Two structurally homologous guanosine triphosphatase (GTPase) domains interact directly during signal recognition particle (SRP)-mediated cotranslational targeting of proteins to the membrane. The 2.05 angstrom structure of a complex of the NG GTPase domains of Ffh and FtsY reveals a remarkably symmetric heterodimer sequestering a composite active site that contains two bound nucleotides. The structure explains the coordinate activation of the two GTPases. Conformational changes coupled to formation of their extensive interface may function allosterically to signal formation of the targeting complex to the signal-sequence binding site and the translocon. We propose that the complex represents a molecular "latch" and that its disengagement is regulated by completion of assembly of the GTPase active site.

The catalytic properties of the hammerhead ribozyme embedded in the (+) strand of the satellite tobacco ringspot viral genome are analyzed with the goal of obtaining the elemental rate constants of the cleavage (k(2)) and ligation (k(-)(2)) steps. Two different chimeras combining the sTRSV (+) hammerhead and the well-characterized hammerhead 16 were used to measure the cleavage rate constant (k(2)), the rate of approach to equilibrium (k(obs) = k(2) + k(-)(2)), and the fraction of full-length hammerhead at equilibrium (k(-)(2)/k(2) + k(-)(2)). When compared to minimal hammerheads that lack the recently discovered loop I-loop II interaction, an extended format hammerhead derived from sTRSV studied here shows at least a 20-fold faster k(2) and a 1300-fold faster k(-)(2) at 10 mM MgCl(2). However, the magnesium dependence of the cleavage rate is not significantly changed. Thus, the enhanced cleavage of this hammerhead observed in vivo is due to its higher intrinsic rate and not due to its tighter binding of magnesium ions. The faster k(-)(2) of this hammerhead suggests that ligation may be used to form circular RNA genomes. This in vitro system will be valuable for experiments directed at understanding the hammerhead mechanism and the role of the loop I-loop II interaction.

Chimeras of the well characterized minimal hammerhead 16 and nine extended hammerheads derived from natural viroids and satellite RNAs were constructed with the goal of assessing whether their very different peripheral tertiary interactions modulate their catalytic properties. For each chimera, three different assays were used to determine the rate of cleavage and the fraction of full length hammerhead at equilibrium and thereby deduce the elemental cleavage (k 2 ) and ligation (k -2 ) rate constants. The nine chimeras were all more active than minimal hammerheads and showed a very broad range of catalytic properties, with values of k 2 varying by 750-fold and k -2 by 100-fold. At least two of the hammerheads showed an altered dependence of k obs on magnesium concentration. Since much less catalytic diversity is observed among minimal hammerheads that lack the tertiary interactions, a possible role for the different tertiary interaction is to modulate the hammerhead cleavage properties in viroids. For example, differing hammerhead cleavage and ligation rates could affect the steady state concentrations of linear, circular, and polymeric genomes in infected cells.

The structural basis for the GTP-dependent co-translational targeting complex between the signal recognition particle (SRP) and its receptor is unknown. The complex has been shown to have unusual kinetics of formation, and association in vivo is likely to be dependent on catalysis by the SRP RNA. We have determined conditions for RNA-independent association of the 'NG' GTPase domains of the prokaryotic homologs of the SRP components, Ffh and FtsY, from Thermus aquaticus. Consistent with previous studies of the Escherichia coli proteins, the kinetics of association and dissociation are slow. The T. aquaticus FtsY is sensitive to an endogenous proteolytic activity that cleaves at two sites--the first in a lengthy linker peptide that spans the interface between the N and G domains, and the second near the N-terminus of the N domain of FtsY. Remarkably, this second cleavage occurs only on formation of the Ffh/FtsY complex. The change in protease sensitivity of this region, which is relatively unstructured in the FtsY but not in the Ffh NG domain, implies that it undergoes conformational change on formation of the complex between the two proteins. The N domain, therefore, participates in the interactions that mediate the GTP-dependent formation of the targeting complex.

The decoding properties of 22 structurally conservative base-pair and base-triple mutations in the anticodon hairpin and tertiary core of Escherichia coli tRNA Ala GGC were determined under single turnover conditions using E. coli ribosomes. While all of the mutations were able to efficiently decode the cognate GCC codon, many showed substantial misreading of near-cognate GUC or ACC codons. Although all the misreading mutations were present in the sequences of other E. coli tRNAs, they were never found among bacterial tRNA Ala GGC sequences. This suggests that the sequences of bacterial tRNA Ala GGC have evolved to avoid reading incorrect codons.