Abstract
Two classes of riboswitches related to the ykkC guanidine-I riboswitch bind phosphoribosyl pyrophosphate (PRPP) and guanosine tetraphosphate (ppGpp). Here we report the co-crystal structure of the PRPP aptamer and its ligand. We also report the structure of the G96A point mutant that prefers ppGpp over PRPP with a dramatic 40,000-fold switch in specificity. The ends of the aptamer form a helix that is not present in the guanidine aptamer and is involved in the expression platform. In the mutant, the base of ppGpp replaces G96 in three-dimensional space. This disrupts the S-turn, which is a primary structural feature of the ykkC RNA motif. These dramatic differences in ligand specificity are achieved with minimal mutations. ykkC aptamers are therefore a prime example of an RNA fold with a rugged fitness landscape. The ease with which the ykkC aptamer acquires new specificity represents a striking case of evolvability in RNA.
Highlights
RNA has diverse functional capabilities, which has driven speculation that the first organisms may have been RNA-based (Breaker, 2012; Crick, 1968; Gilbert, 1986; Orgel, 2004; 1968; Strobel, 2001; Woese et al, 1966)
The present results show that the PRPP aptamer recognizes its ligand through a shifted and extended helical ligand-binding region, allowing for the retention of bound metal ions and extensive hydrogen bond donation to phosphate groups
Just as residue C49 was previously used to distinguish guanidine aptamers from subtype 2 ykkC RNAs, here we show that G96 is the residue that differentiates PRPP and ppGpp aptamers
Summary
RNA has diverse functional capabilities, which has driven speculation that the first organisms may have been RNA-based (Breaker, 2012; Crick, 1968; Gilbert, 1986; Orgel, 2004; 1968; Strobel, 2001; Woese et al, 1966). Evolvability is the propensity of a system to produce a mutated genotype that yields a beneficial phenotype under new selective pressures (Ancel and Fontana, 2000; Kirschner and Gerhart, 1998; Wagner, 2008; Wagner and Altenberg, 1996) Often, this occurs through mutation of an existing gene through divergent evolution. Bacterial b-lactamases demonstrate significant evolvability through mutations in the W-loop This loop determines substrate specificity, but mutation or outright deletion of the loop does not dramatically affect the overall structure of the protein (Banerjee et al, 1998; Hujer et al, 2001; Kurokawa et al, 2000; Wachino et al, 2004). The speed at which an organism adapts is determined by this, as well as the ruggedness of the fitness landscape, which is related to the number of mutations required to reach a new fitness maximum
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