RESUMO
Ribosomal RNA molecules are synthesized as precursors that have to undergo several processing steps to generate the functional rRNA. The 5S rRNA in the archaeon Haloferax volcanii is transcribed as part of a multicistronic transcript containing both large rRNAs and one or two tRNAs. Release of the 5S rRNA from the precursor requires two endonucleolytic cleavages by enzymes as yet not identified. Here we report the first identification of an archaeal 5S rRNA processing endonuclease. The enzyme tRNase Z, which was initially identified as tRNA processing enzyme, generates not only tRNA 3' ends but also mature 5S rRNA 5' ends in vitro. Interestingly, the sequence upstream of the 5S rRNA can be folded into a mini-tRNA, which might explain the processing of this RNA by tRNase Z. The endonuclease is active only at low salt concentrations in vitro, which is in contrast to the 2-4 M KCl concentration present inside the cell in vivo. Electron microscopy studies show that there are no compartments inside the Haloferax cell that could provide lower salt environments. Processing of the 5S rRNA 5' end is not restricted to the haloarchaeal tRNase Z since tRNase Z enzymes from a thermophilic archaeon, a lower and a higher eukaryote, are as well able to cleave the tRNA-like structure 5' of the 5S rRNA. Knock out of the tRNase Z gene in Haloferax volcanii is lethal, showing that the protein is essential for the cell.
Assuntos
Endorribonucleases/metabolismo , Haloferax volcanii/metabolismo , RNA Arqueal/metabolismo , RNA Ribossômico 5S/metabolismo , Sequência de Bases , Endorribonucleases/genética , Genes Arqueais , Haloferax volcanii/genética , Haloferax volcanii/ultraestrutura , Microscopia Eletrônica de Transmissão , Modelos Moleculares , Conformação de Ácido Nucleico , Precursores de RNA/genética , Precursores de RNA/metabolismo , Processamento Pós-Transcricional do RNA , RNA Arqueal/química , RNA Arqueal/genética , RNA Ribossômico 5S/genética , RNA de Transferência/química , RNA de Transferência/genética , RNA de Transferência/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por SubstratoRESUMO
All functional RNAs are generated from precursor molecules by a plethora of processing steps. The generation of mature RNA molecules by processing is an important layer of gene expression regulation catalysed by ribonucleases. Here, we analysed 5S rRNA processing in the halophilic Archaeon Haloferax volcanii. Earlier experiments showed that the 5S rRNA is cleaved at its 5' end by the endonuclease tRNase Z. Interestingly, a tRNA-like structure was identified upstream of the 5S rRNA that might be used as a processing signal. Here, we show that this tRNA-like element is indeed recognised as a processing signal by tRNase Z. Substrates containing mutations in the tRNA-like sequence are no longer processed, whereas a substrate containing a deletion in the 5S rRNA sequence is still cleaved. Therefore, an intact 5S rRNA structure is not required for processing. Further, we used bioinformatics analyses to identify additional sequences in Haloferax containing tRNA-like structures. This search resulted in the identification of all tRNAs, the tRNA-like structure upstream of the 5S RNA and 47 new tRNA-like structural elements. However, the in vitro processing of selected examples showed no cleavage of these newly identified elements. Thus, tRNA-like elements are not a general processing signal in Haloferax.