Molecular Evolution of Multisubunit RNA Polymerases: Structural Analysis

Comprehensive multiple sequence alignments of the multisubunit DNA-dependent RNA polymerase (RNAP) large subunits, including the bacterial β and β′ subunits and their homologs from archaebacterial RNAPs, eukaryotic RNAPs I-III, nuclear-cytoplasmic large double-stranded DNA virus RNAPs, and plant plastid RNAPs, were created [Lane, W. J. and Darst, S. A. (2009). Molecular evolution of multisubunit RNA polymerases: sequence analysis. In press]. The alignments were used to delineate sequence regions shared among all classes of multisubunit RNAPs, defining common, fundamental RNAP features as well as identifying highly conserved positions. Here, we present a systematic, detailed structural analysis of these shared regions and highly conserved positions in terms of the RNAP structure, as well as the RNAP structure/function relationship, when known.     

Analysis of the Open and Closed Conformations of the β Subunits in Thermophilic F1-ATPase by Solution NMR

F₁-ATPase, composed of α, β, γ, δ, and ? subunits, is a unique enzyme in terms of its rotational catalytic activity. The smallest unit showing this function is the α₃β₃γ complex. We have investigated the α₃β₃γ?^ΔC (?^ΔC, truncated ?) complex from thermophilic Bacillus PS3 (TF₁′, 360 kDa) in the solution state by using the combination of extensive deuteration, segmental-labeling, and CRINEPT (cross-correlated relaxation-enhanced polarization transfer) NMR. Well-resolved CRINEPT-HMQC (heteronuclear multiple-quantum correlation) spectra of partially ¹⁵N-labeled TF₁′ were obtained for this huge and asymmetric protein complex. The spectrum of the C-terminal domain of the β subunit revealed that the open form of the β subunit in the TF₁′ complex is similar to that of the free β monomer. The open β subunit in the TF₁′ complex does not exhibit high affinity for nucleotides unlike the monomer, but this is in agreement with the results of single-molecule analysis of TF₁α₃β₃γ. On the other hand, the closed form of the β subunit in the TF₁′ complex was shown to be distinct from that of the nucleotide-bound β monomer. This is consistent with a previous report that the closed form of the TF₁β monomer could be a catalytically activated state. The loop between the N-terminal β-barrel and the central domain is highly flexible in the TF₁′ complex, in contrast to that in the α₃β₃ hexamer, suggesting that it is affected by the presence of the γ subunit in this area.     

Coordinated nuclear import of RNA polymerase III subunits

Eukaryotic RNA polymerases are multisubunit assemblies, whose enzymatic function in the nucleus is intensively studied. However, little is known about the biogenesis of the three RNA polymerases and coupling to nucleo-cytoplasmic transport. Here, we show that Rpc128, the second largest subunit of RNA polymerase III, was mislocalized to the cytoplasm, when a short sequence in the N-terminal domain was deleted. Importantly, nuclear import of other, but not all, RNA polymerase III subunits was impaired in this RPC128DeltaN mutant. These data suggest that RNA polymerase III subunits are not imported independently into the nucleus but may require preassembly into cytoplasmic subcomplexes for coordinated nuclear uptake. We expect these studies to be a starting point to dissect the complex biogenesis pathway of eukaryotic RNA polymerases.     

Conformational coupling,bridge helix dynamics and active site dehydration in catalysis by RNA polymerase

Molecular dynamics simulation of Thermus thermophilus (Tt) RNA polymerase (RNAP) in a catalytic conformation demonstrates that the active site dNMP–NTP base pair must be substantially dehydrated to support full active site closing and optimum conditions for phosphodiester bond synthesis. In silico mutant β R428A RNAP, which was designed based on substitutions at the homologous position (Rpb2 R512) of Saccharomyces cerevisiae (Sc) RNAP II, was used as a reference structure to compare to Tt RNAP in simulations. Long range conformational coupling linking a dynamic segment of the bridge α-helix, the extended fork loop, the active site, and the trigger loop–trigger helix is apparent and adversely affected in β R428A RNAP. Furthermore, bridge helix bending is detected in the catalytic structure, indicating that bridge helix dynamics may regulate phosphodiester bond synthesis as well as translocation. An active site “latch” assembly that includes a key trigger helix residue Tt β′ H1242 and highly conserved active site residues β E445 and R557 appears to help regulate active site hydration/dehydration. The potential relevance of these observations in understanding RNAP and DNAP induced fit and fidelity is discussed.     

Functional Importance of Bacteriophage ?29 DNA Polymerase Residue Tyr148 in Primer-terminus Stabilisation at the 3′-5′ Exonuclease Active Site

Recent crystallographic resolution of ?29 DNA polymerase complexes with ssDNA at its 3′-5′ exonuclease active site has allowed the identification of residues Pro129 and Tyr148 as putative ssDNA ligands, the latter being conserved in the Kx₂h motif of proofreading family B DNA polymerases. Single substitution of ?29 DNA polymerase residue Tyr148 to Ala rendered an enzyme with a reduced capacity to stabilize the binding of the primer terminus at the 3′-5′ exonuclease active site, not having a direct role in the catalysis of the reaction. Analysis of the 3′-5′ exonuclease on primer/template structures showed a critical role for residue Tyr148 in the proofreading of DNA polymerisation errors. In addition, Tyr148 is not involved in coupling polymerisation to strand displacement in contrast to the catalytic residues responsible for the exonuclease reaction, its role being restricted to stabilisation of the frayed 3′ terminus at the exonuclease active site. Altogether, the results lead us to extend the consensus sequence of the above motif of proofreading family B DNA polymerases into Kx₂hxA. The different solutions adopted by proofreading DNA polymerases to stack the 3′ terminus at the exonuclease site are discussed. In addition, the results obtained with mutants at ?29 DNA polymerase residue Pro129 allow us to rule out a functional role as ssDNA ligand for this residue.     

Mapping of Rpb3 and Rpb5 contact sites on two large subunits, Rpb1 and Rpb2, of the RNA polymerase II from fission yeast

[Rpb1 and Rpb2] Mapping of the contact sites␣on two large subunits of the fission yeast Schizosaccharomyces pombe RNA polymerase II with two small subunits, Rpb3 and Rpb5, was carried out using the two-hybrid screening system in the budding yeast Saccharomyces cerevisiae. Rpb5 was found to interact with any fragment of Rpb1 that contained the region H, which is conserved among the subunit 1 homologues of all RNA polymerases, including the β' subunit of prokaryotic RNA polymerases. In agreement with the fact that Rpb5 is shared among all three forms of eukaryotic RNA polymerases, the region H of RNA polymerase I subunit 1 (Rpa190) was also found to interact with Rpb5. On the other hand, two-hybrid screening of Rpb2 fragments from RNA polymerase II indicated the presence of an Rpb3 contact site in the region H which is conserved among the subunit 2 homologues of all RNA polymerases, including the β subunit of prokaryotic RNA polymerases. Possible functions of the regions H in the subunits 1 and 2 are discussed. Received: 10 December 1997 / Accepted: 14 April 1998     

Different Behaviors In Vivo of Mutations in the β Hairpin Loop of the DNA Polymerases of the Closely Related Phages T4 and RB69

The T4 and RB69 DNA replicative polymerases are members of the B family and are highly similar. Both replicate DNA with high fidelity and employ the same mechanism that allows efficient switching of the primer terminus between the polymerase and exonuclease sites. Both polymerases have a β hairpin loop (hereafter called the β loop) in their exonuclease domains that plays an important role in active-site switching. The β loop is involved in strand separation and is needed to stabilize partially strand-separated exonuclease complexes. In T4 DNA polymerase, modification of the β-loop residue G255 to Ser confers a strong mutator phenotype in vivo due to a reduced ability to form editing complexes. Here, we describe the RB69 DNA polymerase mutant with the equivalent residue (G258) changed to Ser but showing only mild mutator activity in vivo. On the other hand, deletion of the tip of the RB69 β loop confers a strong mutator phenotype in vivo. Based on detailed mutational spectral analyses, DNA binding activities, and coupled polymerase/exonuclease assays, we define the differences between the T4 and RB69 polymerases. We propose that their β loops facilitate strand separation in both polymerases, while the residues that form the loop have low structural constraints.     

Identification of Critical Residues for the Tight Binding of Both Correct and Incorrect Nucleotides to Human DNA Polymerase λ

DNA polymerase λ (Pol λ) is a novel X-family DNA polymerase that shares 34% sequence identity with DNA polymerase β. Pre-steady-state kinetic studies have shown that the Pol λ-DNA complex binds both correct and incorrect nucleotides 130-fold tighter, on average, than the DNA polymerase β-DNA complex, although the base substitution fidelity of both polymerases is 10^− 4 to 10^− 5. To better understand Pol λ's tight nucleotide binding affinity, we created single-substitution and double-substitution mutants of Pol λ to disrupt the interactions between active-site residues and an incoming nucleotide or a template base. Single-turnover kinetic assays showed that Pol λ binds to an incoming nucleotide via cooperative interactions with active-site residues (R386, R420, K422, Y505, F506, A510, and R514). Disrupting protein interactions with an incoming correct or incorrect nucleotide impacted binding to each of the common structural moieties in the following order: triphosphate ? base > ribose. In addition, the loss of Watson-Crick hydrogen bonding between the nucleotide and the template base led to a moderate increase in K_d. The fidelity of Pol λ was maintained predominantly by a single residue, R517, which has minor groove interactions with the DNA template.     

Binding of 2′-Amino-2′-Deoxycytidine-5′-Triphosphate to Norovirus Polymerase Induces Rearrangement of the Active Site

Crystal structures of a genogroup II.4 human norovirus polymerase bound to an RNA primer-template duplex and the substrate analogue 2′-amino-2′-deoxycytidine-5′-triphosphate have been determined to 1.8 Å resolution. The alteration of the substrate-binding site that is required to accommodate the 2′-amino group leads to a rearrangement of the polymerase active site and a disruption of the coordination shells of the active-site metal ions. The mode of binding seen for 2′-amino-2′-deoxycytidine-5′-triphosphate suggests a novel molecular mechanism of inhibition that may be exploited for the design of inhibitors targeting viral RNA polymerases.     

Improving the prediction of RNA secondary structure by detecting and assessing conserved stems

The prediction of RNA secondary structure can be facilitated by incorporating with comparative analysis of homologous sequences. However, most of existing comparative methods are vulnerable to alignment errors and thus are of low accuracy in practical application. Here we improve the prediction of RNA secondary structure by detecting and assessing conserved stems shared by all sequences in the alignment. Our method can be summarized by: 1) we detect possible stems in single RNA sequence using the so-called position matrix with which some possibly paired positions can be uncovered; 2) we detect conserved stems across multiple RNA sequences by multiplying the position matrices; 3) we assess the conserved stems using the Signal-to-Noise; 4) we compute the optimized secondary structure by incorporating the so-called reliable conserved stems with predictions by RNAalifold program. We tested our method on data sets of RNA alignments with known secondary structures. The accuracy, measured as sensitivity and specificity, of our method is greater than predictions by RNAalifold.     

Mechanism of U-Insertion RNA Editing in Trypanosome Mitochondria: Characterization of RET2 Functional Domains by Mutational Analysis

3′-Terminal uridylyl transferases (TUTases) selectively bind uridine 5′-triphosphate (UTP) and catalyze the addition of uridine 5′-monophosphate to the 3′-hydroxyl of RNA substrates in a template-independent manner. RNA editing TUTase 1 and RNA editing TUTase 2 (RET2) play central roles in uridine insertion/deletion RNA editing, which is an essential part of mitochondrial RNA processing in trypanosomes. Although the conserved N-terminal (catalytic) domain and C-terminal (nucleotide base recognition) domain are readily distinguished in all known TUTases, nucleotide specificity, RNA substrate preference, processivity, quaternary structures, and auxiliary domains vary significantly among enzymes of divergent biological functions. RET2 acts as a subunit of the RNA editing core complex to carry out guide-RNA-dependent U-insertion into mitochondrial mRNA. By correlating mutational effects on RET2 activity as recombinant protein and as RNA editing core complex subunit with RNAi-based knock-in phenotypes, we have assessed the UTP and RNA binding sites in RET2. Here we demonstrate functional conservation of key UTP-binding and metal-ion-coordinating residues and identify amino acids involved in RNA substrate recognition. Invariant arginine residues 144 and 435 positioned in the vicinity of the UTP binding site are critical for RET2 activity on single-stranded and double-stranded RNAs, as well as function in vivo. Recognition of a double-stranded RNA, which resembles a guide RNA/mRNA duplex, is further facilitated by multipoint contacts across the RET2-specific middle domain.     

P. aeruginosa PilT Structures with and without Nucleotide Reveal a Dynamic Type IV Pilus Retraction Motor

Type IV pili are bacterial extracellular filaments that can be retracted to create force and motility. Retraction is accomplished by the motor protein PilT. Crystal structures of Pseudomonas aeruginosa PilT with and without bound β,γ-methyleneadenosine-5′-triphosphate have been solved at 2.6 Å and 3.1 Å resolution, respectively, revealing an interlocking hexamer formed by the action of a crystallographic 2-fold symmetry operator on three subunits in the asymmetric unit and held together by extensive ionic interactions. The roles of two invariant carboxylates, Asp Box motif Glu163 and Walker B motif Glu204, have been assigned to Mg²⁺ binding and catalysis, respectively. The nucleotide ligands in each of the subunits in the asymmetric unit of the β,γ-methyleneadenosine-5′-triphosphate-bound PilT are not equally well ordered. Similarly, the three subunits in the asymmetric unit of both structures exhibit differing relative conformations of the two domains. The 12° and 20° domain rotations indicate motions that occur during the ATP-coupled mechanism of the disassembly of pili into membrane-localized pilin monomers. Integrating these observations, we propose a three-state “Ready, Active, Release” model for the action of PilT.     

A Structural Basis for the Recognition of 2′-Deoxyguanosine by the Purine Riboswitch

Riboswitches are noncoding RNA elements that are commonly found in the 5′-untranslated region of bacterial mRNA. Binding of a small-molecule metabolite to the riboswitch aptamer domain guides the folding of the downstream sequence into one of two mutually exclusive secondary structures that directs gene expression. The purine riboswitch family, which regulates aspects of purine biosynthesis and transport, contains three distinct classes that specifically recognize guanine/hypoxanthine, adenine, or 2′-deoxyguanosine (dG). Structural analysis of the guanine and adenine classes revealed a binding pocket that almost completely buries the nucleobase within the core of the folded RNA. Thus, it is somewhat surprising that this family of RNA elements also recognizes dG. We have used a combination of structural and biochemical techniques to understand how the guanine riboswitch could be converted into a dG binder and the structural basis for dG recognition. These studies reveal that a limited number of sequence changes to a guanine-sensing RNA are required to cause a specificity switch from guanine to 2′-deoxyguanosine, and to impart an altered structure for accommodating the additional deoxyribose sugar moiety.