The regulation of RNA polymerase subunit synthesis and its relationship to the expression of ribosome component genes have been investigated in strains of Escherichia coli having a mutation in one of the genes specifying either the β or β' subunit of the core enzyme.' Particular attention has been focused on the L10 transcriptional unit (organization: P[sub= L10] – rp1J(LlO) - rplL(L7/L12) - attenuator - rpoB(β) -rpoC(β ') ). The mutant strain XH56 produces a defective β ' subunit which renders the RNA polymerase inactive in transcription initiation at 42°C; at somewhat lower temperatures, the RNA polymerase activity is only partially restricted. A temperature shift of this strain from 30°C to 39°C resulted in a rapid 5-fold increase in the transcription of the rpo β and CI genes and in the synthesis rates of the β and β' subunits, indicating that β and β' synthesis is regulated primarily at the transcriptional level. Transcription of the α subunit gene, located in the spc-str region of the chromosome, was also enhanced. Transcription of the lacZ gene (coding for β -galactosidase) was decreased to undetectable levels, indicating that the dramatic increase of rpo β and C. transcription occurred at the expense of transcription of other operons. The mutant strains Ts4 and A2R7 produce defective β' and β subunits respectively which are unable to assemble into core RNA polymerase at the nonpermissive temperature. In these strains RNA polymerase assembled prior to a temperature shift from 30°C to 42°C retains its activity but little or no enzyme is assembled after the shift. Prolonged incubation of these strains after such a shift produced a gradual 1.5- to 2-fold increase in the transcription of the rpoβ and C genes and in the synthesis rates of the β and β' subunits. During the restrictions, transcription of ribosome component genes was essentially unchanged. RNA polymerase assembly was also inhibited in strains carrying both a temperature-sensitive amber suppressor mutation and an amber mutation in the rpoβ gene. Under permissive conditions these amber mutations are suppressed by insertion of serine into the β protein at the UAG codon. After a temperature shift to 42°C, core RNA polymerase synthesis is restricted due to the failure to produce 3 in the non-polar amber strain MX515 and both β and β' in the polar amber strain MX515. Core enzyme synthesized prior to the shift retains its activity. Inhibition of core enzyme synthesis in this manner resulted in a gradual stimulation of rpoβ and C transcription; in the polar strain this was accompanied by a concomitant increase in the synthesis rate of the β' subunit protein. The increase of rpoβ and C transcription involved both increased initiation at P[sub L10] and relaxed termination in the rp1L-rpoβ intergenic space. It was also observed that transcription of the a subunit gene was specifically stimulated during the restriction, suggesting that the regulatory mechanisms are specific for genetic units containing core RNA polymerase genes. These results therefore indicate that the mechanisms which govern the transcriptional frequency of operons containing RNA polymerase genes are coupled to the demand for active RNA polymerase; a sudden restriction of enzyme activity produces a rapid and dramatic increase of rpoβ and C transcription whereas a slow restriction results in only a gradual and less extensive induction. The regulatory mechanisms operating within the L10 transcription unit were accentuated by introducing the composite colEl plasmid pJC701 into the RNA polymerase activity mutant strain XH56.. All of the genes in the L10 transcription unit except the distally located rpoC are present on this plasmid and therefore were amplified in the transformed bacteria. The partial temperature inactivation of RNA polymerase activity in this strain allowed us to modulate the transcription of the proximal rp1J-rp1L genes and the distal rpoβ gene over a 10-fold and 30-fold range respectively. The observed imbalance in transcription between the proximal and distal portions of the L10 transcription unit strongly. suggest that the restriction has :two distinct effects: (i) it stimulates initiation at the major L10 promotor and (ii) it reduces termination at the attenuator located within or near the rp1L-rpoβ intergenic space. The synthesis rates of L7/L12 and β subunit proteins were also measured and compared to their respective mRNA levels under these conditions. The synthesis rate of L7/L12 protein and β protein varied by less than 2-fold and by 15-fold respectively. These measurements clearly indicate that translation of excess L7/L12 ribosomal protein mRNA is severely restricted and contributes to maintaining the balanced synthesis of ribosome components. The translational efficiency of 3 mRNA was also reduced by about 50%. Under the above conditions, β protein is produced in large excess relative to β' subunit protein. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

Synthesis of the CCA end of essential tRNAs is performed either by CCA-adding enzymes or as a collaboration between enzymes restricted to CC- and A-incorporation. While the occurrence of such tRNA nucleotidyltransferases with partial activities seemed to be restricted to Bacteria, the first example of such split CCA-adding activities was reported in Schizosaccharomyces pombe. Here, we demonstrate that the choanoflagellate Salpingoeca rosetta also carries CC- and A-adding enzymes. However, these enzymes have distinct evolutionary origins. Furthermore, the restricted activity of the eukaryotic CC-adding enzymes has evolved in a different way compared to their bacterial counterparts. Yet, the molecular basis is very similar, as highly conserved positions within a catalytically important flexible loop region are missing in the CC-adding enzymes. For both the CC-adding enzymes from S. rosetta as well as S. pombe, introduction of the loop elements from closely related enzymes with full activity was able to restore CCA-addition, corroborating the significance of this loop in the evolution of bacterial as well as eukaryotic tRNA nucleotidyltransferases. Our data demonstrate that partial CC- and A-adding activities in Bacteria and Eukaryotes are based on the same mechanistic principles but, surprisingly, originate from different evolutionary events.

Thèse de doctorat : Biologie moléculaire et cellulaire : Strasbourg 1 : 2008. / Titre provenant de l'écran-titre. Bibliogr. p. 273-279.

The mitochondrial genome of the nematode Romanomermis culicivorax encodes for miniaturized hairpin-like tRNA molecules that lack D- as well as T-arms, strongly deviating from the consensus cloverleaf. The single tRNA nucleotidyltransferase of this organism is fully active on armless tRNAs, while the human counterpart is not able to add a complete CCA-end. Transplanting single regions of the Romanomermis enzyme into the human counterpart, we identified a beta-turn element of the catalytic core that—when inserted into the human enzyme—confers full CCA-adding activity on armless tRNAs. This region, originally identified to position the 30 -end of the tRNA primer in the catalytic core, dramatically increases the enzyme’s substrate affinity. While conventional tRNA substrates bind to the enzyme by interactions with the T-arm, this is not possible in the case of armless tRNAs, and the strong contribution of the beta-turn compensates for an otherwise too weak interaction required for the addition of a complete CCA-terminus. This compensation demonstrates the remarkable evolutionary plasticity of the catalytic core elements of this enzyme to adapt to unconventional tRNA substrates.

For flawless translation of mRNA sequence into protein, tRNAs must undergo a series of essential maturation steps to be properly recognized and aminoacylated by aminoacyl-tRNA synthetase, and subsequently utilized by the ribosome. While all tRNAs carry a 30 -terminal CCA sequence that includes the site of aminoacylation, the additional 50 -G-1 position is a unique feature of most histidine tRNA species, serving as an identity element for the corresponding synthetase. In eukaryotes including yeast, both 30 -CCA and 50 -G-1 are added post-transcriptionally by tRNA nucleotidyltransferase and tRNAHis guanylyltransferase, respectively. Hence, it is possible that these two cytosolic enzymes compete for the same tRNA. Here, we investigate substrate preferences associated with CCA and G-1-addition to yeast cytosolic tRNAHis, which might result in a temporal order to these important processing events. We show that tRNA nucleotidyltransferase accepts tRNAHis transcripts independent of the presence of G-1; however, tRNAHis guanylyltransferase clearly prefers a substrate carrying a CCA terminus. Although many tRNA maturation steps can occur in a rather random order, our data demonstrate a likely pathway where CCA-addition precedes G-1 incorporation in S. cerevisiae. Evidently, the 30 -CCA triplet and a discriminator position A73 act as positive elements for G-1 incorporation, ensuring the fidelity of G-1 addition.

Background: To allow an immediate treatment of an infection with suitable antibiotics and bactericides or fungicides, there is an urgent need for fast and precise identification of the causative human pathogens. Methods based on DNA sequence comparison like 16S rRNA analysis have become standard tools for pathogen verification. However, the distinction of closely related organisms remains a challenging task. To overcome such limitations, we identified a new genomic target sequence located in the single copy gene for tRNA nucleotidyltransferase fulfilling the requirements for a ubiquitous, yet highly specific DNA marker. In the present study, we demonstrate that this sequence marker has a higher discriminating potential than commonly used genotyping markers in pro- as well as eukaryotes, underscoring its applicability as an excellent diagnostic tool in infectology. Results: Based on phylogenetic analyses, a region within the gene for tRNA nucleotidyltransferase (CCA-adding enzyme) was identified as highly heterogeneous. As prominent examples for pro- and eukaryotic pathogens, several Vibrio and Aspergillus species were used for genotyping and identification in a multiplex PCR approach followed by gel electrophoresis and fluorescence-based product detection. Compared to rRNA analysis, the selected gene region of the tRNA nucleotidyltransferase revealed a seven to 30-fold higher distinction potential between closely related Vibrio or Aspergillus species, respectively. The obtained data exhibit a superb genome specificity in the diagnostic analysis. Even in the presence of a 1,000-fold excess of human genomic DNA, no unspecific amplicons were produced. Conclusions: These results indicate that a relatively short segment of the coding region for tRNA nucleotidyltransferase has a higher discriminatory potential than most established diagnostic DNA markers. Besides identifying microbial pathogens in infections, further possible applications of this new marker are food hygiene controls or metagenome analyses.

Background: To allow an immediate treatment of an infection with suitable antibiotics and bactericides or fungicides, there is an urgent need for fast and precise identification of the causative human pathogens. Methods based on DNA sequence comparison like 16S rRNA analysis have become standard tools for pathogen verification. However, the distinction of closely related organisms remains a challenging task. To overcome such limitations, we identified a new genomic target sequence located in the single copy gene for tRNA nucleotidyltransferase fulfilling the requirements for a ubiquitous, yet highly specific DNA marker. In the present study, we demonstrate that this sequence marker has a higher discriminating potential than commonly used genotyping markers in pro- as well as eukaryotes, underscoring its applicability as an excellent diagnostic tool in infectology. Results: Based on phylogenetic analyses, a region within the gene for tRNA nucleotidyltransferase (CCA-adding enzyme) was identified as highly heterogeneous. As prominent examples for pro- and eukaryotic pathogens, several Vibrio and Aspergillus species were used for genotyping and identification in a multiplex PCR approach followed by gel electrophoresis and fluorescence-based product detection. Compared to rRNA analysis, the selected gene region of the tRNA nucleotidyltransferase revealed a seven to 30-fold higher distinction potential between closely related Vibrio or Aspergillus species, respectively. The obtained data exhibit a superb genome specificity in the diagnostic analysis. Even in the presence of a 1,000-fold excess of human genomic DNA, no unspecific amplicons were produced. Conclusions: These results indicate that a relatively short segment of the coding region for tRNA nucleotidyltransferase has a higher discriminatory potential than most established diagnostic DNA markers. Besides identifying microbial pathogens in infections, further possible applications of this new marker are food hygiene controls or metagenome analyses.