Insights into how RNase R degrades structured RNA: analysis of the nuclease domain

Abstract

RNase R readily degrades highly structured RNA, whereas its paralogue, RNase II, is unable to do so. Furthermore, the nuclease domain of RNase R, devoid of all canonical RNA-binding domains, is sufficient for this activity. RNase R also binds RNA more tightly within its catalytic channel than does RNase II, which is thought to be important for its unique catalytic properties. To investigate this idea further, certain residues within the nuclease domain channel of RNase R were changed to those found in RNase II. Among the many examined, we identified one amino acid residue, R572, that has a significant role in the properties of RNase R. Conversion of this residue to lysine, as found in RNase II, results in weaker substrate binding within the nuclease domain channel, longer limit products, increased activity against a variety of substrates and a faster substrate on-rate. Most importantly, the mutant encounters difficulty in degrading structured RNA, pausing within a double-stranded region. Additional studies show that degradation of structured substrates is dependent upon temperature, suggesting a role for thermal breathing in the mechanism of action of RNase R. On the basis of these data, we propose a model in which tight binding within the nuclease domain allows RNase R to capitalize on the natural thermal breathing of an RNA duplex to degrade structured RNAs.

Figure 1. Distribution of Variant Residues in the Nuclease Domain Channel of RNase R

All panels were generated using PyMOL (DeLano, W. L. The PyMOL Molecular Graphics System (2002). DeLano Scientific, San Carlos, CA. CA.[Online.] http://www.pymol.org) based on the crystal structure of RNase II (PDB code: 2IX1). (a) A cartoon representation of RNase R clearly showing the path that the RNA takes between the cold-shock and S1 domains into the nuclease domain. The cold-shock domains are colored in cyan and blue for CSD1 and CSD2, respectively, the nuclease domain is green with the catalytic magnesium ion represented by an orange sphere and the S1 domain is red. The RNA substrate is shown as yellow sticks. (b) An alternative perspective of RNase R obtained by rotation of panel (a). Coloring is the same as in panel (a). (c) The same as panel (b) with residues comprising the nuclease domain channel wall shown in a surface representation. Residues that are conserved between RNase R and RNase II remain in green and residues that differ are in magenta. (d) An enlarged surface representation of the residues which form the nuclease domain channel wall. As in panel (c), residues that are conserved between RNase R and RNase II are shown in green and residues that differ are in magenta. (The black areas are where amino acids have been removed to allow visualization of the channel). The approximate positions of the RNase R mutants constructed in this study are indicated. For orientation purposes, the entrance and exit of the channel are labeled.

Figure 2. Activity of Nuclease Domain Channel Mutant Extracts on Structured RNA

Assays were carried out as described under “Materials and Methods” with 10 μM ds17-A₁₇ substrate and 2.5 μg wild-type RNase R, 0.5 μg wild-type RNase II, 2.5 μg uninduced, 2.5 μg RNase R (273-279), 2.5 μg RNase R (428-433), 5 μg RNase R H456N, 2.5 μg RNase R (523/545), 10 μg RNase R H565T, 2.5 μg RNase R R572K and 2.5 μg RNase R (620-627) cell extract. Aliquots were taken at the indicated times and analyzed by denaturing PAGE. A schematic representation of the substrate is shown at the top of the figure with the position of the ³²P label denoted by an asterisk. Non-specific product bands that do not appear to originate from RNase R activity are indicated on the left of the wild-type RNase R panel by arrowheads.

Figure 3. Activity of RNase R H565T R572K and RNase R H456N H565T R572K on Structured RNA

Assays were carried out as described under “Materials and Methods” with 10 μM ds17-A₁₇ substrate and 2.5 μg wild-type RNase R, 10 μg H565T, 2.5 μg R572K, 2.5 μg H565T R572K and 5 μg H456N H565T R572K cell extract. Aliquots were taken at the indicated times and analyzed by denaturing PAGE. A schematic representation of the substrate is shown at the top of the figure with the position of the ³²P label denoted by an asterisk.

Figure 4. Purity of RNase R Mutants

One μg of purified wild-type RNase R, RNase R R572K, RNase R H456N H565T R572K and RNase II were resolved by 10% SDS-PAGE and visualized by Coomassie blue staining. The molecular masses, in kDa, of protein standards (M) are indicated on the left. Pure RNase II was obtained from Dr. A. Malhotra (University of Miami, Miami, Florida).

Figure 5. Activity of Purified RNase R R572K and RNase R H456N H565T R572K

Activity assays were carried out as described under “Materials and Methods”. Aliquots were taken at the indicated times and analyzed by denaturing PAGE. (a) ss17-A₁₇ was present at 10 μM with 0.1 μM wild-type RNase R, 0.01 μM R572K, 0.02 μM H456N H565T R572K and 0.002 μM RNase II. (b) ds17-A₁₇ was present at 10 μM with 0.2 μM wild-type RNase R, 0.03 μM RNase R R572K, 0.1 μM RNase R H456N H565T R572K and 0.002 μM RNase II. A schematic representation of the substrates is shown above each panel with the position of the ³²P label indicated by an asterisk.

Figure 6. Activity of Purified Wild-Type RNase R and RNase R R572K Under Single-Turnover Conditions

Single-turnover experiments were carried out as described under “Materials and Methods”. Reactions were allowed to proceed for the indicated times and analyzed by denaturing PAGE. A schematic representation of the substrate is shown at the top of the figure with the position of the ³²P label indicated by an asterisk.

Figure 7. Effect of Temperature on the Ability of RNase R to Degrade Structured RNA

Assays were carried out as described under “Materials and Methods” except they were performed at the indicated temperatures. ss17-A₁₇ was present at 10 μM with 0.07 μM wild-type RNase R or ds17-A₁₇ was present at 10 μM with 0.2 μM RNase R. Aliquots were taken during an appropriate time-course to measure linear degradation of the substrate and analyzed by denaturing PAGE. Results are plotted as a ratio of the rate on double-stranded RNA to the rate on single-stranded RNA. The values represent the mean of two experiments and the standard deviation is indicated.

Figure 8. A Model for the Mechanism of Degradation of Double-Stranded RNA by RNase R

(a) RNase R. (b) RNase II. The RNA-binding domains are shown in cyan, blue and red for the CSD1s, CSD2s and S1 domains, respectively. The nuclease domains are in green with the catalytic centers shown as magenta triangles. In the binding step, RNase R or RNase II bind to the 3′ single-stranded overhang of a structured RNA molecule. The 3′ terminal nucleotide is then cleaved and released. Translocation of both enzymes is blocked by the double-stranded region of the RNA. RNase R remains bound to the RNA substrate as the end of the duplex is opened by thermal breathing and/or a wedge at the entrance to the nuclease domain channel. RNase R translocates forward to position the RNA for the cleavage of the next nucleotide. In contrast, following nucleotide cleavage, the RNA dissociates from RNase II preventing further degradation of the substrate.