Peripheral regions of natural hammerhead ribozymes greatly increase their self-cleavage activity

Abstract

Natural hammerhead ribozymes are mostly found in some viroid and viroid-like RNAs and catalyze their cis cleavage during replication. Hammerheads have been manipulated to act in trans and assumed to have a similar catalytic behavior in this arti®cial context. However, we show here that two natural cis-acting hammerheads self-cleave much faster than trans-acting derivatives and other reported arti®cial hammerheads. Moreover, modi®cations of the peripheral loops 1 and 2 of one of these natural hammerheads induced a >100-fold reduction of the self-cleavage constant, whereas engineering a trans-acting arti®cial hammerhead into a cis derivative by introducing a loop 1 had no effect. These data show that regions external to the central conserved core of natural hammerheads play a role in catalysis, and suggest the existence of tertiary interactions between these peripheral regions. The interactions, determined by the sequence and size of loops 1 and 2 and most likely of helices I and II, must result from natural selection and should be studied in order to better understand the hammerhead requirements in vivo.

Efficient Ligation of the Schistosoma Hammerhead Ribozyme

“…The cleavage reaction for several natural hammerhead ribozymes has been studied (28,29,32), but the ligation kinetics have only been characterized for the sTRSV hammerhead (32).…”

“…In addition, Scott and coworkers have solved the X-ray structure of a construct of the Schistosoma mansoni hammerhead ribozyme, which contains the natural loop-loop tertiary interaction (40). This structure shows large conformational changes in the catalytic core compared to the structure of the minimal hammerhead ribozyme and represents an improved model for understanding the catalytic mechanism of the hammerhead ribozyme (30, 40).The cleavage reaction for several natural hammerhead ribozymes has been studied (28,29,32), but the ligation kinetics have only been characterized for the sTRSV hammerhead (32).Here we report on the ligation kinetics and internal equilibrium of the best characterized hammerhead, the Schistosoma ribozyme. This Schistosoma construct is an efficient ligase and yields up to 23% ligated product starting from the fully cleaved substrate, which is the highest level of ligation reported for any unmodified hammerhead.…”

Thermozymes

“…[18][19][20] In our opinion, ribozyme-based devices have the advantage of almost universal applicability for controlling RNA functions. Apart from regulating mRNA translation in bacteria and mRNA integrity in mammalian cells, 21,22 they can be utilized in order to control the activity of tRNAs, 23 16S rRNA, 24 as well as RNAi in mammalia.…”

“…The position was chosen in order to preserve stem I/II interactions that facilitate fast cleavage kinetics at physiological Mg 2+ concentrations in naturally occurring HHRs. [18][19][20] To screen for sequences that respond to changing temperatures with altered gene expression, we randomized six nucleotides (Fig. 1C, communication module highlighted in green) located at the junction between the ribozyme core and the fourU hairpin.…”

Intronic hammerhead ribozymes in mRNA biogenesis

“…The HHR is made up of three double helices (helix I to III) that intersect at a three-way junction containing the catalytic core of 15 highly conserved nucleotides (Figure 1 ). Originally described as a hammerhead-like fold because of its predicted secondary structure, the motif actually adopts in solution a ' Y ' -shaped fold, where helix III coaxially stacks with helix II, and helix I is parallel to the coaxial stack interacting with helix II through tertiary interactions required for efficient self-cleavage in vivo (De la Pe ñ a et al, 2003 ;Khvorova et al , 2003 ;Scott , 2006 : Chi et al , 2008 ) ( Figure 1A). Three different topologies have been described for this ribozyme, named type I, II or III according to the open-ended helix that connects the HHR motif with the flanking sequences ( Figure 1B).…”