RESUMEN
Ribosomal RNA from all organisms contains post-transcriptionally modified nucleotides whose function is far from clear. To gain insight into the molecular interactions of modified nucleotides, we investigated the modification status of Thermus thermophilus 5 S and 23 S ribosomal RNA by mass spectrometry and chemical derivatization/primer extension. A total of eleven modified nucleotides was found in 23 S rRNA, of which eight were singly methylated nucleotides and three were pseudouridines. These modified nucleotides were mapped into the published three-dimensional ribosome structure. Seven of the modified nucleotides located to domain IV, and four modified nucleotides located to domain V of the 23 S rRNA. All posttranscriptionally modified nucleotides map in the center of the ribosome, and none of them are in contact with ribosomal proteins. All except one of the modified nucleotides were found in secondary structure elements of the 23 S ribosomal RNA that contact either 16 S ribosomal RNA or transfer RNA, with five of these nucleotides physically involved in intermolecular RNA-RNA bridges. These findings strongly suggest that the post-transcriptional modifications play a role in modulating intermolecular RNA-RNA contacts, which is the first suggestion on a specific function of endogenous ribosomal RNA modifications.
Asunto(s)
Procesamiento Postranscripcional del ARN/fisiología , ARN Ribosómico 23S/metabolismo , Thermus thermophilus/genética , Sitios de Unión , Espectrometría de Masas , Nucleótidos/análisis , Etiquetado in Situ Primed , ARN Ribosómico 16S/metabolismo , ARN de Transferencia/metabolismoRESUMEN
Human serum amyloid P component (SAP) was expressed in the methylotrophic yeast Pichia pastoris. SAP cDNA was placed under control of regulatory sequences derived from the alcohol oxidase gene (AOX1), and its protein product was secreted using the Saccharomyces cerevisiae alpha-mating factor signal sequence. Recombinant SAP (r-SAP) was produced in a bioreactor with computer controlled fed-batch mode and purified by use of a C-terminal histidine tag. The yield of purified r-SAP was 3-4mg from 1L supernatant and 5-6mg from 1L cell paste, indicating that the majority of the produced SAP was not secreted. Treatment of the cell paste with EDTA increased the yield further by about 30%. The N-terminal of r-SAP purified from the supernatant showed non-complete cleavage of the alpha-mating factor signal sequence. Purified r-SAP, analyzed under native conditions, was shown to be a decamer, like purified human SAP (h-SAP), with monomers of 27kDa. Each monomer had one N-glycosylation site, positioned at the same site as for h-SAP. r-SAP bound to antibodies produced against h-SAP. Furthermore, r-SAP bound to ds DNA and influenza A virus subunits in a Ca(2+)-dependent manner and inhibited influenza A virus hemagglutination. These results indicate that r-SAP produced in P. pastoris has the same biological activity as purified h-SAP.
Asunto(s)
Componente Amiloide P Sérico/genética , Secuencia de Aminoácidos , Secuencia de Bases , Cartilla de ADN , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Humanos , Datos de Secuencia Molecular , Pichia/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Componente Amiloide P Sérico/química , Componente Amiloide P Sérico/aislamiento & purificaciónRESUMEN
Finding rate constants from experimental data is often difficult because of offset and noise. A computer program was developed to average experimental data points, reducing the effect of noise, and to produce a loge of slope plot--a plot of the natural logarithm of the slope of a curve--eliminating the effect of any offset. If y-values depend exponentially on x-values the loge of slope plot is rectilinear and the slope is equal to the first order rate constant. Therefore the loge of slope plot provides easy identification of exponential sections of any experimental or calculated data, corresponding rate constants, and small changes in the rate constant as exemplified by analysis of titrant added to a batch culture of Aspergillus niger. The loge of slope plot was easily applicable and superior to conventional methods of analysis of exponential decreasing or increasing data.