TAILIEUCHUNG - Báo cáo khoa học: Characterization of the tRNA and ribosome-dependent pppGpp-synthesis by recombinant stringent factor from Escherichia coli

Stringent factor is a ribosome-dependent ATP:GTP pyrophosphoryl trans-ferase that synthesizes (p)ppGpp upon nutrient deprivation. It is activated by unacylated tRNA in the ribosomal amino-acyl site (A-site) but it is unclear how activation occurs. A His-tagged stringent factor was isolated by affinity-chromatography and precipitation. This procedure yielded a protein of high purity that displayed (a) a low endogenous pyrophosphoryl transferase activity that was inhibited by the antibiotic tetracycline; . | ềFEBS Journal Characterization of the tRNA and ribosome-dependent pppGpp-synthesis by recombinant stringent factor from Escherichia coli Rose-Marie Knutsson Jenvert1 2 and Lovisa Holmberg Schiavone1 1 Cell Biology Unit NaturalScience Section Sodertorns Hogskola Huddinge Sweden 2 Department of Cell Biology Arrhenius Laboratories E5 Stockholm University Sweden Keywords pppGpp RelA ribosome stringent response tRNA Correspondence L. Holmberg Schiavone Cell Biology Unit NaturalScience Section Sodertorns University College S-141 89 Huddinge Sweden Fax 46 8608 4510 Tel 46 8608 4597 E-mail Received 25 August 2004 revised 4 November 2004 accepted 25 November 2004 doi Stringent factor is a ribosome-dependent ATP GTP pyrophosphoryl transferase that synthesizes p ppGpp upon nutrient deprivation. It is activated by unacylated tRNA in the ribosomal amino-acyl site A-site but it is unclear how activation occurs. A His-tagged stringent factor was isolated by affinity-chromatography and precipitation. This procedure yielded a protein of high purity that displayed a a low endogenous pyrophosphoryl transferase activity that was inhibited by the antibiotic tetracycline b a low ribosome-dependent activity that was inhibited by the A-site specific antibiotics thiostrepton micrococcin tetracycline and viomycin c a tRNA- and ribosome-dependent activity amounting to 4500 pmol pppGpp per pmol stringent factor per minute. Footprinting analysis showed that stringent factor interacted with ribosomes that contained tRNAs bound in classical states. Maximal activity was seen when the ribosomal A-site was presaturated with unacylated tRNA. Less tRNA was required to reach maximal activity when stringent factor and unacylated tRNA were added simultaneously to ribosomes suggesting that stringent factor formed a complex with tRNA in solution that had higher affinity for the ribosomal A-site. However tRNA-saturation curves performed at .

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