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HflX GTPases are found in all three domains of life, the Bacteria, Archaea, and Eukarya. HflX from Escherichia coli has been shown to bind to the 50S ribosomal subunit in a nucleotide-dependent manner, and this interaction strongly stimulates its GTPase activity. We recently determined the structure of an HflX ortholog from the archaeon Sulfolobus solfataricus (SsoHflX). It revealed the presence of a novel HflX domain that might function in RNA binding and is linked to a canonical G domain. This domain arrangement is common to all archaeal, bacterial, and eukaryotic HflX GTPases. This paper shows that the archaeal SsoHflX, like its bacterial orthologs, binds to the 50S ribosomal subunit. This interaction does not depend on the presence of guanine nucleotides. The HflX domain is sufficient for ribosome interaction. Binding appears to be restricted to free 50S ribosomal subunits and does not occur with 70S ribosomes engaged in translation. The fingerprint ¹H-15N heteronuclear correlation nuclear magnetic resonance (NMR) spectrum of SsoHflX reveals a large number of well-resolved resonances that are broadened upon binding to the 50S ribosomal subunit. The GTPase activity of SsoHflX is stimulated by crude fractions of 50S ribosomal subunits, but this effect is lost with further high-salt purification of the 50S ribosomal subunits, suggesting that the stimulation depends on an extrinsic factor bound to the 50S ribosomal subunit. Our results reveal common properties but also marked differences between archaeal and bacterial HflX proteins. [ABSTRACT FROM AUTHOR]

BtuCD is a member of the ATP-binding cassette transporters in Escherichia coli that imports vitamin B12 ino the cell by utilizing the energy of ATP hydrolysis. Crystal structures of BtuCD and its homologous protein HI1470/1 in various conformational states support the ''alternating access'' mechanism which proposes the conformational transitions of the substrate translocation pathway at transmembrane domain (TMD) between the outward-facing and inward-facing states. The conformational transition at TMD is assumed to couple with the movement of the cytoplasmic nucleotide-binding domains (NBDs) driven by ATP hydrolysis/binding. In this study, we performed targeted molecular dynamics (MD) simulations to explore the atomic details of the conformational transitions of BtuCD importer. The outward-facing to inward-facing (O→I) transition was found to be initiated by the conformational movement of NBDs. The subsequent reorientation of the substrate translocation pathway at TMD began with the closing of the periplasmic gate, followed by the opening of the cytoplamic gate in the last stage of the conformational transition due to the extensive hydrophobic interactions at this region, consistent with the functional requirement of unidirectional transport of the substrates. The reverse inward-facing to outward-facing (I→O) transition was found to exhibit intrinsic diversity of the conformational transition pathways and significant structural asymmetry, suggesting that the asymmetric crystal structure of BtuCD-F is an intermediate state in this process. [ABSTRACT FROM AUTHOR]

Transhydrogenase catalyses the transfer of reducing equivalents between NAD(H) and NADP(H) coupled to the translocation of protons across a membrane. The NAD(H)-binding domain of transhydrogenase (domain I protein) from Rhodospirillum rubrum and from Escherchia coli were overexpressed and purified. Nucleoude binding to the domain I proteins was determined by equilibrium dialysis. NADH and its analogue, acetylpyridine adenine dinucleotide (reduced form), bound with relatively high affinity (Kd = 32 μM and 120 μM). respectively, for the R. rubrum protein). The binding affinity was similar at pH 8.0 and pH 9.0 in zwitterionic buffers and at pH 7.5 in sodium phosphate buffer, NAD+hound with lower affinity (Kd = 300 μM). NADPH bound only very weakly (Kd1> 1 mM). Using a centrifugation procedure, Yamaguchi and Hatefi [Yamaguchi, M & Hatefi, Y. (1993) J. Biol. Chem, 268, 1781-17877 ] found that mitochondrial transhydrogenase, and a proteolytically derrived domain I fragment from that exnzyme, bound one NADH per dimer. They suggested that this result implied half-of-the-sites reactivity for the interaction between the nucleotide ligand and the protein. However our studies on both the E. coli and the R. rubrum recombinant transhydrogenase domain I protein using equilibrum dialysis show that the binding stoichiometry for both NADH and the reduced form of acetylpyridine adenine dinucleotide (AcPdADH) is two nucleotides per dimer: no interaction between the monochondrial transhydrogenases are discussed. [ABSTRACT FROM AUTHOR]

The sequence of the tufA gene from the extreme thermophilic eubacterium Thermus aquaticus EP 00276 was determined. The GC content in third positions of codons is 89.5%, with an unusual predominance of guanosine (60.7%). The derived protein sequence differs from tufA- and tufB- encoded sequences for elongation factor Tu (EF-Tu) of Thermus thermophilus HB8, another member of the genus Thermus, in 10 of the 405 amino acid residues. Three exchanges are located in the additional loop of ten amino acids (182–191 ). The loop, probably involved in nucleotide binding, is absent in EF-Tu of the mesophile Escherichia coli. Since EF-Tu from E. coil is quite unstable, the protein is well-suited for analyzing molecular changes that lead to thermostabilization. Comparison of the EF-Tu domain I from E. coli and Thermus strains revealed clustered amino acid exchanges in the C-terminal part of the first helix and in adjacent residues of the second loop inferred to interact with the ribosome. Most other exchanges in the guanine nucleotide binding domain are located in loops or nearest vicinity of loops suggesting their importance for thermostability. The T. aquaticus EF-Tu was overproduced in E. coil using the tac expression system. Identity of the recombinant T. aquaticus EF-Tu was verified by Western blot analysis, N-terminal sequencing and GDP binding assays. [ABSTRACT FROM AUTHOR]

cAMP receptor protein from Escherichia coli has been purified on a large scale. Analogues of cAMP modified on the 6-NH2 group of the adenosine ring, the ribose 2'OH group or the cyclic phosphate are able to displace cAMP from its binding site with dissociation constants of similar magnitude to that of cAMP. More extensive modification produces weaker binding. Ultraviolet/visible difference spectroscopy and fluorescence spectroscopy show that the environment of the bound adenosine moiety is considerably less polar than that in aqueous solvent, while an anthraniloyl group substituted on the 2'OH position remains accessible to solvent. The 2-NH2 group of cGMP appears to be protonated in the bound form, while no change in the charge state of cAMP is apparent. [ABSTRACT FROM AUTHOR]

The replication of M13 single-stranded DNA by the 9S DNA polymerase α from calf thymus has been studied in vitro. Priming conditions, the nature of the replication products and conditions for optimal elongation have been investigated. Oligonucleotides comprising only four nucleotides can serve as primers. Both ribo and deoxy oligonucleotides can be elongated. Priming by the short oligonucleotides occurs at multiple sites on the M13 genome. If replication is primed at single sites with a specific pentadecamer or with RNA in the origin of replication, specific pausing sites are observed. These pausing sites can partly be correlated with secondary structures in the template DNA. Addition of Escherichia coli single- stranded DNA binding protein leads to a weakening of pausing sites and to the synthesis of longer products. The 9S enzyme is able to proceed through most of the pausing sites resulting in the synthesis of product molecules as long as 6600 nucleotides. The 9S DNA polymerase α contains a potent DNA primase activity which enables it to initiate replication on a single-stranded template in the presence of the four NTPs . However, priming is also possible in the presence of ATP alone. The priming sites are not randomly distributed over the M13 DNA. [ABSTRACT FROM AUTHOR]

Limited digestion of the Escherichia coli 50-S subunit permits the isolation of two large fragments of the 23-S RNA. One arises from the 5' end of the 23-S RNA and contains about 1200 nucleotides. The other arises from the 3' end of the 23-S RNA and contains about 2000 nucleotides. Each of the ten 50-S proteins known to bind specifically and independently to the 23-S RNA were tested for their ability to interact with these two RNA fragments. It was determined that the 5' terminal segment contains the specific binding sites for proteins L4, L20 and L24, whereas the 3' terminal segment contains the specific binding sites for proteins LI, L2, L3, L6, L13, L16 and L23. [ABSTRACT FROM AUTHOR]