Bacterial Ribonuclease P Holoenzyme in Complex with tRNA
Created by Mackenzie Long
Bacterial Ribonuclease P holoenzyme in complex with tRNA (3Q1Q) from Thermotoga maritima is a universal ribozyme responsible for the endonucleolytic cleavage of the precursor transfer RNA’s (pre-tRNA) 5’ leader sequence. Modification of the 5’ and 3’ ends of the pre-tRNA molecules is required to form mature tRNA that can function in protein translation (Chamberlain, 1678). Ribonuclease P (RNase P) is a holoenzyme containing an RNA subunit and one or more protein subunits. The essential RNA component (P RNA) recognizes and cleaves the substrate. The protein subunit (P protein) acts as a cofactor and catalyst increasing the efficiency of cleavage by a magnitude of two to three times. While P RNA can process RNA in the absence of the protein subunit in vitro, the protein is required for RNA processing in vivo. Despite extensive knowledge about the mechanism of the RNA subunit of the holoenzyme, little is known about the way the protein interacts with the pre-tRNA leader and the specific interactions in the active site. Understanding these reaction mechanisms will enhance current knowledge about the protein subunit’s role in the holoenzyme, which will allow for a better understanding of the RNA enzyme mechanism and the essential role of the protein subunit in vivo (Reiter 2012, 1).
The crystallized structure of the holoenzyme is complexed with mature tRNAPHE; therefore, the structure exists as a ribozyme/product complex containing three domains: two RNA subunits and one protein subunit. Subunit A refers to the P protein component that is 118 residues in length, subunit B refers to the RNA P component, which contains an S and C domain and consists of 347 residues, and subunit C refers to the tRNA component of the complex, which is the shortest of the subunits containing 86 residues. Hydropathy calculations of the protein subunit reveal its largely hydrophilic character with the exception of a primarily hydrophobic region spanning from residues 76 to 87 (Berman). Additionally, the protein's core contains primarily hydrophobic residues most notably Val, Ile, and Leu (Kasantsev 2003, 7499). The components of the RNase P are largely unchanged when bound to tRNA (Reiter 2010, 785). The molecular weight of the bacterial RNase P holoenzyme in complex with tRNA is 274.28 Da and its isoelectric point (pI) is 5.52 (Expasy, 1). The secondary structure of the protein subunit is 43% helical (4 alpha helices and 2 3/10 helices), 19% beta sheet (4 beta strands), and also possesses several random coils (Bernstein).
Bacterial RNase P holoenzyme in complex with tRNA and in the presence of the 5’ leader (3Q1R) is an alternate conformation of the RNase P complex. This conformation contains an additional subunit, the 5’ tRNA leader sequence, and so contains four subunits as opposed to three. The three remaining subunits have the same structure and sequence as that of RNase P and thus, have the same function, to cleave the 5’ pre-tRNA leader to form the mature 5’ tRNA terminus (Bernstein). P RNase recognizes its pre-tRNA substrate in the trans conformation (Reiter 2012, 1). The function of pre-tRNA modification by RNase P is governed by two factors: substrate specificity and the chemical mechanism of cleavage. The tertiary fold of the P RNA employs intermolecular base stacking, A-minor, and base pairing interactions for efficient tRNA recognition.
The universally conserved residues of the tertiary fold spanning from Arg-59 to Arg-65 play a structural role rather than a catalytic role by forming H-bonds and electrostatic interactions, which stabilize the RNA subunit. This observation supports the belief that the conservation of sequence is imposed by structural constraints rather than for catalysis (Reiter 2010, 787). Intermolecular base stacking occurs between the unstacked nucleotide bases (G19 and C56) of the tRNA’s D and TΨC loops and the unstacked nucleotide bases (A112 and G147) in the CR-II and CR-III regions of the P RNA’s S-domain. The tRNA’s interaction with the CR-II and CR-III regions also serves as a form of quality control by ensuring that the length of the acceptor stem is approximately seven base pairs. The A minor interaction involves the unstacked A189 nucleotide of the P RNA’s P11 stem that contacts the minor groove of the tRNA acceptor stem. Base pairing and A-minor interactions allow for shape complementarity and convey the major role of the RNA P’s S-domain in substrate recognition (Reiter 2010, 787).
At the 3' terminus of the tRNA subunit, C84, C85, and A86 form canonical base pairs with G264, G263, and G262, respectively in the P15 loop of the P RNA subunit. These interactions separate the 3’ from the 5’ terminus of the tRNA, allowing for the 5’ terminus to be cleaved independently. The 3’ end extends through a tunnel constructed by P6/P15/P16/P17 of the P RNA. This separation of the tRNA 5’ and 3’ ends enables them to interact with P RNA in the active site. One magnesium ligand binding pocket lies adjacent to the site of P RNA-tRNA recognition (Reiter 2010, 787).
The RNase P ribozyme is a multiple turnover rate enzyme that illustrates a conserved RNA-based mechanism of RNA cleavage. The P protein component of the holoenzyme enhances the efficiency of the reaction. Situated between the P15 and P3 stems, the protein subunit of the complex interacts with the CR-IV and CR-V regions of the P RNA. The protein is located in close proximity to the mature 5’ end of the tRNA, but too distant to interact with it. However, the pre-tRNA leader interacts significantly more with the protein than the P RNA subunit of the holoenzyme interacts with the pre-tRNA leader. The P protein directs the 5′ leader pre-tRNA substrate and contacts the conserved regions of the P RNA structure for the purposes of stabilization. Thus, the protein subunit functions both in substrate recognition and catalysis (Reiter 2010, 786).
The 5’ leader sequence of the tRNA contacts Phe-17, Phe-21, Lys-51, Arg-52, and Lys-90 and forms interactions with Ser-26, Gln-28, Lys-56, and Arg-89 on the protein (Reiter 2010, 786). More specifically, Arg-89 of the protein binds to the phosphate backbone of the 3’ leader’s N5 nucleotide, which is positioned adjacent to Lys-90 and Lys-53. Phe-17 interacts with the N4 nucleobase of the pre-tRNA leader in proximity to Phe-21. This positioning of residues results in hydrophobic base interactions and hydrogen bonding that permits substrate binding and catalysis (Reiter 2012, 8-9).
The general mechanism of cleavage involves the metal dependent hydrolysis of the leader sequence’s phosphodiester bond to form the mature tRNA product. In the active site, the tRNA 5’ end is located between the P4 helix of the holoenzyme, a conserved U52 nucleotide, and two magnesium ligands. The P RNA subunit serves as a scaffold for the metal ions, coordinating the metal ions, which serve a crucial role in catalysis by stabilizing the transition state. The metals coordinate with the 5’ phosphorus atom of the tRNA leader (Reiter 2012, 1). Though both stabilized by the phosphate ligands in the complex, the magnesium ions have different binding affinities. The highest affinity magnesium ion activates the nucleophilic hydroxyl ion enabling it to engage in an SN2 nucleophilic substitution (Reiter 2012, 2). The second, lower affinity, magnesium ion stabilizes the transition state and activates the 3’ oxygen by neutralizing its negative charge. The complex contains four metal ions in total, one of which activates the leaving group by lowering the pKa of the water molecule with which it coordinates enabling the proton transfer to the 3’ oxygen of the phosphate. The transition state is shown in Figure 1. The remaining ligand is thought to stabilize pre-tRNA binding (Kazantsev 2006, 731).
Bacterial Ribonuclease P possesses many differences in structure and function from the eukaryotic versions such as Human Pancreatic Ribonuclease (2K11) and serves a function essential for cell life. These characteristics render bacterial RNase P a potential drug target for disarming bacterial invasion by inactivating the RNase activity of the bacteria without harming the eukaryotic or host’s RNase P activity. (Berchanski, 1896). Arginine-Aminoglycoside conjugates (AACs) and the more potent aminoglycoside-polyarginine conjugates (APACs) are competivie inhibitors of RNAse P containing Arg residues that mimic the positively charged, highly conserved RNR region of the RNase P protein preventing the protein subunit and pre-tRNA from binding to the P1P4 and J19/4 regions (Berchanski, 1904). Another potential drug model employing antisense oligonucleotide inhibitors is being explored to inhibit substrate binding at the tRNA 3’-CCA binding (P15 loop) and P10/11-J11/12 regions of the RNA subunit (Willkomm, 1041).
RNase P Protein (1D6T) from Staphylococcus Aureus has a sequence that overlaps with 13% of the query sequence as determined employing the PSI-BLAST database. The E value of 0.002 is less than the threshold E value of 0.05 for the comparison protein indicating significant similarity in the sequences of the two proteins (Cates). The Dali Server is another database utilized to compare proteins; however, it evaluates tertiary or three-dimensional as opposed to primary structure. The comparison protein’s Z-score of 11.9 is greater than 2.0, indicating that the similarities in the folds of the two proteins are significant (Holm).
Despite the fact that the two proteins arise from organisms with significant phylogenetic diversity, most bacterial RNase P proteins contain a similar structure unlike those of eukaryotes and archea. This is expected as the two proteins function to cleave the 5’ end leader sequence from the pre-tRNA to form the mature 5’ end product in bacteria. The root mean square (rms) deviations determined by superimposing the carbon backbones of the two proteins are small. Additionally, the positively charged, highly conserved, RNR regions found in both proteins contribute to their comparable electropositive surfaces that facilitate their interactions with RNA (Kazantsev 2003, 7499). Unlike Thermotoga Maritima, the protein of Staphylococcus Aureus is not crystallized in complex with its tRNA and pre-tRNA subunits and contains a smaller percentage of helices and beta sheets (Bernstein).