Sequence elements outside the hammerhead ribozyme catalytic core enable intracellular activity

AbstractHairpin ribozymes occur naturally only in the satellite RNAs of tobacco ringspot virus (TRsV), chicory yellow mottle virus (CYMoV) and arabis mosaic virus (ArMV). The catalytic centre of the predominantly studied sTRsV hairpin ribozyme, and of sArMV is organised around a four-way helical junction. We show here that sCYMoV features a five-way helical junction instead. Mutational analysis indicates that the fifth stem does not influence kinetic parameters of the sCYMoV hairpin ribozymein vitroreactions, and therefore seems an appendix to that junction in the other ribozymes. We report further that all three ribozymes feature a three-way helical junction outside the catalytic core in stem A, with Watson-Crick complementarity to loop nucleotides in stem B. Kinetic analyses of cleavage and ligation reactions of several variants of the sTRsV and sCYMoV hairpin ribozymesin vitroshow that the presence of this junction interferes with their reactions, particularly the ligation. We provide evidence that this is not due to a presumed interaction of the afore-mentioned elements in stems A and B. The evolutionary survival of thiscis-inhibiting element seems rather to be caused by the coincidence of its position with that of the hammerhead ribozyme in the other RNA polarity.

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Prediction of hammerhead ribozyme intracellular activity with the catalytic core fingerprint

Biochemical Journal ◽

10.1042/bj20121761 ◽

2013 ◽

Vol 451 (3) ◽

pp. 439-451 ◽

Cited By ~ 1

Author(s):

Marta Magdalena Gabryelska ◽

Eliza Wyszko ◽

Maciej Szymański ◽

Mariusz Popenda ◽

Jan Barciszewski

Keyword(s):

Gene Expression ◽

Tertiary Structure ◽

Hammerhead Ribozyme ◽

Regulation Of Gene Expression ◽

Flanking Sequence ◽

Catalytic Core ◽

Model Calculations ◽

Intracellular Activity

Hammerhead ribozyme is a versatile tool for down-regulation of gene expression in vivo. Owing to its small size and high activity, it is used as a model for RNA structure–function relationship studies. In the present paper we describe a new extended hammerhead ribozyme HH-2 with a tertiary stabilizing motif constructed on the basis of the tetraloop receptor sequence. This ribozyme is very active in living cells, but shows low activity in vitro. To understand it, we analysed tertiary structure models of substrate–ribozyme complexes. We calculated six unique catalytic core geometry parameters as distances and angles between particular atoms that we call the ribozyme fingerprint. A flanking sequence and tertiary motif change the geometry of the general base, general acid, nucleophile and leaving group. We found almost complete correlation between these parameters and the decrease of target gene expression in the cells. The tertiary structure model calculations allow us to predict ribozyme intracellular activity. Our approach could be widely adapted to characterize catalytic properties of other RNAs.

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Diffusely Bound Mg2+Ions Slightly Reorient Stems I and II of the Hammerhead Ribozyme To Increase the Probability of Formation of the Catalytic Core†

Biochemistry ◽

10.1021/bi0347757 ◽

2003 ◽

Vol 42 (33) ◽

pp. 9924-9936 ◽

Cited By ~ 31

Author(s):

David Rueda ◽

Katrin Wick ◽

S. Elizabeth McDowell ◽

Nils G. Walter

Keyword(s):

Hammerhead Ribozyme ◽

Catalytic Core

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A Conformational Change in the Catalytic Core of the Hammerhead Ribozyme upon Cleavage of an RNA Substrate†

Biochemistry ◽

10.1021/bi9620520 ◽

1997 ◽

Vol 36 (3) ◽

pp. 518-525 ◽

Cited By ~ 28

Author(s):

Jean-Pierre Simorre ◽

Pascale Legault ◽

Arlene B. Hangar ◽

Paul Michiels ◽

Arthur Pardi

Keyword(s):

Conformational Change ◽

Hammerhead Ribozyme ◽

Catalytic Core

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Zinc-dependent cleavage in the catalytic core of the hammerhead ribozyme: evidence for a pH-dependent conformational change

Nucleic Acids Research ◽

10.1093/nar/gkg349 ◽

2003 ◽

Vol 31 (10) ◽

pp. 2595-2600 ◽

Cited By ~ 10

Author(s):

E. J. Borda

Keyword(s):

Conformational Change ◽

Hammerhead Ribozyme ◽

Catalytic Core ◽

Ph Dependent

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Inhibition of HIV-1 gp41 expression with hammerhead ribozymes

Biochemical Journal ◽

10.1042/bj20150398 ◽

2015 ◽

Vol 471 (1) ◽

pp. 53-66 ◽

Cited By ~ 1

Author(s):

Agnieszka Fedoruk-Wyszomirska ◽

Maciej Szymański ◽

Paweł Głodowicz ◽

Marta Gabryelska ◽

Eliza Wyszko ◽

...

Keyword(s):

Hammerhead Ribozyme ◽

In Vitro Studies ◽

Hammerhead Ribozymes ◽

Intracellular Activity ◽

Highly Effective ◽

Hiv 1

We propose a new highly effective hammerhead ribozyme against HIV-1 gp41 that can be potentially used as a basis in HIV-1 therapy. We demonstrate that the ribozyme's intracellular activity cannot be inferred solely from in vitro studies.

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