Figure 1.
Scheme for the synthesis of rRNA and ribosomal proteins.
The scheme shows the parameters of of the rRNA and protein components. The synthesis of rRNA is the rate limiting step in the synthesis of ribosomes. The synthesis of rRNA involves transcription of rRNA (rrn) operons as shown in Figure 2; the synthesis of ribosomal proteins is achieved by coupled transcription/translation (see Figure 3). The parameters used are defined in Table 1 and the required equations are listed in Table 2. The cartoon of a bacterium includes the genome and ribosomes that are represented in blue (newly synthesised ribosomes are shown in a lighter blue).
Table 1.
Definitions of variables.
Table 2.
Equations used to evaluate 16S rRNA, rpsL and rplL chain elongation rates (see table 4 and tables S1 and S2).
Figure 2.
Schematic representation of the two rrn operons of M. fortuitum.
Each operon comprises, in the order 5′ to 3′, the genes for 16S rRNA (rrs), 23S rRNA (rrl) and 23S rRNA (rrf). The 5′-end of the operon is presented. Horizontal bars indicate the regions of the transcripts analyzed using qRT-PCR: rrs (mature 16SrRNA) (blue); rrnA operon PCL1 (red) and rrnB operon P1-B (green). The detection of rrnA PCL1 includes all transcripts derived from all the four rrnA promoters (namely, P1 to P3 and PCL1). The rate εrrs nucleotides h−1 of 16S rRNA synthesis was calculated by means of equation (6). The analysis is based on two assumptions; first, that there is one precursor-16S rRNA per RNAP [34]; and secondly, that the synthesis of precursor-16S rRNA is completed before the synthesis of precursor-23S rRNA begins [33].
Table 3.
Evaluation of 16S rRNA chain elongation rates (nucleotides h−1) during exponentially growth of M. fortuitum.
Figure 3.
Scheme for coupled gene transcription/translation.
The diagram represents a snapshot of an ORF of a population-average cell synthesizing protein. The size of the coding region (400 base-pairs) is close to that of rpsL (375 base-pairs) and rplL (393 base-pairs). The transcription start point (tsp), the 5′- and 3′-nontranslated regions and the terminus (ter) of transcription are indicated; RNAPs and ribosomes are not drawn to scale. RNAPs are spaced at one RNAP per 80 base-pairs and ribosomes at one ribosome per 80 nucleotides. The nascent polypeptide chains are shown as curly lines ending in an ‘*’. The instantaneous value of the specific synthesis rate of the encoded protein is the product of the number of ribosomes translating transcripts of the ORF and the polypeptide chain elongation rate (see equation (9)) The numbers of transcripts were measured by qRT-PCR and the numbers of associated proteins were deduced by means of the parameter nR(i)/tr(i), the average number of ribosomes per transcript, which is by given by equation (18). The polypeptide chain elongation rate is given by equation (13). There is an upper limit to the numbers of transcripts and their associated ribosomes per ORF; for the example shown above the limit is five or so transcripts and fifteen or so ribosomes.
Table 4.
Evaluation of r-proteins polypeptide chain elongation rates (amino acids h−1) during exponential growth of M. fortuitum.
Table 5.
Comparison of the average numbers of transcripts of the genes studied per 1,000 copies of 16S rRNA and of the numbers of ribosomes per transcript.
Table 6.
Synthesis of ribosomes of M. fortuitum and comparison with E. coli B/r.
Table 7.
Primers and amplification conditions used in this study for qRT-PCR.