Post-transcriptional regulation of the steady-state levels of mitochondrial tRNAs in HeLa cells

Abstract

In human mitochondrial DNA (mtDNA), the tRNA genes are located in three different transcription units that are transcribed at three different rates. To analyze the regulation of tRNA formation by the three transcription units, we have examined the steady-state levels and metabolic properties of the tRNAs of HeLa cell mitochondria. DNA excess hybridization experiments utilizing separated strands of mtDNA and purified tRNA samples from exponential cells long term labeled with [32P]orthophosphate have revealed a steady-state level of 6 x 10(5) tRNA molecules/cell, with three-fourths being encoded in the H-strand and one-fourth in the L-strand. Hybridization of the tRNAs with a panel of M13 clones of human mtDNA containing, in most cases, single tRNA genes and a quantitation of two-dimensional electrophoretic fractionations of the tRNAs have shown that the steady-state levels of tRNA(Phe) and tRNA(Val) are two to three times higher than the average level of the other H-strand-encoded tRNAs and three to four times higher than the average level of the L-strand-encoded tRNAs. Similar experiments carried out with tRNAs isolated from cells labeled with very short pulses of [5-3H]uridine have indicated that the rates of formation of the individual tRNA species are proportional to their steady-state amounts. Therefore, the approximately 25-fold higher rate of transcription of the tRNA(Phe) and tRNA(Val) genes relative to the other H-strand tRNA genes and the 10-16-fold higher rate of transcription of the L-strand tRNA genes relative to the H-strand tRNA genes are not reflected in the steady-state levels or the rates of formation of the corresponding tRNAs. A comparison of the steady-state levels of the individual tRNAs with the corresponding codon usage for protein synthesis, as determined from the DNA sequence and the rates of synthesis of the various polypeptides, has not revealed any significant correlation between the two parameters.