Figure 1: Schematic of the partitioning of the Azoarcus ribozyme into fragments. (A) The 197-nt source molecule was partitioned into five fragments (U, gray; V, red; X; yellow; Y, blue; Z, green) such that catalytic activity in trans, plus covalent self-assembly could be assayed. Removal of the U fragment from the system at the location indicated by the arrow leaves an L–30 construct which can then be divided into four fragments that can exhibit activity when 5-nt head groups (h = GGCAU) are appended to the 5′ portions of X, Y and Z (see text). A substrate oligomer (lower-case letters = head; black line = tail) binds via a 5′-CAU-3′ to the IGS (GUG) of the ribozyme, which catalyzes phosphotransfer of the tail to its own 3′ end. The native IGS of the ribozyme is denoted by GUG1, while the four exogenous GUG triplets that occur naturally in the remainder of the ribozyme are gray boxes denoted 2–5 (in circles). The tertiary interactions that hold the IGS and the IGS complement into the active site as determined by X-ray crystallography (24) are denoted using the hydrogen-bonding symbolism of Leontis et al. (41). (B) Schematic of how a ribozyme complex (either as a covalently contiguous molecule or as several fragments cooperating in trans) can perform catalysis in the absence of the U fragment that contains the native IGS. Here, the use of an exogenous IGS (GUG5) present in the h•Z fragment to bind to the IGS complement CAU is depicted.

Mentions: To create an IGS-less (‘blind’) version, of the L–8 Azoarcus ribozyme, the first 30 nt of the naturally occurring form of the 205-nt intron were removed, resulting in an L–30 construct lacking the first occurrence of the GUG triplet (termed GUG1), which is the canonical IGS for the ribozyme. Without this triplet, transesterification activity should be abolished. Previously it has been shown that the Azoarcus ribozyme can covalently self-assembly through recursive and autocatalytic recombination reactions after having been fragmented into four roughly equally sized pieces termed W, X, Y and Z (21). The removal of the first 22 nt in W results in a new fragment, here termed V (Figure 1A). Of interest is that the remaining 175 nt of the wild-type version of this ribozyme contain four additional GUG triplets, denoted GUG2, GUG3, GUG4 and GUG5 (Figure 1A and B). By chance, any given triplet such as GUG should appear fewer than three times in 175 nt. Regardless, these ‘exogenous’ GUGs could potentially complement the missing canonical IGS if they were to work in trans. Such activity would be similar to the manner in which fragments of the Tetrahymena ribozyme can non-covalently assemble to restore activity, although with each RNA presumably only adopting one role (9,10).

Vaidya N, Lehman N - Nucleic Acids Res. (2009)

Bottom Line: However, a single RNA genotype has the potential to adopt two or perhaps more distinct phenotypes as a result of differential folding and/or catalytic activity.Such multifunctionality would be particularly significant if the phenotypes were functionally inter-related in a common biochemical pathway.This property of RNA to be multifunctional in a single reaction pathway bolsters the probability that a system of self-replicating molecules could have existed in an RNA world during the origins of life on the Earth.

Affiliation: Department of Chemistry, Portland State University, PO Box 751, Portland, OR 97207, USA.

Abstract: Catalytic RNA molecules possess simultaneously a genotype and a phenotype. However, a single RNA genotype has the potential to adopt two or perhaps more distinct phenotypes as a result of differential folding and/or catalytic activity. Such multifunctionality would be particularly significant if the phenotypes were functionally inter-related in a common biochemical pathway. Here, this phenomenon is demonstrated by the ability of the Azoarcus group I ribozyme to function when its canonical internal guide sequence (GUG) has been removed from the 5' end of the molecule, and added back exogenously in trans. The presence of GUG triplets in non-covalent fragments of the ribozyme allow trans-splicing to occur in both a reverse splicing assay and a covalent self-assembly assay in which the internal guide sequence (IGS)-less ribozyme can put itself together from two of its component pieces. Analysis of these reactions indicates that a single RNA fragment can perform up to three distinct roles in a reaction: behaving as a portion of a catalyst, behaving as a substrate, and providing an exogenous IGS. This property of RNA to be multifunctional in a single reaction pathway bolsters the probability that a system of self-replicating molecules could have existed in an RNA world during the origins of life on the Earth.