RESUMEN
VemP ( Vibrio protein export monitoring polypeptide) is a secretory protein comprising 159 amino acid residues, which functions as a secretion monitor in Vibrio and regulates expression of the downstream V.secDF2 genes. When VemP export is compromised, its translation specifically undergoes elongation arrest at the position where the Gln156 codon of vemP encounters the P-site in the translating ribosome, resulting in up-regulation of V.SecDF2 production. Although our previous study suggests that many residues in a highly conserved C-terminal 20-residue region of VemP contribute to its elongation arrest, the exact role of each residue remains unclear. Here, we constructed a reporter system to easily and exactly monitor the in vivo arrest efficiency of VemP. Using this reporter system, we systematically performed a mutational analysis of the 20 residues (His138-Phe157) to identify and characterize the arrest motif. Our results show that 15 residues in the conserved region participate in elongation arrest and that multiple interactions between important residues in VemP and in the interior of the exit tunnel contribute to the elongation arrest of VemP. The arrangement of these important residues induced by specific secondary structures in the ribosomal tunnel is critical for the arrest. Pro scanning analysis of the preceding segment (Met120-Phe137) revealed a minor role of this region in the arrest. Considering these results, we conclude that the arrest motif in VemP is mainly composed of the highly conserved multiple residues in the C-terminal region.
Asunto(s)
Proteínas Bacterianas/metabolismo , Modelos Moleculares , Mutación , Terminación de la Cadena Péptídica Traduccional , Ingeniería de Proteínas , Ribosomas/metabolismo , Vibrio/metabolismo , Secuencias de Aminoácidos , Sustitución de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Secuencia Conservada , Eliminación de Gen , Genes Reporteros , Cinética , Operón Lac , Mutagénesis Sitio-Dirigida , Oligopéptidos/genética , Oligopéptidos/metabolismo , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Estabilidad Proteica , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Ribosomas/químicaRESUMEN
Among various Z-form DNA inducers, such as transition metal complexes, polyamines and high ionic concentrations, 8-methylguanine have received attention as efficient chemical modifications. Although it is clear that m8-modified guanine base markedly stabilizes the Z conformation of short oligonucleotides under physiological salt conditions, how sequence composition affects the preference of Z-DNA is still not well established. In this study, various oligomers of d(CG)n or d(GC)n containing either 8-methylguanine in a different position were synthesized and their capacity of stabilizing Z-DNA were evaluated by CD spectra and then compared with each other. It is was found out that the Z-DNA stabilizing effect depend on the order of arrangement of m(8)G and m(8)rG in DNA strands and the center position is the most effective to stabilize the Z-DNA and promote the B to Z transition.