Ribosomal Hibernation-Associated Factors in .

Bacteria convert active 70S ribosomes to inactive 100S ribosomes to survive under various stress conditions. This state, in which the ribosome loses its translational activity, is known as ribosomal hibernation. In gammaproteobacteria such as , ribosome modulation factor and hibernation-promoting factor are involved in forming 100S ribosomes.

Functional Sites of Ribosome Modulation Factor (RMF) Involved in the Formation of 100S Ribosome.

One of the important cellular events in all organisms is protein synthesis, which is catalyzed by ribosomes. The ribosomal activity is dependent on the environmental situation of the cell. Bacteria form 100S ribosomes, lacking translational activity, to survive under stress conditions such as nutrient starvation.

Coordinated Regulation of Rsd and RMF for Simultaneous Hibernation of Transcription Apparatus and Translation Machinery in Stationary-Phase .

Transcription and translation in growing phase of , the best-studied model prokaryote, are coupled and regulated in coordinate fashion. Accordingly, the growth rate-dependent control of the synthesis of RNA polymerase (RNAP) core enzyme (the core component of transcription apparatus) and ribosomes (the core component of translation machinery) is tightly coordinated to keep the relative level of transcription apparatus and translation machinery constant for effective and efficient utilization of resources and energy. Upon entry into the stationary phase, transcription apparatus is modulated by replacing RNAP core-associated sigma (promoter recognition subunit) from growth-related RpoD to stationary-phase-specific RpoS.

YqjD is an inner membrane protein associated with stationary-phase ribosomes in Escherichia coli.

Here, we provide evidence that YqjD, a hypothetical protein of Escherichia coli, is an inner membrane and ribosome binding protein. This protein is expressed during the stationary growth phase, and expression is regulated by stress response sigma factor RpoS. YqjD possesses a transmembrane motif in the C-terminal region and associates with 70S and 100S ribosomes at the N-terminal region.

Structure of the 100S ribosome in the hibernation stage revealed by electron cryomicroscopy.

In the stationary growth phase of bacteria, protein biosynthesis on ribosomes is suppressed, and the ribosomes are preserved in the cell by the formation of the 100S ribosome. The 100S ribosome is a dimer of the 70S ribosome and is formed by the binding of the ribosome modulation factor and the hibernation promoting factor. However, the binding mode between the two 70S ribosomes and the mechanism of complex formation are still poorly understood.

Solution structure of the E. coli ribosome hibernation promoting factor HPF: Implications for the relationship between structure and function.

The 70S Escherichia coli ribosome dimerizes to form an inactive 100S ribosome during stationary phase, which is called "ribosome hibernation". The hibernation promoting factor HPF plays a crucial role in 100S ribosome formation. However, YfiA, a known paralog of HPF inhibits 100S formation, although it shares high sequence similarity.

Activities of Escherichia coli ribosomes in IF3 and RMF change to prepare 100S ribosome formation on entering the stationary growth phase.

The canonical ribosome cycle in bacteria consists of initiation, elongation, termination, and recycling stages. After the recycling stage, initiation factor 3 (IF3) stabilizes ribosomal dissociation by binding to 30S subunits for the next round of translation. On the other hand, during the stationary growth phase, it has been elucidated that Escherichia coli ribosomes are dimerized (100S ribosome formation) by binding ribosome modulation factor (RMF) and hibernation promoting factor (HPF), leading to a hibernation stage.

Neither helix in the coiled coil region of the axle of F1-ATPase plays a significant role in torque production.

F(1)-ATPase is an ATP-driven rotary molecular motor in which the central gamma-subunit rotates inside the cylinder made of alpha(3)beta(3) subunits. The amino and carboxy termini of the gamma-subunit form the axle, an alpha-helical coiled coil that deeply penetrates the stator cylinder. We previously truncated the axle step by step, starting with the longer carboxy terminus and then cutting both termini at the same levels, resulting in a slower yet considerably powerful rotation.

Axle-less F1-ATPase rotates in the correct direction.

F1-adenosine triphosphatase (ATPase) is an ATP-driven rotary molecular motor in which the central gamma subunit rotates inside a cylinder made of three alpha and three beta subunits alternately arranged. The rotor shaft, an antiparallel alpha-helical coiled coil of the amino and carboxyl termini of the gamma subunit, deeply penetrates the central cavity of the stator cylinder. We truncated the shaft step by step until the remaining rotor head would be outside the cavity and simply sat on the concave entrance of the stator orifice.

Role of HPF (hibernation promoting factor) in translational activity in Escherichia coli.

During the stationary phase of growth in Escherichia coli, ribosome modulation factor (RMF) and hibernation promoting factor (HPF) dimerize most 70S ribosomes to form 100S ribosomes. The process of 100S formation has been termed 'ribosomal hibernation'. Here, the contributions of HPF to 100S formation and translation were analysed in vitro.

Three binding sites for stalk protein dimers are generally present in ribosomes from archaeal organism.

Ribosomes have a characteristic protuberance termed the stalk, which is indispensable for ribosomal function. The ribosomal stalk has long been believed to be a pentameric protein complex composed of two sets of protein dimers, L12-L12, bound to a single anchor protein, although ribosomes carrying three L12 dimers were recently discovered in a few thermophilic bacteria. Here we have characterized the stalk complex from Pyrococcus horikoshii, a thermophilic species of Archaea.

TOR regulates late steps of ribosome maturation in the nucleoplasm via Nog1 in response to nutrients.

The protein kinase TOR (target of rapamycin) controls several steps of ribosome biogenesis, including gene expression of rRNA and ribosomal proteins, and processing of the 35S rRNA precursor, in the budding yeast Saccharomyces cerevisiae. Here we show that TOR also regulates late stages of ribosome maturation in the nucleoplasm via the nuclear GTP-binding protein Nog1. Nog1 formed a complex that included 60S ribosomal proteins and pre-ribosomal proteins Nop7 and Rlp24.

The rotor tip inside a bearing of a thermophilic F1-ATPase is dispensable for torque generation.

F(1)-ATPase is an ATP-driven rotary molecular motor in which the central gamma-subunit rotates inside a stator cylinder made of alpha(3)beta(3) subunits. To elucidate the role of rotor-stator interactions in torque generation, we truncated the gamma-subunit at its carboxyl terminus, which forms an alpha helix that penetrates deeply into the stator cylinder. We used an alpha(3)beta(3)gamma subcomplex of F(1)-ATPase derived from thermophilic Bacillus PS3 and expressed it in Escherichia coli.

In vitro reconstitution of the GTPase-associated centre of the archaebacterial ribosome: the functional features observed in a hybrid form with Escherichia coli 50S subunits.

We cloned the genes encoding the ribosomal proteins Ph (Pyrococcus horikoshii)-P0, Ph-L12 and Ph-L11, which constitute the GTPase-associated centre of the archaebacterium Pyrococcus horikoshii. These proteins are homologues of the eukaryotic P0, P1/P2 and eL12 proteins, and correspond to Escherichia coli L10, L7/L12 and L11 proteins respectively. The proteins and the truncation mutants of Ph-P0 were overexpressed in E.

A mode of assembly of P0, P1, and P2 proteins at the GTPase-associated center in animal ribosome: in vitro analyses with P0 truncation mutants.

Ribosomal P0, P1, and P2 proteins, together with the conserved domain of 28 S rRNA, constitute a major part of the GTPase-associated center in eukaryotic ribosomes. We investigated the mode of assembly in vitro by using various truncation mutants of silkworm P0. When compared with wild type (WT)-P0, the C-terminal truncation mutants CDelta65 and CDelta81 showed markedly reduced binding ability to P1 and P2, which was offset by the addition of an rRNA fragment covering the P0.

Comparative analysis of the ribosomal components of the hydrogenosome-containing protist, Trichomonas vaginalis.

The ribosomes of the amitochondriate but hydrogenosome-containing protist lineage, the trichomonads, have previously been reported to be prokaryotic or primitive eukaryotic, based on evidence that they have a 70S sedimentation coefficient and a small number of proteins, similar to prokaryotic ribosomes. In order to determine whether the components of the trichomonad ribosome indeed differ from those of typical eukaryotic ribosomes, the ribosome of a representative trichomonad, Trichomonas vaginalis, was characterized. The sedimentation coefficient of the T.

The ribosome modulation factor (RMF) binding site on the 100S ribosome of Escherichia coli.

During the stationary growth phase, Escherichia coli 70S ribosomes are converted to 100S ribosomes, and translational activity is lost. This conversion is caused by the binding of the ribosome modulation factor (RMF) to 70S ribosomes. In order to elucidate the mechanisms by which 100S ribosomes form and translational inactivation occurs, the shape of the 100S ribosome and the RMF ribosomal binding site were investigated by electron microscopy and protein-protein cross-linking, respectively.

Normal assembly of 60 S ribosomal subunits is required for the signaling in response to a secretory defect in Saccharomyces cerevisiae.

A secretory defect leads to transcriptional repression of both ribosomal protein and rRNA genes in yeast. To elucidate the mechanism of the signaling, we previously isolated rrs mutants that were unable to respond to a secretory defect, and we cloned RRS1 encoding a nuclear protein that was required for ribosome biogenesis (Tsuno, A., Miyoshi, K.