P4-P6 RNA Ribozyme Domain
Created by Hyeon Jin Lee
Synthetically constructed P4-P6 RNA ribozyme domain (1GID) is one of the three domains (Figure 1) that make up the group I intron, a ribozyme, found in the protozoa Tetrahymena thermophilia (1). Group I introns are mobile genetic elements that are inserted into a variety of ribonucleic acid genes with a preference for conserved regions (2). They are typically found in the nuclei, mitochondria, and chloroplasts (2) of bacteria, simple eukaryotes, and plants (3). Group I introns are catalytically active RNAs capable of splicing themselves out of precursor RNAs during post-transcriptional processing (3). Most active introns are inserted after a uracil residue in RNA, end with a guanine residue (2), and are flanked with a 5’- and 3’-exons (3). Consequently, group I intron ribozymes are their own substrate as part of the precursor RNA and product as the excised intron. It was previously believed that all enzymes were proteins, but the discovery of this protein-like RNA with enzymatic activity and mobility led to the redefinition of an enzyme. Thus, the P4-P6 domain, a class of ribozymes, is of interest to scientists (2).
Self-splicing is aided by magnesium ions and takes place via a two-step transesterification mechanism initiated by an exogenous guanosine nucleotide binding to the guanosine-binding site (Figure 2) (4). First, the bound exogenous guanosine at G-264 cleaves the 5’-splice site, completing the first transesterification step (5). The intron then goes through a conformational change, replacing the exogenous guanosine (5). The 5’-exon, still base paired to the intron, cleaves the 3’-splice site at G-414, completing the second transesterification step (5). The 5’- and 3’-exons are thus ligated and the intron excised (5). Due to the active exogenous guanosine still present in the excised intron, the intron is capable of acting like a true catalyst that can act intermolecularly on other precursor RNAs in vitro (5). In vivo, however, the intron usually acts only on itself (1).
The P4-P6 ribozyme domain contains 158 of the 247 nucleotides, roughly half of the active intron (6), but does not contain the catalytic sites (4). It weighs 103331.18 Da and has an undetermined isoelectric point (7). It is hypothesized that it initiates the tertiary folding of the entire intron into a catalytically active one (2). This may explain its highly conserved nucleotide sequences, as well as the intricate and extensive hydrogen bond interactions that take place within this domain (6). The domain also adopts a similar globular conformation as an independent unit and as a part of the entire intron, which indicates its folding is important for the intron as a whole (2). Additionally, experiments that mutated certain nucleotides in the domain revealed that parts of the domain are critical for the intron’s catalytic function, stability, and folding (6).
The synthesized P4-P6 domain crystal contains two identical chains of the domain, labeled as that act independently (4). The domain contains base-paired (P) segments and joining (J) regions that overall divide the domain into thirteen sections (Figure 1) (6). The base-paired segments are formed intramolecularly (1) in both a Watson-Crick and noncanonical manner (6). The J3/4 and J6/7 regions each have highly conserved triple bases that link the P4-P6 domain to the P3-P9 domain (4). The J4/5 region contains adenosines (A206, A207, A115, A114, and A113) that crosslink to the guanosine in the G·U wobble base pair (6) involved in the 5’-splice site recognition during splicing (8). It is hypothesized that J4/5 is involved in correctly orienting the exogenous guanosine at G-264 for proper catalytic activity (6). P5a, P5b, and P5c, collectively referred to as the P5abc extension, are the first portions of the whole intron to fold (2) and provide the ribozyme with stability and catalytic function (6). The J5/J5a region is vital for the catalytic function of the intron and also provides structural support (4).
The P4-P6 domain contains many secondary elements commonly found in RNA molecules: coaxially stacked helices (6), a metal ion core (9), bulges, hairpin loops joined at a two-stem junction, a tetraloop-receptor motif (1), A-minor motifs (6), and ribose zippers (1). Many of these secondary structures contain important residues that play key roles in folding the domain into its tertiary structure. There are two major helical segments in the domain: one made of coaxially stacked helices of the P4, P5, and P6 segments and the other composed of coaxially stacked helices of the P5abc extension segments (10). A 150° sharp bend in the J5/J5a region aligns two major coaxially stacked helical regions side by side, allowing important regions on both helices to interact with one another to form its tertiary structure (6). The polyanionic phosphate backbones are able to closely approach each other due to the domain’s metal ion core of magnesium ions (2) that alleviate and stabilize the electrostatic repulsion (6). The first two magnesium ions are coordinated by A-184, A-186, and A-187 in the A-rich bulge; a third magnesium ion is coordinated by G-188; a fourth by A-140 and G-163; and the fifth magnesium ion by A-171 (9).
There are a couple of bulges in the domain, with the most important being the A-rich bulge (6). The A-rich bulge is a corkscrew turn with its bases rotated outwards and its phosphate groups rotated and tightly packed inwards (6). Adenosines 183, 184, 186, and 187 are highly conserved and form long-range hydrogen bonds with the base pairs in the P4 segment (Table 1) (6). There are two major hairpin loops that meet at a two-stem junction (6). One of the hairpin loops is a GNRA tetraloop composed of the sequence 5’-GAAA-3’, whose adenosines form specific hydrogen bonds with residues (U-224, A-248, C-223, and G-250) in the tetraloop receptor (Table 2) (6). The extensive hydrogen bonding interactions between the two helical halves via the A-rich bulge and GNRA tetraloop bring the two helical halves together to form the tertiary structure (6). These adenosine interactions create a few of the many A-minor motifs commonly found in RNA molecules (10). “Unpaired adenosine residues [that] dock into the minor groove of a receptor helix” characterizes the A-minor motif that is important for the conformation of RNA (10). Additionally, the hydrogen bond networks formed between the helical halves via the A-rich bulge and tetraloop regions create what is known as ribose zippers (6). Hydrogen bonding between the 2’-OH or the pyrimidine O2 (purine N3) of one base and the 2’-OH of the second base characterize ribose zippers (6).
Metal ligands associated with the P4-P6 ribozyme domain also aid the catalysis of the splicing activity in addition to the folding of the domain previously mentioned. The ligands associated with this domain consist of magnesium ions and cobalt hexammine (III) ions (7). Like most ribozymes, the group I intron is a metalloenzyme that has a higher preference for magnesium ions (2). A recent model proposes that at least three magnesium ions are needed for activationof , stabilization of, and proper orientation during the splicing reaction (5). The cobalt hexammine (III) ion is believed to be able to substitute magnesium ions functionally: it is able to aid in the folding of the ribozyme (11) as well as initiate and aid in the splicing activity (12).
The residues involved in the extensive hydrogen bonding and magnesium ion coordination (Table 3) are the functionally important residues in this domain as they dictate how this domain folds. The ionic interactions between the residues and the magnesium ions as well as the hydrogen bonds that bring the helical halves together (Tables 1, 2, and 3) are the important interactions in this domain. These residues and ligands with their respective interactions regulate how the P4-P6 ribozyme domain folds, whose folding activity initiates the folding of the entire intron into an active intron.
There are a few instances of alternate conformations of this domain. The crystallized form of this whole ribozyme contains two identical chains with the exception of a slight difference in their 3’-terminuses (4). In an intact domain, the base triples in J3/4 and J6/7 form a triple helix with the P4, P6, P3, and P7 helices; this triple helical array is absent in the independent crystal unit (4). Nucleotide base mutations have been made to the domain that resulted in slight structural changes (13). The P4-P6 ribozyme domain with C109G and G212C mutations (1L8V) has one less hydrogen bond and a slightly carbon atom burying between the two helical halves (10). A deletion of C209 (1HR2) resulted in an adenine residue more fully docked into a minor groove in the P4 segment (14). This resulted in greater tertiary stability and a faster crystallization rate of the P4-P6 domain (14).
PSI-BLAST was used to find a comparison protein based on primary structure (13) while Dali was used to find a comparison protein based on tertiary structure (15). ExPASy's compute pI/Mw tool was used to determine the isoelectric point based on amino acid sequence (16). Unfortunately, all three servers were designed to process proteins using their amino acid sequences. Since the P4-P6 ribozyme domain is a nucleic acid and solely consists of nucleotide bases, the servers were incapable of processing the ribozyme domain.
Individual research, however, led to a functional comparison ribozyme – the group II intron ribozyme. Both group I and group II intron ribozymes are mobile, self-splicing metalloenzymes found in the various RNA precursors found in plant mitochondria and chloroplasts (5). They also have conserved secondary structures necessary for catalysis and identification, and locate the 5’-splice site by internal guide sequences that properly align the 5’-exon (17). With respect to nucleotide sequences, conformations, and splicing pathways, the two groups have very little in common (17). The group II intron (3BWP) is larger at 412 residues and has only one chain in its crystallized structure (7). In comparison, the group I intron (1X8W) is smaller at 247 residues and has two identical chains in its crystallized structure (4). They proceed through a different pathway that is more representative of the pathway taken by spliceosomes, a ribonucleoprotein enzyme (Figure 3).