Mapping contacts of the S12–S7 intercistronic region of str operon mRNA with ribosomal protein S7 of E. coli

AbstractIn E. coli, S7 initiates 30S ribosome assembly by binding to 16S rRNA. It also regulates translation of the S12 and S7 cistrons of the ‘streptomycin’ operon transcript by binding to the S12–S7 intercistronic region. Here, we describe the contacts of N-terminally His6-tagged S7 with this region as mapped by UV-induced cross-linking. The cross-links are located at U(−34), U(−35), quite distant from the start codons of the two cistrons. In order to explain the mechanism of translational repression of S12–S7, we consider a possible conformational rearrangement of the intercistronic RNA structure induced by S7 binding

Different duplex/quadruplex junctions determine the properties of anti-thrombin aptamers with mixed folding.

Mixed duplex/quadruplex oligonucleotides have attracted great interest as therapeutic targets as well as effective biomedical aptamers. In the case of thrombin-binding aptamer (TBA), the addition of a duplex motif to the G-quadruplex module improves the aptamer resistance to biodegradation and the affinity for thrombin. In particular, the mixed oligonucleotide RE31 is significantly more effective than TBA in anticoagulation experiments and shows a slower disappearance rate in human plasma and blood. In the crystal structure of the complex with thrombin, RE31 adopts an elongated structure in which the duplex and quadruplex regions are perfectly stacked on top of each other, firmly connected by a well-structured junction. The lock-and-key shape complementarity between the TT loops of the G-quadruplex and the protein exosite I gives rise to the basic interaction that stabilizes the complex. However, our data suggest that the duplex motif may have an active role in determining the greater anti-thrombin activity in biological fluids with respect to TBA. This work gives new information on mixed oligonucleotides and highlights the importance of structural data on duplex/quadruplex junctions, which appear to be varied, unpredictable, and fundamental in determining the aptamer functional properties

Several structural motifs cooperate in determining the highly effective anti-thrombin activity of NU172 aptamer

Despite aptamers are very promising alternative to antibodies, very few of them are under clinical trials or are used as drugs. Among them, NU172 is currently in Phase II as anticoagulant in heart disease treatments. It inhibits thrombin activity much more effectively than TBA, the best-known thrombin binding aptamer. The crystal structure of thrombin-NU172 complex reveals a bimodular duplex/quadruplex architecture for the aptamer, which binds thrombin exosite I through a highly complementary surface involving all three loops of the G-quadruplex module. Although the duplex domain does not interact directly with thrombin, the features of the duplex/quadruplex junction and the solution data on two newly designed NU172 mutants indicate that the duplex moiety is important for the optimization of the protein-ligand interaction and for the inhibition of the enzyme activity. Our work discloses the structural features determining the inhibition of thrombin by NU172 and put the basis for the design of mutants with improved properties

Mapping of the second tetracycline binding site on the ribosomal small subunit of E.coli

Tetracycline blocks stable binding of aminoacyl-tRNA to the bacterial ribosomal A-site. Various tetracycline binding sites have been identified in crystals of the 30S ribosomal small subunit of Thermus thermophilus. Here we describe a direct photo- affinity modification of the ribosomal small subunits of Escherichia coli with 7-[(3)H]-tetracycline. To select for specific interactions, an excess of the 30S subunits over tetracycline has been used. Primer extension analysis of the 16S rRNA revealed two sites of the modifications: C936 and C948. Considering available data on tetracycline interactions with the prokaryotic 30S subunits, including the presented data (E.coli), X-ray data (T.thermophilus) and genetic data (Helicobacter pylori, E.coli), a second high affinity tetracycline binding site is proposed within the 3′-major domain of the 16S rRNA, in addition to the A-site related tetracycline binding site

Reacción de condensación de di-2-piridilmetano con co

Se estudió la reacción de condensación de di-2-piridilmatano con los compuestos carbonílicos: benzaldehido, 2, piridilcarboxaldehido, acetona y di-2-piridilcetona, catalizada por sales de los metales de la primera serie de transición: cobalto, níquel cinc, cobre. El producto de condensación, en todos los casos, corresponde a las especies R2CH-COHR'R", donde R'=Py; R'=H, CH3,Py; R"=CH3, fenil, Py. Los productos fueron aislados en la forma de los respectivos complejos con el ion bivalente del correspondiente metal de transición. La fórmula general es [ML2'(ClO4)2 (solvente)x, en que L es el producto de condensación. En todos los casos se postula una estructura octahédrica. Se corrobora dicha estructura en el caso del complejo de cobre(II) con 1,1-di-(2-piridil)-2-hidroxil-2-metilpropano, mediante difracción de rayos X