RESUMEN

Eukaryotic eIF5A and its bacterial orthologue EF-P are translation elongation factors whose task is to rescue ribosomes from stalling during the synthesis of proteins bearing particular sequences such as polyproline stretches. Both proteins are characterized by unique post-translational modifications, hypusination and lysinylation, respectively, which are essential for their function. An orthologue is present in all Archaea but its function is poorly understood. Here, we show that aIF5A of the crenarchaeum Sulfolobus solfataricus is hypusinated and forms a stable complex with deoxyhypusine synthase, the first enzyme of the hypusination pathway. The recombinant enzyme is able to modify its substrate in vitro resulting in deoxyhypusinated aIF5A. Moreover, with the aim to identify the enzyme involved in the second modification step, i.e. hypusination, a set of proteins interacting with aIF5A was identified.

Asunto(s)

Proteínas Arqueales/metabolismo , Factores de Iniciación de Péptidos/metabolismo , Procesamiento Proteico-Postraduccional , Sulfolobus solfataricus/metabolismo , Lisina/análogos & derivados , Lisina/metabolismo

RESUMEN

In this work, we have studied the in vitro translational features of a bicistronic mRNA of the extremely thermophilic Archaeon Sulfolobus solfataricus, with the aim of determining the nature of the cis-acting signals controlling the recognition of the translation initiation sites in the Archaea. We found that the most important feature for efficient initiation was the presence of a Shine-Dalgarno (SD)-like ribosome-binding motif, whose disruption entirely abolished the translation of the corresponding cistron. The influence of other features, such as the type of initiation codon, was variable and depended upon the gene and its position in the mRNA. However, the translational block caused by the disruption of the SD sequences could be removed by deleting the 5' untranslated region altogether, thereby creating a 'leaderless' mRNA. This suggests that 'leaderless' initiation operates by a default mechanism that does not require a specific mRNA-rRNA interaction and may be common to all three primary domains of life.

Asunto(s)

Biosíntesis de Proteínas , Sulfolobus/genética , Sulfolobus/metabolismo , Regiones no Traducidas 5'/metabolismo , Codón Iniciador , Eliminación de Gen , Regulación de la Expresión Génica Arqueal , Genes/genética , Mutagénesis Sitio-Dirigida , Sistemas de Lectura Abierta , ARN de Archaea/genética , ARN de Archaea/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ribosomas/metabolismo , Transcripción Genética