Preferential binding of bacteriophage Mu repressor to supercoiled Mu DNA

It was shown, using a relatively simple assay, that Mu repressor, cI, binds specifically to a region which spans the leftmost HindIII cleavage site on the phage genome. This extends the observations of Kwoh and Zipser [Nature (London) 277, 489-491 (1979)], who were able to define a binding region to the left of this site. These results provide support for the idea that the eight blocks of repeated DNA sequences, which also span the HindIII cleavage site, are involved in repressor binding. These results also indicate that cI repressor has a marked preference for supercoiled DNA.     

Use of bacteriophage Mu to isolate deletions in the his-nif region of Klebsiella pneumoniae.

Klebsiella pneumoniae M5a1 is naturally resistant to infection by bacteriophage Mu. Mutants of K. pneumoniae sensitive to Mu infection were isolated and found to support both lytic and lysogenic development of Mu. K. pneumoniae lysogens containing a heat-inducible Mu prophage integrated in his were isolated. Strains carrying deletions extending from his into nif were obtained after heat treatment of these lysogens. Such deletions should be useful for determining the map order and cistronic organization of the nif genes.     

DNA transposition of bacteriophage Mu. A quantitative analysis of target site selection in vitro.

The Mu transpositional DNA recombination machinery selects target sites by assembling a protein-DNA complex that interacts with the target DNA and reacts whenever it locates a favorable sequence composition. Splicing of a transposon into the target generates a 5-bp duplication that reflects the original target site. Preferential usage of different target pentamers was examined with a minimal Mu in vitro system and quantitatively compiled consensus sequences for the most preferred and the least preferred sites were generated. When analyzed as base steps, preferences toward certain steps along the 5-bp target site were detected. We further show that insertion sites can be predicted on the basis of additively calculated base step values. Also surrounding sequences influence the preference of a given pentamer; a symmetrical structural component was revealed, suggesting potential hinges at and around the target site.     

Transduction of bacteriophage Mu by bacteriophage T1.

Phage T1 transduces phage Mu PFU from Mu-lysogenic donor cells to sensitive recipient cells. The efficiency of transduction depends on the chromosomal location of the Mu prophage. T1, therefore, appears to package different regions of the bacterial chromosome with different efficiencies. Although T1 transduces bacterial markers with different efficiencies, there is no direct correlation between the efficiency of transduction of a bacterial marker and the efficiency of transduction of Mu PFU from donor cells with the Mu prophage located in that marker.     

Site-specific recombination in bacteriophage Mu: characterization of binding sites for the DNA invertase Gin.

Site-specific DNA inversion in phage Mu is catalysed by the phage-encoded DNA invertase Gin and a host factor FIS. We demonstrate that purified Gin protein binds specifically to 34-bp sequences that flank the G segment as inverted repeats. Each inverted repeat (IR) contains two binding sites for Gin which have to be arranged in a specific configuration to constitute a recombinogenic site. While one of these sites is bound when present alone, the other site is bound only in conjunction with the first one, suggesting cooperative binding. In addition to the sites within the IR, Gin binds with lower affinity to AT-rich sequences adjacent to the IR. We demonstrate that these sites do not participate in the inversion reaction. The IR itself can be shortened to 25 bp without effect on inversion frequency. Using gel mobility shift experiments on circular permuted fragments containing the IR we show that Gin bends DNA upon binding. We discuss the possibility that DNA bending is related to the formation of a productive synaptic complex.     

Bending of the origin of replication of E. coli by binding of IHF at a specific site

In studies of DNA replication in Escherichia coli, an important question concerns the role of the initiator protein DnaA. This protein is known to bind to a specific 9-bp sequence in the origin of replication, but it is not understood how it can recognize another, relatively distant, 13-bp sequence that has no homology to the binding site but is where the DnaA protein serves its catalytic function in the initiation of DNA replication. This effect of DnaA might be achieved by bending of DNA in this region. I have searched for putative binding sites for integration host factor (IHF), a protein known to bend DNA. Here I report the finding of an IHF binding site in the E. coli origin and present direct evidence that IHF binds and causes DNA bending in this region. On the basis of these results I propose a model wherein formation of a higher-order nucleoprotein structure would facilitate the action of DnaA protein in the initiation events.     

The phiX174-type primosome promotes replisome assembly at the site of recombination in bacteriophage Mu transposition.

Initiation of Escherichia coli DNA synthesis primed by homologous recombination is believed to require the phiX174-type primosome, a mobile priming apparatus assembled without the initiator protein DnaA. We show that this primosome plays an essential role in bacteriophage Mu DNA replication by transposition. Upon promoting transfer of Mu ends to target DNA, the Mu transpososome undergoes transition to a pre-replisome that permits initiation of DNA synthesis only in the presence of primosome assembly proteins PriA, DnaT, DnaB and DnaC. These assembly proteins promote the engagement of primase and DNA polymerase III holoenzyme, initiating semi-discontinuous replication preferentially at the Mu left end. The results indicate that these proteins play a crucial role in promoting replisome assembly on a recombination intermediate.     

The RNA binding site of bacteriophage MS2 coat protein.

The coat protein of the RNA bacteriophage MS2 binds a specific stem-loop structure in viral RNA to accomplish encapsidation of the genome and translational repression of replicase synthesis. In order to identify the structural components of coat protein required for its RNA binding function, a series of repressor-defective mutants has been isolated. To ensure that the repressor defects were due to substitution of binding site residues, the mutant coat proteins were screened for retention of the ability to form virus-like particles. Since virus assembly presumably requires native structure, this approach eliminated mutants whose repressor defects were secondary consequences of protein folding or stability defects. Each of the variant coat proteins was purified and its ability to bind operator RNA in vitro was measured. DNA sequence analysis identified the nucleotide and amino acid substitutions responsible for reduced RNA binding affinity. Localization of the substituted sites in the three-dimensional structure of coat protein reveals that amino acid residues on three adjacent strands of the coat protein beta-sheet are required for translational repression and RNA binding. The sidechains of the affected residues form a contiguous patch on the interior surface of the viral coat.     

Translational regulatory signals within the coding region of the bacteriophage lambda cIII gene.

Six independent mutations which enhance the lysogenic response were analyzed. The mutations cause single-base substitutions at three sites within the cIII coding sequence, one of which does not change the amino acid code. The mutations allow for elevated translation of the cIII gene, possibly via changes in the mRNA secondary structure.     

Properties of bacteriophage T4 thymidylate synthase following mutagenic changes in the active site and folate binding region

Amino acid replacements have been introduced in specific sites of bacteriophage T4 thymidylate synthase (T4-TS) to assess the role that these changes have on enzyme activity. Each of the conserved amino acids in the active-site region of T4-TS was modified, and the effects that these changes had on the kinetic and physical properties of this enzyme were measured. The mutations introduced were Pro-155-Ala (P155A), Cys-156-Ser (C156S), and His-157-Val (H157V) with the resulting synthases possessing kcat's of 10.3, 0.008, and 2.70 s-1, respectively, relative to that of the wild-type enzyme of 11.8 s-1. Equilibrium dialysis was performed on the wild-type and mutant enzymes to determine the binding constants for 2'-deoxyuridylate and 5-fluoro-2'-deoxyuridylate, and while in most cases the extent of binding of these nucleotides to the mutant proteins was reduced when compared with wild-type TS, the number of binding sites involved remained about 1 or less for the binary complex and almost 2 for the ternary complex. Heat and urea stability studies revealed that the mutant with the highest enzyme activity, P155A, was the most unstable, while spectrofluorometric analyses revealed that the structures of P155A and H157V were perturbed relative to the C156S and wild-type TSs. These studies are in agreement with others implicating the phylogenetically conserved active-site cysteine as playing an essential mechanistic role in the catalytic process promoted by TS. The proximal amino acids on either side of this cysteine, although also highly conserved, do not appear to affect the catalytic mechanism directly, but may do so indirectly through their influence on the conformation at the active site as well as other regions of the enzyme. Amino acids replacements were introduced also into the folate and deoxynucleotide 5'-phosphate binding sites of the T4-phage TS to ascertain the potential role that these amino acids play in the catalytic process. These positions were selected on the basis of previous chemical modification and X-ray crystallographic studies on Lactobacillus casei TS. Amino acid residues 48 and 49, which are in the putative folate binding site, were converted from lysines to arginines; in the former case, the mutated enzyme had less than 7% of the wild-type activity while in the latter, the mutated enzyme still retained about 60% of its activity.(ABSTRACT TRUNCATED AT 400 WORDS)     

Identification of the bacteriophage D108 kil gene and of the second region of sequence nonhomology with bacteriophage Mu

To identify the second region of sequence nonhomology between the genomes of the transposable bacteriophages Mu and D108 originally observed by electron-microscopic analysis of DNA heteroduplexes and to localize functions ascribed to the 'accessory' or 'semi-essential' early regions of the phages between genes B and C, a 0.9-kb fragment of each genome located immediately beyond the B gene was cloned and sequenced. Three open reading frames (ORFs) were identified in each. The region of nonhomology is located within the 3' portion of the third ORF. D108 is shown to possess a Kil function similar to that previously shown for Mu, and that function is encoded by the first ORF.     

Analysis of the ends of bacteriophage Mu using site-directed mutagenesis

We showed previously that two regions at the left end (L1 and L3) and one at the right end (R2) of bacteriophage Mu are essential for transposition. These regions all contain a 22 base-pair sequence with the consensus YGtTTCAYtNNAARYRCGAAAR, where Y and R represent any pyrimidine and purine, respectively. The Mu A protein binds to these regions in vitro, and weakly to sequences between nucleotides 1 and 30 of the right end (R1) and between nucleotides 110 and 135 of the left end (L2). These weak A binding sites contain the sequence AARYRCGAAAR. Here we show, using site-directed mutagenesis, that the weak A binding sites are essential for transposition. Mutations in these weak A binding sites have a greater effect on transposition than mutations of corresponding base-pairs in the stronger A binding sites, located adjacent to these weak A binding sites. We confirm the importance of several of the conserved base-pairs in the consensus sequence YGtTTCAYtNNAARYRCGAAAR. The base-pairs in the A binding sites that are shown to be essential for transposition are all conserved in the ends of the related bacteriophage D108. Furthermore, it is shown that the distance of 90 base-pairs between the two regions at the left end (L1 and L2L3) is essential.     

The binding site for coat protein on bacteriophage Qbeta RNA.

The site of interaction of phage Qbeta coat protein with Qbeta RNA was determined by ribonuclease T1 degradation of complexes of coat protein and [32P]-RNA obtained by codialysis of the components from urea into buffer solutions. The degraded complexes were recovered by filtration through nitrocellulose filters, and bound [32P]RNA fragments were extracted and separated by polyacrylamide gel electrophoresis. Fingerprinting and further sequence analysis established that the three main fragments obtained (chain lengths 88, 71 and 27 nucleotides) all consist of sequences extending from the intercistronic region to the beginning of the replicase cistron. These results suggest that in the replication of Qbeta, as in the case of R17, coat protein acts as a translational repressor by binding to the ribosomal initiation site of the replicase cistron.