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.

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 doublestranded 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 proliferating cell nuclear antigen (PCNA) 3 is a homotrimeric, ring-shaped protein that forms a closed circle around double-stranded DNA. The protein serves as a processivity factor for the eukaryotic replicative polymerases ␦ and ⑀ by tethering them to the DNA (1). Additionally, PCNA interacts with a large number of replication, repair, and signaling factors to coordinate enzymatic processes at sites of replication and repair (2). This recruitment of nucleic-acid enzymes to a topological clamp around the DNA is a strategy employed in organisms ranging from bacteriophage to humans. The remarkable similarity of the ring-shaped structures of the Escherichia coli and bacteriophage T4 sliding clamps to PCNA underscores the evolutionary success of this molecular approach.PCNA is a homotrimer consisting of 37-kDa subunits, each of which comprises two similar globular domains. The PCNA monomers are arrange in head-to-tail fashion, forming a ring with pseudo 6-fold symmetry. The central channel has a diameter of 34 Å, large enough to accommodate double-stranded DNA (3, 4). PCNA forms stable ring-shaped trimers in solution (5) that need to be opened to load onto DNA (6). The clamp loader, replication factor C (RFC), mediates the assembly of PCNA onto DNA at primer-template junctions (7) or at nicks in the DNA backbone (8) in a process that is dependent on ATP. The PCNA⅐DNA complex is very stable, exhibiting a half-life of tens of minutes (9, 10).Although the interactions of various replication and repair proteins with PCNA are well studied (recently reviewed in Ref.2), the interactions between PCNA and DNA are less well understood. Structural studies reveal that the central channel of the clamp is lined with highly conserved, positively charged residues (3, 4). Mutational analysis indicates that these residues may be more important for PCNA loading onto DNA than for sliding along DNA (11). Molecular dynamics simulations suggest that the positively charged residues interact with the phosphodiester backbone but that these interactions are highly dynamic and are frequently displaced by ions from solution (12).The ring struc...

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.

The ring-shaped helicase of bacteriophage T7 (gp4), the product of gene 4, has basic β-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 β-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.beta hairpin | DNA replication | leading-strand synthesis | oligomerization | sliding helicase T he replisome of bacteriophage T7 can be reconstituted using T7 gp5 (DNA polymerase), Escherichia coli thioredoxin (trx) (processivity factor), T7 gp2.5 (ssDNA-binding protein) and T7 gp4 (helicase-primase) (1). The helicase domain of gp4 places it within the SF4 family of helicases (2). Like other members of this family, gp4 assembles onto ssDNA as a hexamer, an oligomerization that is facilitated by dTTP (3, 4). The inherent dTTPase of gp4 is stimulated approximately 40-fold by ssDNA, a stimulation that is required for its unidirectional translocation on ssDNA (4, 5). However, the molecular mechanism by which ssDNA enhances the hydrolysis of dTTP is not well understood. Although gp4 alone translocates on ssDNA and catalyzes limited unwinding of dsDNA, its major role in replication is manifest when it functions with T7 DNA polymerase within the replisome (1, 6). DNA synthesis activity of gp5/trx provides the driving force to accelerate DNA unwinding by gp4 (7). The association of gp5/trx with gp4 during leading-strand DNA synthesis increases the processivity for the 800 nucleotides observed using ssDNA templates to greater than 17 kb per binding event (1).The crystal structure of the hexameric gp4 shows a 6-fold symmetric ring with a central core of 25-30 Å, dimensions that resemble the size and shape of the gp4 hexameric rings seen in electron micrographs (3,8). Electron microscopy combined with other studies provides strong evidence that the ssDNA passes through the central core that provides the DNA-binding site. It is proposed that ssDNA transfers from one subunit to the adjacent subunit sequentially (8, 9). The crystal structure of gp4 also revealed the presence of three loops (loops I, II, and III) that protrude into the central cavity (8). Earlier mutational data had suggested that residues ar...

To locate its target site on DNA, a transcription factor (TF) must recognize its site amongst millions to billions of alternative sites on DNA. Studies suggested that TFs in order to facilitate their search process alternate between 3D diffusion in solution and 1D diffusion along DNA. The duration of such a search depends on the rate at which a TF slides along DNA and the frequency with which it alternates between 1D and 3D diffusion. We are interested in the 1D searching mechanism of p53, a transcription factor that functions as a tumor suppressor in human cells. We are using single-molecule techniques to observe diffusion of the fluorescently labeled p53 proteins along individual, stretched DNA molecules. In our previous studies, we determined the 1D diffusion coefficient of p53 protein. By measuring the 1D diffusion of the p53 protein as a function of ionic strength, we determined that the p53 protein maintains close contact with the DNA duplex and tracks the helical pitch. Current work involves the characterization of the role of the different protein domains in sliding. The C-terminus of p53 is suggested to be responsible for keeping the protein in contact with DNA by non-specifically interacting with the negatively charged backbone of DNA, while the core domain is suggested to be responsible for specifically binding the target site. We will present single-molecule data on the diffusional mobility along DNA of the C-terminal domain of p53, the p53 lacking its C-terminus, and the core domain of p53.

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