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Phage display is a laboratory technique for the study of protein—protein , protein — peptide , and protein— DNA interactions that uses bacteriophages viruses that infect bacteria to connect proteins with the genetic information that encodes them. These displaying phages can then be screened against other proteins, peptides or DNA sequences, in order to detect interaction between the displayed protein and those other molecules. In this way, large libraries of proteins can be screened and amplified in a process called in vitro selection, which is analogous to natural selection. Phage display was first described by George P. Smith in , when he demonstrated the display of peptides on filamentous phage long, thin viruses that infect bacteria by fusing the virus's capsid protein to one peptide out of a collection of peptide sequences. In , Stephen Parmley and George Smith described biopanning for affinity selection and demonstrated that recursive rounds of selection could enrich for clones present at 1 in a billion or less. Restriction Site Mapping Of Λ Phage Dna.

The authors wish it to be known that, in their opinion, the first five authors should be regarded as Joint First Authors.

The genome packaging motor of tailed bacteriophages and herpesviruses is a powerful nanomachine built by several copies of a large TerL and a small TerS terminase subunit. The motor assembles transiently at the portal vertex of an empty precursor capsid or procapsid to power genome encapsidation.

Terminase subunits have been studied in-depth, especially in classical bacteriophages that infect Escherichia coli or Salmonellayet, less is known about the packaging motor of Pseudomonas -phages that have increasing biomedical relevance. Here, we investigated the small terminase subunit from three Podoviridae phages that infect Pseudomonas aeruginosa. We found TerS is polymorphic in solution but assembles into a nonamer in its high-affinity heparin-binding conformation.

X-ray scattering and molecular modeling Resyriction TerS adopts an open conformation in solution, characterized by dynamic HTHs that move around an oligomerization core, generating discrete binding crevices for DNA.

INTRODUCTION

We propose a model for sequence-specific recognition of packaging initiation Dns by lateral interdigitation of DNA. The packaging motor is formed by several copies of two non-structural proteins, TerL and TerS, which assemble onto a unique vertex of procapsid occupied by the dodecameric portal protein. At this vertex, the portal protein replaces a single penton, forming a channel for the passage of DNA, as well as a sensor for genome-packaging 5—7 and an anchoring site for the terminase complex.

In certain phages, small nuclease-associated proteins called HNH-proteins facilitate the packaging reaction, possibly by interacting with TerL 8. Terminase subunits play a vital role in the life cycle of bacteriophages and herpesviruses. In contrast, TerS is a DNA recognition subunit that binds packaging initiation sites referred to as pac or cos in preparation for genome packaging TerS also stimulates the ATPase activity of TerL 21—23while repressing the large terminase nuclease activity 24 Despite decades of research, Resfriction mechanisms of TerS binding to DNA and its role in motor initiation are not fully understood.

The substrate for genome packaging is a concatemer DNA molecule that consists of multiple genome units covalently linked together. Terminase subunits use different packaging strategies to process concatemeric DNA and insert a single genome inside a procapsid. A major difference exists between Reztriction that use a cos versus a pac sequence As a result, cos packagers package accurately one genome unit at a time, without terminal duplications.

In contrast, the pac sequence is found in viruses that use the head-full packaging mechanism, among which P22 is perhaps the best-characterized example In these phages, which typically also result in generalized transduction, the pac site is the recognition site for TerS. The termination cut is also the start of the packaging for the next chromosome along the concatemer, which results in viral chromosomes that have a terminal redundancy and individual chromosomes that are circular permutations of the unique viral sequence. Also, in SPP1, the pac site is Phate Restriction Site Mapping Of Λ Phage Dna be used only once every four packaging events These phages typically lack terminase sequence-specificity, and their Article source usually associate weakly with DNA in vitro.

This effect was inhibited by distamycin, a minor groove binder that causes local distortion of the minor groove of DNA In T4, TerS is dispensable for packaging in a defined packaging assay carried out with non-physiological concentrations of purified T4 components 40 Also, the physical association of terminase subunits, and whether TerL and TerS remain assembled into a complex during genome-packaging, is poorly understood. In many phages, the two subunits fail to interact stably in solution, with two noticeable exceptions. In P22, the terminase complex can be isolated from Salmonella infected cells 43 or formed in Escherichia coli Mappping and purified to homogeneity.

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Thus, although the general architecture of terminase subunits is conserved in the virosphere, the specific mechanisms by which TerS and TerL interact to promote genome-packaging may have diverged significantly in different phages. PaP3 TerL, previously named p03, shares the classical domain signature of large viral terminases, with an N-terminal ATPase and a C-terminal nuclease domain In this paper, we present the first three-dimensional structure of a Restriction Site Mapping Of Λ Phage Dna -phage TerS that we studied using hybrid structural methods.

All plasmids were sequenced to confirm the fidelity of the DNA sequence. Heparin-chromatography was omitted during the purification of Ala-mutants because of the decreased binding of these mutants to More info. PaP3 TerL M. TerL was purified by metal affinity chromatography on Ni-beads Genscript. Eluted fractions containing TerL were concentrated using a 30 kDa Millipore concentrator. PaP3 TerS from peak 2 was used in both assays. The intensity of the bands was quantified using ImageJ The fraction of TerS bound to DNA was calculated by dividing the total intensity here all bands in each lane by that of the bands representing protein—DNA complexes.]