Dissertation (Metadaten)
Titel:Untersuchungen zur Regulation der Transkription bei Methanosarcina mazei
Autor:Jens Thomsen
Fakultät:Mathematisch-Naturwissenschaftliche Fakultät
DDC Sachgebiet:570 Biowissenschaften, Biologie
Datum der mdl. Prüfung:17.02.2004
Referent(in):Prof. Dr. M. Thomm
Korreferent(en) Korreferentin:Prof. Dr. P. Schönheit
Beschreibung (original):(Keine Zusammenfassung in deutscher Sprache vorhanden.) (No summary in German language.)
(übersetzt):To investigate the Methanosarcina mazei regulation of transcription, an in vitro transcription system with purified Methanococcus thermolithotrophicus RNA polymerase and recombinant TBP and TFB from Methanosarcina mazei was established. First, the tfb gene was identified in a genomic lambda-genebank of Methanosarcina mazei S-6, then the gene was sequenced and cloned. Analysis of TBP and TFB amino acid sequences revealed characteristic archaeal features for these proteins. Then TBP and TFB were expressed in E. coli and purified by means of column-chromatography. The functional activity of recombinant TBP and TFB was demonstrated in the in vitro transcription system of Methanococcus thermolithotrophicus. The purified RNA polymerase from Methanosarcina mazei showed no specific activity, therefore the RNA polymerase from Methanococcus thermolithotrophicus was purified for the use in the cell-free transcription system. Analysis of in vitro transcription of the Methanosarcina mazei heat shock genes grpE, dnaK, and dnaJ, which have the in vivo organization 5’-grpE-dnaK-dnaJ-3’, showed only transcription of grpE. The in vitro transcriptional start site of grpE, determined by means of primer extension, is identical with the in vivo transcriptional start site. In transcription of the dnaK gene there is only a weak transcript detectable, which is possibly an unspecific one, and there is no transcription of dnaJ. In vivo there are hints for a monocistronic mode of transcription for these genes, so probably activation for transcription of dnaK and dnaJ is required, which is also suggested by the weakly conserved TATA-box of these genes. A noticeable fivefold direct repeat upstream of dnaK had no influence on in vitro transcription. The gene of the archaeal transcription factor TFE was also cloned, afterwards the protein was expressed in E. coli and purified by means of column-chromatography. Analysis of the amino acid sequence revealed the typical characteristics of archaeal homologues. The functional activity of the recombinant protein was demonstrated in the established cell-free transcription system. It was shown that there is a strong stimulation of transcription when sub-optimal TBP concentrations were used, whereas at optimal TBP concentration only a weak stimulatory effect is detectable. It was not possible to show a clear specific in vitro transcription inhibition for the glnK gene from Methanosarcina mazei by the potential repressor AmtR, because of the inhibitory effect of the protein on transcription of other genes as well, which is probably caused by an unspecific DNA affinity. Nevertheless the inhibitory effect on transcription of glnK is stronger, and it was shown that there is probably a binding of AmtR to a potential binding site 495 bp upstream of the transcriptional start site of the glnK gene. Preincubation of AmtR and TBP/TFB, respectively, with promoter DNA of glnK, followed by in vitro transcription or gel retardation assays showed that there is no specific binding of AmtR to another potential binding site in the TATA-box region. AmtR shifts unaffected by the used DNA the DNA-TBP-TFB complex, and AmtR even can form a DNA-TBP-AmtR complex. So in the presence of DNA there is an interaction between TBP and AmtR, but without DNA, there was no direct interaction detectable. These results indicate a regulatory function of AmtR.
Schlagworte:Methanosarcina mazei ; Genregulation ;
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