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. 2016 Jan 29;44(4):1813–1832. doi: 10.1093/nar/gkw050

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© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 6. — Recombinant LACTB2 cleaves ssRNA but not dsRNA or ssDNA. (A) LACTB2 does not cleave ssDNA. LACTB2 was incubated with 5΄ [³²P] 30 nt-long RNA or ssDNA of the same sequence for 15, 30 and 60 min at 37°C. Lane (−): RNA or ssDNA incubated for 60 min, with no added protein. Lanes under the triangle: incubation for 15, 30 and 60 min. (B) A stem-loop structured RNA is cleaved by LACTB2 at the loop. LACTB2 was incubated with 5΄ [³²P] 30 nt stem-loop-shaped RNA, as presented below the figure. The reaction was incubated at 25°C, a temperature in which the stem is formed, for 15, 30 and 60 min in the presence of yeast tRNA. Lane (L): nucleotide ladder of the 30 nt RNA. Lane (−): incubation for 60 min, with no added protein. Lanes under the triangle: incubation for 15, 30 and 60 min. The sequence and predicted stem-loop structure formed at 25°C is shown at the bottom. (C) LACTB2 cleaves the substrate at a temperature in which the stem-loop structure is not formed. LACTB2 was incubated at 25°C in the presence of yeast tRNA with a linear 16 nt RNA corresponding to the nucleotide sequence of the stem of the molecule used in (B), in order to show the presence of cleavage sites in this sequence. In addition, the same reaction presented in panel B, was repeated at 37°C, where the stem-loop structure is not predicted to be formed. The sequence and structure of the RNA at 37°C are shown below the autoradiogram. Black arrows indicate the cleavage sites.