title: Measurement of the rates of synthesis of three components of ribosomes of Mycobacterium fortuitum: a theoretical approach to qRT-PCR experimentation.
abstract: BACKGROUND
  Except for the ribosomal protein L12 (rplL), ribosomal proteins are present as one copy per ribosome; L12 (rplL) is unusual because it is present as four copies per ribosome. Thus, the strategies used by Mycobacterium fortuitum to regulate ribosomal protein synthesis were investigated, including evaluations of the rates of chain elongations of 16S rRNA, rplL and ribosomal protein S12 (rpsL).
  
  
  METHODS
  RNA was isolated from cell cultures and cDNA was prepared. The numbers of cDNA copies of 16S rRNA, precursor-16S rRNA and transcripts of rpsL and rplL were quantified by qRT-PCR and then related to the rates of 16S rRNA, rpsL and rplL chain elongations by means of a mathematical framework for coupled transcription/translation.
  
  
  RESULTS
  The rates of synthesis of 16S rRNA, rpsL and rplL respectively were found to be approximately 50 x 10(3) nucleotides h(-1), 1.6 x 10(3) amino acid residues h(-1) and 3.4 x 10(3) amino acid residues h(-1). The number of transcripts of rplL was approximately twice that of rpsL. These data account for the presence of one copy of rpsL and four copies of rplL per ribosome, and reveal that the rate of M. fortuitum ribosome synthesis was closer to that of M. tuberculosis than to E. coli. Except for rplJ, the elongation rate obtained for rpsL was inferred to be appropriate for all other proteins present as one copy per ribosome.
  
  
  CONCLUSIONS
  The results obtained provide the basis for a comprehensive view of the kinetics of ribosome synthesis, and of the ways that bacterial cells utilize genes encoding ribosomal proteins. The methodology also applies to proteins involved in transcription, energy generation and to bacterial proteins in general. The method proposed for measuring the fidelity of cDNA preparations is intrinsically much more sensitive than procedures that measure the integrity of 16S rRNA.
authors.affiliation: Departamento de Medicina Preventiva, Facultad de Medicina, Universidad Autónoma, Madrid, Spain.
authors.forename: Maria Jesus
authors.initials: MJ
authors.lastname: Garcia
authors.forename: Maria Carmen
authors.initials: MC
authors.lastname: Nuñez
authors.forename: Robert Ashley
authors.initials: RA
authors.lastname: Cox
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics*; D029968:Escherichia coli Proteins; D008962:Models, Theoretical; D019907:Mycobacterium fortuitum / Q000235:genetics*; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D000091243:Ribosomal Protein S9; D012269:Ribosomal Proteins / Q000235:genetics*; D012270:Ribosomes / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D012336:RNA, Ribosomal, 16S; D000091243:Ribosomal Protein S9; D012269:Ribosomal Proteins; C000717190:RpsI protein, E coli; C034212:ribosomal protein S12