Escherichia coli and Pseudomonas putida RNA polymerases display identical contacts with promoters

Summary Methylation protection experiments with four promoters (P1 and P2 of the pBR322 plasmid, lacUV5 and lambda P₀) have shown that the RNA polymerases from Escherichia coli and Pseudomonas putida, while differing in the primary structure of the subunits involved in DNA binding, display identical patterns of DNA contacts. Nor do these enzymes differ in covalent cross-linking patterns with a partially apurinized promoter. We conclude that the two RNA polymerases have very similar structures of DNA binding centers. The evolutionary conservation of this structure may account for the fact that diverse RNA polymerases often recognize and efficiently use promoters of distant bacterial species.     

Fibulin-5 binds urokinase-type plasminogen activator and mediates urokinase-stimulated β1-integrin-dependent cell migration

uPA (urokinase-type plasminogen activator) stimulates cell migration through multiple pathways, including formation of plasmin and extracellular metalloproteinases, and binding to the uPAR (uPA receptor; also known as CD87), integrins and LRP1 (low-density lipoprotein receptor-related protein 1) which activate intracellular signalling pathways. In the present paper we report that uPA-mediated cell migration requires an interaction with fibulin-5. uPA stimulates migration of wild-type MEFs (mouse embryonic fibroblasts) (Fbln5+/+ MEFs), but has no effect on fibulin-5-deficient (Fbln5-/-) MEFs. Migration of MEFs in response to uPA requires an interaction of fibulin-5 with integrins, as MEFs expressing a mutant fibulin-5 incapable of binding integrins (Fbln(RGE/RGE) MEFs) do not migrate in response to uPA. Moreover, a blocking anti-(human β1-integrin) antibody inhibited the migration of PASMCs (pulmonary arterial smooth muscle cells) in response to uPA. Binding of uPA to fibulin-5 generates plasmin, which excises the integrin-binding N-terminal cbEGF (Ca2+-binding epidermal growth factor)-like domain, leading to loss of β1-integrin binding. We suggest that uPA promotes cell migration by binding to fibulin-5, initiating its cleavage by plasmin, which leads to its dissociation from β1-integrin and thereby unblocks the capacity of integrin to facilitate cell motility.     

Synthesis of Dioxolane Analogues of Dideoxynucleotides and Their Substrate Properties in DNA Synthesis Reactions

Abstract

Dioxolane derivatives of dNTP were prepared and their substrate properties were investigated in DNA synthesis reactions.     

Stability of cloned promoter-containing fragments

Strong bacteriophages lambda and T7 promoters for Escherichia coli RNA polymerase were cloned in a multicopy plasmid. To achieve this result, two variants of the promoter-probe vectors were constructed. It was found that (i) modifications of the nucleotide sequence, apart from the commonly accepted promoter region, both upstream and downstream of the RNA initiation point greatly influenced the efficiency of promoters in vivo, (ii) a recombinant DNA composed of one of the promoter-probe plasmids and a tandem of A1, A2, and A3 promoters of T7 bacteriophage DNA induced a reproducible secondary change in plasmid DNA upon cloning. This change was substitution of the part of the recombinant that originated as T7 by a large portion of the host DNA.     

2'',3''-Dideoxy-3'' aminonucleoside 5''-triphosphates are the terminators of DNA synthesis catalyzed by DNA polymerases.

It is shown that 2',3'-dideoxy-3'-aminonucleoside 5'-triphosphates with adenine, guanine, cytosine and thymine bases are effective inhibitors of DNA polymerase I, calf thymus DNA polymerase alpha and rat liver DNA polymerase beta. The effect of the above-mentioned compounds is markedly higher than corresponding action of the well-known DNA synthesis inhibitors arabinonucleoside 5'-triphosphates and 2',3'-dideoxynucleoside 5'-triphosphates. 2',3'-dideoxy-3'-aminonucleoside 5'-monophosphate residues incorporate into the 3'-terminus of the primer and terminate the DNA chain elongation. The possibility of using 2',3'-dideoxy-3'-aminonucleoside 5'-triphosphates as terminators for DNA sequencing by the polymerization method is demonstrated.     

Sequence-specific inhibition of RNA elongation by actinomycin D

We have developed a method to localize specific sites where RNA elongation is arrested due to DNA-bound ligands. The method was used to determine apparent binding sites for actinomycin D. We have found 14 strong RNA hindrance sites along nucleotide sequence of T7 and D111 T7 DNA of 380 nucleotides full length under low actinomycin D concentration conditions. Nucleotide sequence of all the sites is described by general formula XGCY where X ‡ G and Y ‡ C.     

Mycoplasma gallisepticum 16S rRNA genes

Abstract The genome of Mycoplasma gallisepticum A5969 contains a truncated pseudogene for 16S rRNA in addition to a single unsplit rRNA-operon and a second discontinuous set of rRNA genes. Other M. gallisepticum strains tested do not posses the truncated gene. This gene is almost identical to full-size isolated 16S rRNA gene starting from at least 500 nucleotides upstream of the coding sequence and ending at the 977th nucleotide within the structural part of 16S rRNA.