ABSTRACT
Trichosanthin is a ribosome-inactivating protein with multiple pharmacological properties. By a yeast two-hybrid system, ribosomal phosphoproteins P0 and P1 and a putative mitotic checkpoint protein, MAD2B, were found to interact with an active-site mutated trichosanthin (TCS). The interactions were verified by an in vitro binding assay of recombinant wild-type TCS and target proteins. The interaction domain of P0 was mapped to amino acids 220-273, which had been previously reported to be involved in the interaction with P1 and P2 in yeast. Consistent with our previous finding that the last seven residues of TCS are not essential for an active conformation, the same deletion did not affect the interaction with P0. Our present study suggests that TCS may disrupt the binding of elongation factors to the P-complex, in addition to the well-known N-glycosidase activity for ribosome inactivation.
Subject(s)
Phosphoproteins/metabolism , Proteins/metabolism , Ribosomal Proteins/metabolism , Trichosanthin/metabolism , Binding Sites , Cell Line , Humans , Mad2 Proteins , Mutagenesis, Site-Directed , Peptide Elongation Factor 2/metabolism , Protein Binding , Structure-Activity Relationship , Trichosanthin/genetics , YeastsABSTRACT
A cytidinyl derivative, N(4)-(6-aminopyridin-2-yl)- 2'-deoxycytidine ((p)C), could interact with a CG base pair to support the triple-helix (triplex) formation of oligodeoxyribonucleotides. Characteristics of (p)C in the formation of both intramolecular triplex, i.e., a "paper clip type" triplex ((P)CT) and intermolecular triplex, i.e., a "linear type" triplex (LT) was monitored by optical methods and isothermal titration calorimetric measurements. Experimental results revealed that the LT with (p)C*CG internally was independent of the solution pH. Only single substitution of (p)C, situated internally but not terminally, facilitated the (P)CT formation by the UV thermal melting study at the neutral pH. However, the best stabilization of the PCT in acidic conditions occurred when (p)C at the end of the triplex rather than internally. In addition, an LT, but not a (P)CT, containing an alternating (p)CT(p)CT(p)C sequence, could be formed in the conditions of 20 mM MgCl(2) and/or 5 mM spermine. Thus, the presence of several nucleotides of (p)C in proximity along the Hoogsteen strand may lead to structural distortion such that the more flexible LT with multiple substitutions is formed in favor of the more rigid PCT.
Subject(s)
Aminopyridines/chemistry , DNA/chemistry , DNA/chemical synthesis , Deoxycytidine/chemistry , Base Composition , Base Pairing , Base Sequence , Calorimetry , Cations , Circular Dichroism , Deoxycytidine/analogs & derivatives , Ethidium/chemistry , Fluorescence , Fluorescent Dyes/chemistry , Hydrogen Bonding , Hydrogen-Ion Concentration , Nucleic Acid Conformation , Nucleic Acid Denaturation , Oligodeoxyribonucleotides/chemistry , Spermine/pharmacology , TemperatureABSTRACT
This paper examines two issues concerning the acquisition of grammatical morphemes: (1) How is the acquisition of grammatical morphemes influenced by prosodic and phonological characteristics of the language being learned? and (2) What sorts of prosodic and phonological properties do grammatical morphemes have that might aid children in applying particular segmentation strategies? To address these issues, we compared the acquisition of grammatical morphemes in a pair of morphosyntactically similar but prosodically different languages, namely Taiwan Mandarin Chinese (TMC) and Taiwanese (TW). We analyse the patterns of realization and omission of a highly frequent subset of grammatical morphemes in six children's speech, recorded between the ages 1:6 and 2:3. The results from the between-language comparisons suggest that rhythmic characteristics of languages can affect segmentation by providing different kinds of prosodic handles for children to grasp at.
