in DNA unwinding. However, they complement for the growth of T7 bacteriophage lacking gene 4. Single-molecule studies show that these three altered helicases support rates of leading-strand DNA synthesis comparable to that observed with wild-type gp4. Gp4-K467A, devoid of unwinding activity alone, supports leading-strand synthesis in the presence of T7 DNA polymerase. We propose that DNA polymerase limits the backward movement of the helicase during unwinding as well as assisting the forward movement necessary for strand separation.

Proliferating cell nuclear antigen (PCNA) plays an important role in eukaryotic genomic maintenance by topologically binding DNA and recruiting replication and repair proteins. The ring-shaped protein forms a closed circle around double-stranded DNA and is able to move along the DNA in a random walk. The molecular nature of this diffusion process is poorly understood. We use single-molecule imaging to visualize the movement of individual, fluorescently labeled PCNA molecules along stretched DNA. Measurements of diffusional properties as a function of viscosity and protein size suggest that PCNA moves along DNA using two different sliding modes. Most of the time, the clamp moves while rotationally tracking the helical pitch of the DNA duplex. In a less frequently used second mode of diffusion, the movement of the protein is uncoupled from the helical pitch, and the clamp diffuses at much higher rates.

The recent demonstration and utilization of fluorescent proteins whose fluorescence can be switched on and off has greatly expanded the toolkit of molecular and cell biology. These photoswitchable proteins have facilitated the characterization of specifically tagged molecular species in the cell and have enabled fluorescence imaging of intracellular structures with a resolution far below the classical diffraction limit of light. Applications are limited, however, by the fast photobleaching, slow photoswitching, and oligomerization typical for photoswitchable proteins currently available. Here, we report the molecular cloning and spectroscopic characterization of mKikGR, a monomeric version of the previously reported KikGR that displays high photostability and switching rates. Furthermore, we present single-molecule imaging experiments that demonstrate that individual mKikGR proteins can be localized with a precision of better than 10 nanometers, suggesting their suitability for super-resolution imaging.

Peptide bond formation and peptide release are catalyzed in the active site of the large subunit of the ribosome where universally conserved nucleotides surround the CCA ends of the peptidyl- and aminoacyl-tRNA substrates. Here, we describe the use of an affinity-tagging system for the purification of mutant ribosomes and analysis of four universally conserved nucleotides in the innermost layer of the active site: A2451, U2506, U2585, and A2602. While pre-steady-state kinetic analysis of the peptidyl transferase activity of the mutant ribosomes reveals substantially reduced rates of peptide bond formation using the minimal substrate puromycin, their rates of peptide bond formation are unaffected when the substrates are intact aminoacyl-tRNAs. These mutant ribosomes do, however, display substantial defects in peptide release. These results reveal a view of the catalytic center in which an inner shell of conserved nucleotides is pivotal for peptide release, while an outer shell is responsible for promoting peptide bond formation.