Crystal structure combined with genetic analysis of the Thermus thermophilus ribosome recycling factor shows that a flexible hinge may act as a functional switch.
Ribosome recycling factor (RRF), in concert with elongation factor EF-G, is required for disassembly of the posttermination complex of the ribosome after release of polypeptides. The crystal structure of Thermus thermophilus RRF was determined at 2.6 A resolution. It is a tRNA-like L-shaped molecule consisting of two domains: a long three-helix bundle (domain 1) and a three-layer beta/alpha/beta sandwich (domain 2). Although the individual domain structures are similar to those of Thermotoga maritima RRF (Selmer et al., Science, 1999, 286:2349-2352), the interdomain angle differs by 33 degrees in two molecules, suggesting that the hinge between two domains is potentially flexible and responsive to different conditions of crystal packing. The hinge connects hydrophobic junctions of domains 1 and 2. The structure-based genetic analysis revealed the strong correlation between the hinge flexibility and the in vivo function of RRF. First, altering the hinge flexibility by making alanine or serine substitutions for large-size residues conserved at the hinge loop and nearby in domain 1 frequently gave rise to gain of function except a Pro residue conserved at the hinge loop. Second, the hinge defect resulting from a too relaxed hinge structure can be compensated for by secondary alterations in domain 1 that seem to increase the hydrophobic contact between domain 1 and the hinge loop. These results show that the hinge flexibility is vital for the function of RRF and that the steric interaction between the hinge loop and domains 1 and 2 restricts the interdomain angle and/or the hinge flexibility. These results indicate that RRF possesses an architectural difference from tRNA regardless of a resemblance to tRNA shape: RRF has a "gooseneck" elbow, whereas the tRNA elbow is rigid, and the direction of flex of RRF and tRNA is at a nearly right angle to each other. Moreover, surface electrostatic potentials of the two RRF proteins are dissimilar and do not mimic the surface potential of tRNA or EF-G. These properties will add a new insight into RRF, suggesting that RRF is more than a simple tRNA mimic.
Toyoda
T
T
Department of Tumor Biology, The Institute of Medical Science, The University of Tokyo, Japan.
Tin
O F
OF
Ito
K
K
Fujiwara
T
T
Kumasaka
T
T
Yamamoto
M
M
Garber
M B
MB
Nakamura
Y
Y
D000483:Alleles; D000595:Amino Acid Sequence; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics; D005816:Genetic Complementation Test; D008958:Models, Molecular; D018716:Molecular Mimicry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation / Q000235:genetics*; D018583:Pliability; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012343:RNA, Transfer / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012269:Ribosomal Proteins; D016415:Sequence Alignment; D055672:Static Electricity; D013329:Structure-Activity Relationship; D013489:Suppression, Genetic / Q000235:genetics; D013824:Thermus / Q000737:chemistry; D016963:Thermus thermophilus / Q000737:chemistry* / Q000235:genetics
D011506:Proteins; D012269:Ribosomal Proteins; C087267:ribosome releasing factor; D012343:RNA, Transfer