Structural and Biochemical Properties of Novel Self-Cleaving Ribozymes

Abstract

Fourteen well-defined ribozyme classes have been identified to date, among which nine are site-specific self-cleaving ribozymes. Very recently, small self-cleaving ribozymes have attracted renewed interest in their structure, biochemistry, and biological function since the discovery, during the last three years, of four novel ribozymes, termed twister, twister sister, pistol, and hatchet. In this review, we mainly address the structure, biochemistry, and catalytic mechanism of the novel ribozymes. They are characterized by distinct active site architectures and divergent, but similar, biochemical properties. The cleavage activities of the ribozymes are highly dependent upon divalent cations, pH, and base-specific mutations, which can cause changes in the nucleotide arrangement and/or electrostatic potential around the cleavage site. It is most likely that a guanine and adenine in close proximity of the cleavage site are involved in general acid-base catalysis. In addition, metal ions appear to play a structural rather than catalytic role although some of their crystal structures have shown a direct metal ion coordination to a non-bridging phosphate oxygen at the cleavage site. Collectively, the structural and biochemical data of the four newest ribozymes could contribute to advance our mechanistic understanding of how self-cleaving ribozymes accomplish their efficient site-specific RNA cleavages.

Keywords: catalytic mechanism; hatchet; novel ribozymes; pistol; structure; twister; twister-sister.

Figure 1

Secondary and tertiary structures of two representatives of twister ribozymes. (A) The structure of the twister ribozyme from O. sativa [30]. Additional stem-loop segments, P3 and/or P5, can be generated, as shown in black dotted lines; (B) The structure of the env22 twister ribozyme [29]. In (A) and (B), red arrowhead indicates the U-A cleavage site. On the secondary structure, highly conserved nucleotides (>97%) are marked by asterisks. Stems (P1-P4) and pseudoknots (T1 and T2) are colour-coded in the tertiary structure. In particular, two nucleotides at the cleavage site and bound magnesium ions are coloured in cyan and yellow, respectively. Protein Data Bank (PDB) accession codes for (A) and (B) are 4OJI and 4RGE, respectively.

Figure 2

Sequence and secondary structure model of the TS-1 twister sister ribozyme [9] (A) and the Ht-1 hatchet ribozyme [11] (B). Highly conserved and non-native nucleotides are coloured in red and grey, respectively. The cleavage sites are indicated by red arrowheads.

Figure 3

Secondary and tertiary structures of the env25 pistol ribozyme [34]. On the secondary structure (left); highly conserved nucleotides are marked by asterisks, and red arrowhead indicates the G53-U54 cleavage site. Stems (P1-P3) and pseudoknots (T1) are colour-coded in the tertiary structure (right). In particular, two nucleotides at the cleavage site and bound magnesium ions are coloured in cyan and yellow, respectively. PDB accession code is 5K7C.

Figure 4

Internal phosphoester transfer mechanism for RNA cleavage. The 2′ oxygen nucleophile of a sugar attacks its adjacent phosphorus atom, together with subsequent protonation and departure of the 5′ oxygen of a sugar.