The Obg/CgtA subfamily is a conserved group of monomeric GTP-binding proteins found in the genomes of all organisms sequenced thus far. The evolutionary relationships of the GTPase superfamily suggested a role in ribosome function (6
). This prediction appears accurate, since all mitochondrial (8
), nuclear (12
), and prokaryotic (29
) Obg/CgtA proteins examined thus far are associated with ribosomes. Moreover, these proteins are also involved in the assembly of the large ribosomal subunit (28
). Recently, it has become clear that, in addition to a role in the late assembly of the large ribosomal subunit, the bacterial Obg/CgtA proteins are also directly involved in stress response (30
In Escherichia coli
, amino acid starvation leads to uncharged tRNAs binding to the ribosomal acceptor site (A-site) in a codon-dependent manner (18
) which is detected by RelA, a (p)ppGpp synthetase. The increase in (p)ppGpp levels leads to the “stringent response” that provides the cell with the regulatory means to control gene expression and thereby cope with starvation. The levels of (p)ppGpp are kept low in nutrient-rich media by SpoT, a bifunctional enzyme related to RelA that has both (p)ppGpp synthetase and hydrolase activity (19
). The hydrolase activity of SpoT is inhibited under nutrient-limiting conditions, allowing intracellular (p)ppGpp levels to increase during the stringent response, as RelA produces (p)ppGpp. In E. coli
and Vibrio cholerae
, the GTP-binding protein CgtA (also called YhbZ or Obg) interacts with SpoT (48
) on the large ribosomal particle (30
). Depletion of CgtA results in an increase in (p)ppGpp levels (30
), raising the possibility that during exponential growth CgtA directly inhibits the hydrolase activity of SpoT.
It has been proposed that, in Saccharomyces cerevisiae
, amino acid starvation also leads to uncharged tRNAs binding to the A-site but that the output is not RelA synthesis of (p)ppGpp (21
). The effector protein Gcn1 detects the uncharged tRNA and relays the A-site occupancy information to the protein kinase Gcn2. Gcn2 then phosphorylates the translation initiation factor 2α (eIF-2α), leading to reduced global protein synthesis and increased expression of amino acid biosynthetic enzymes (21
). The signal transduction pathway governing Gcn2 is called general amino acid control. Like RelA, Gcn2 and Gcn1 bind to ribosomes, and previous findings support the idea that Gcn1 affects A-site function (53
In S. cerevisiae
there are four Obg/CgtA subfamily members that also appear to play roles in ribosome function: Nog1 (Ypl093w), Mtg2 (Yhr168w), Yal036c, and Ygr173w. Nog1 is a nucleolar protein that plays a key role in assembly of the large ribosomal subunit; depletion of Nog1 leads to a decrease in 60S subunit assembly and formation of halfmer polysomes (12
). Mtg2 associates with the large mitochondrial ribosomal subunit, is critical for mitochondrial translation, and is required for the maintenance of proper ribosomal subunit ratios (8
The two remaining S. cerevisiae Obg/CgtA proteins, Yal036c and Ygr173w (hereafter called Rbg1 and Rbg2 for ribosome binding GTPase), belong to the DRG subgroup of Obg/CgtA proteins. Rbg1 and Rbg2 are tripartite proteins that are 52% similar to each other (Fig. ) and ubiquitously found in all eukaryotes and archaea sequenced to date. These proteins are similar to the other Obg/CgtA proteins only in the guanine nucleotide-binding domain (amino acids [aa] 69 to 275 and aa 67 to 275 for Rbg1 and Rbg2, respectively). Within this conserved GTPase domain, the Rbg proteins also have a 67-aa insertion of unknown function between the conserved G3 and G4 motifs (Fig. ). This insertion sequence is unique to the eukaryotic and archaeal DRG subfamily (as determined by PSI BLAST searches).
FIG. 1. Rbg1 and Rbg2 are tripartite proteins, here illustrated as a diagram. Shaded regions represent regions of high similarity. The universally conserved G1 through G4 motifs of the guanine nucleotide binding domain are indicated by overlines. The C-terminal (more ...)
The N and C termini of the Rbg proteins are distinct from those of the nucleolar, mitochondrial, and bacterial Obg/CgtA proteins. The N-terminal amino acids of the Rbg proteins (aa 1 to 68 and aa 1 to 66 for Rbg1 and Rbg2, respectively), predicted to contain two adjacent helices of unknown function, are strongly conserved (45% identical). The C terminus of the Rbg proteins (aa 290 to 368) contains a TGS domain (69
), a sequence of ~50 aa that forms a β-sheet structure (32
; Fig. ). The function of the TGS domain is currently unknown, although a regulatory role has been suggested (3
). TGS domains are found in a limited number of proteins including threonyl-tRNA synthetases, DRG-like GTPases and, interestingly, in the (p)ppGpp synthetases SpoT and RelA.
Several lines of evidence suggest that the cytoplasmic Rbg proteins are involved in ribosome function. First, the expression pattern RBG1
displays under various conditions clusters with genes involved in ribosome and rRNA biosynthesis (67
). In addition, the RBG1
promoter has a conserved element found in many genes involved in ribosome function (67
). Moreover, Rbg1, as well as other proteins involved in translation initiation, was copurified in a complex with eIF4G1-TAP (14
). The related gene, RBG2
, is synthetically sick in combination with a deletion of the large ribosomal subunit genes rpl22a
(N. J. Krogan, unpublished data).
We show here that Rbg1 associates with polyribosomes but not with the 40S or 60S subunits or with 80S monosomes, indicating that Rbg1 specifically associates with translating ribosomes. Interacting partners of Rbg1 were identified by using a yeast two-hybrid screen. Among the interaction partners was a protein of unknown function, Gir2, which has sequence similarity to the N-terminal GI (Gcn2 and Impact) domain of Gcn2 that is involved in Gcn1 binding. We found that Gir2 also associates with ribosomes, and we have several lines of evidence showing that Gir2 binds to Gcn1 via its GI domain. Gir2 overexpression diminishes Gcn2 function, and this could be reverted by Gcn2 overexpression, suggesting that Gir2 competes with Gcn2 for Gcn1 binding. The polyribosome association of Gir2 was not dependent on Rbg1, although its association with polysomes was in part dependent on Gcn1. Based on the connection between Rbg1 and Gir2 and between Gir2 and the components of the general amino acid control pathway, Rbg1 and Gir2 may play a role in adjusting the cell to stress conditions.