Association of the halobacterial 7S RNA to the polysome correlates with expression of the membrane protein bacterioopsin.

Abstract

The sedimentation behavior of the halobacterial 7S RNA and bacterioopsin mRNA was ass after application of total cell lysates to sucrose gradients. These two RNAs cosedimented predominantly with membrane-bound polysomes, and the quantity of 7S RNA bound to the ribosomes was directly correlated with the expression of bacterioopsin. Puromycin treatment released the 7S RNA from the polysomes, indicating that it is transiently associated with protein translation. We suggest that halobacteria contain a signalrecognition-like particle involved in translation of membraneassociated proteins.In mammalian cells the 7S RNA is part of the signal recognition particle (SRP), which mediates cotranslational processing mechanisms for membrane and secretory proteins (1,2). SRP is a ribonucleoparticle that recognizes the leader sequence ofthe nascent polypeptide chain as it emerges from the ribosome. The interaction causes a translational arrest that is only released if the complex is targeted to a receptor in the membrane (3). These mechanisms prevent synthesis of hydrophobic proteins in a hydrophilic environment and direct membrane and secretory proteins to their final destinations in the cell. Small ribonucleoproteins resembling SRP have also been found in yeast (Schizosaccharomyces pombe and Yarrowia lipolytica) and in Escherichia coli (4-6), but in contrast to mammalian cells they translocate many proteins posttranslationally; genetic studies do not as yet corroborate a SRP-coupled translocation mechanism (7-9). Genetic analysis in E. coli has shown that the 4.5S RNA, which has been identified as a component of the ribonucleoparticle, is acting on translating ribosomes and perhaps in concert with elongation factor G GTP (10). However, it remains unclear whether it is generally obligatory for translation or only participates in translation of a subset of proteins.Like other archaebacteria, Halobacterium halobium possesses a 7S RNA with a possible secondary structure almost identical to that of the mammalian SRP RNA (11-13). Its function is unknown, but as archaebacterial 7S RNA genes can replace the 4.5S RNA gene in E. coli (14), it is likely that a signal recognition-like particle exists in halobacteria. In this report, we demonstrate that the halobacterial 7S RNA is associated with ribosomes during translation, and our data suggest that it is specifically involved in translation of membrane proteins. (18,19). The precursor is processed in a two-step mechanism (20, 21), but processing is not necessary for correct folding of the protein. Both forms integrate into the-purple membrane with no conformational differences from the mature protein (22). The presequence is unusual and lacks similarities to other prokaryotic and eukaryotic signal sequences (23). This observation raises questions about its functional significance. In this report we show that the N-terminal sequences of six halobacterial integral membrane proteins share a common motif that may be of functional importance. MATERIALS AND METHODSRNAzol was purchase...

Ordered membrane insertion of an archaeal opsin in vivo

“…The presequence may be recognized by the signal recognition particle, which in other systems initiates cotranslational insertion by the secretory translocase (18). In accord with this hypothesis, the RNA component of the signal recognition particle is localized to the membrane on induction of BO synthesis (19). The model also is supported by our previous work, in which we used an in vivo kinetic assay to show that the N terminus of BO is translocated cotranslationally, indicating that the first transmembrane segment is inserted cotranslationally (20).…”

Post-translational Secretion of Fusion Proteins in the Halophilic Archaea Haloferax volcanii

“…However, not all archaeal membrane proteins may rely on the SRP pathway for their insertion, as possibly exemplified by studies addressing the homologous (20,41) and heterologous (22) expression of bacterioopsin. Indeed, the SRP pathway is implicated in the membrane insertion of only a subset of bacterial plasma membrane proteins (42).…”

“…Based on the cosedimentation of 7 S RNA and bacterioopsin mRNA with membrane-bound polysomes, as well as more recent in vivo kinetic analysis, a cotranslational SRP-dependent mode of protein translocation is suggested in Archaea (20,21). In contrast, studies following the membrane insertion of a chimeric version of bacterioopsin expressed in Haloferax volcanii reported that the fusion protein is first synthesized in the cytoplasm and only then inserted into the membrane (22).…”

“…In contrast, studies following the membrane insertion of a chimeric version of bacterioopsin expressed in Haloferax volcanii reported that the fusion protein is first synthesized in the cytoplasm and only then inserted into the membrane (22). Studies relying on newly synthesized bacterioopsin as a reporter of the relation between translation and translocation in Archaea must be viewed, however, with caution, given this protein's unusually short 13-residue signal peptide that lacks a hydrophobic core and contains negatively charged glutamate residues, rather than the positively charged amino acids found in most signal peptides (20), as well as the possibility of a dedicated system for bacterioopsin membrane insertion (22). Moreover, the interaction between translation and translocation of a membrane protein may differ in the case of protein secretion.…”

Transcription in vitro and in vivo of the 7S RNA gene associated with the ribosomal RNA operon in the hyperthermophilic archaeon Methanothermus fervidus

“…The essential function of the 4.5S RNA in translation in E. coli can be replaced by an archaeal 7S RNA [6][7][8]. It seems likely, therefore, that the 7S RNAs in the Archaea play a role in translation and a 7S RNA has, in fact, been isolated from Halobacterium halobium physically associated with polysomes [9]. The discovery that, in the genome of Methanothermus fervidus, the gene encoding the 7S RNA is located immediately upstream of a tRNA Ser gene and rRNA Operon ( [3]; see Fig.…”