X-ray analysis does not provide quantitative estimates of the relative importance of the molecular contacts it reveals or of the relative contributions of specific and nonspecific interactions to the total affinity of specific DNA to enzymes. Stepwise increase of DNA ligand complexity has been used to estimate the relative contributions of virtually every nucleotide unit of 8-oxoguanine-containing DNA to its total affinity for Escherichia coli 8-oxoguanine DNA glycosylase (Fpg protein). Fpg protein can interact with up to 13 nucleotide units or base pairs of single- and double-stranded ribo- and deoxyribo-oligonucleotides of different lengths and sequences through weak additive contacts with their internucleotide phosphate groups. Bindings of both single-stranded and double-stranded oligonucleotides follow similar algorithms, with additive contributions to the free energy of binding of the structural components (phosphate, sugar, and base). Thermodynamic models are provided for both specific and nonspecific DNA sequences with Fpg protein. Fpg protein interacts nonspecifically with virtually all of the base-pair units within its DNA-binding cleft: this provides approximately 7 orders of magnitude of affinity (Delta G degrees approximately equal to -9.8 kcal/mol) for DNA. In contrast, the relative contribution of the 8-oxoguanine unit of the substrate (Delta G degrees approximately equal to -0.90 kcal/mol) together with other specific interactions is <2 orders of magnitude (Delta G degrees approximately equal to -2.8 kcal/mol). Michaelis complex formation of Fpg protein with DNA containing 8-oxoguanine cannot of itself provide the major part of the enzyme specificity, which lies in the k(cat) term; the rate is increased by 6-8 orders of magnitude on going from nonspecific to specific oligodeoxynucleotides.
Affinity labeling of human placental 80S ribosomes with mRNA analogs of up to 12 uridyl residues, i.e. alkylating derivatives of oligouridylates bearing either 4-(N-2-chloroethyl-N-methylamino)benzylmethylphosphamide group at the 5'-termini or 2',3'-O-[4-(N-2-chloroethyl-N-methylamino)]benzylidene residue attached to the 3'-termini, in the presence of cognate Phe-tRNA(Phe) has been investigated. All the mRNA analogs modified only the 40S subunit. The fraction of 18S rRNA modified by the mRNA analogs with the alkylating group at the 5'-end decreased dramatically with extension of the reagent oligouridylate moiety. Nucleotides of 18S rRNA alkylated with the mRNA analogs were determined using a reverse transcription technique. For the mRNA analogs with the alkylating groups at the 3'-termini, G1702 and G1763/G1764 were identified as the cross-linking sites. The intensities of the bands corresponding to reverse transcriptase stops depended on the length of the reagent oligouridylate moieties. Cross-linking sites of the mRNA analogs with the alkylating group at the 5'-termini on 18S rRNA were A1023, C1026, C1057 and A1058 for the (pU)3 and (pU)4 derivatives and a single nucleotide C1057 for the (pU)6 one. Ribosomal protein S26 was found as the main target of modification with the same derivatives of (pU)6 and (pU)12.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.