In vitro assembly of an archaeal D-L-N RNA polymerase subunit complex reveals a eukaryote-like structural arrangement.

Archaeal RNA polymerases (RNAPs) resemble the eukaryotic nuclear RNAPs in complexity, and many of their subunits display a high degree of sequence similarity to their eukaryotic counterparts. Here we describe specific protein-protein contacts present between individual recombinant RNAP subunits from the archaeon Methanococcus jannaschii. Subunits D and L interact specifically with each other in two-hybrid assays. D also interacts under the same conditions with the RPB11 and AC19 subunits from the yeast Saccharomyces cerevisiae, suggesting that essential elements of the binding surface between these proteins have been conserved across the archaeal/eukaryotic evolutionary domain boundary. Interactions between L and RPB3 or AC40 were, however, not detectable. Recombinant D and L subunits associate under in vitro conditions and copurify with each other during size-exclusion chromatography. Addition of an another recombinant subunit (N) to the D-L complex results in the formation of a triple complex. This D-L-N complex resembles the RPB3-RPB11-RPB10 or AC40-AC19-RPB10 complexes in eukaryotic RNAPIIand RNAPI/RNAPIII, respectively. Our data provide evidence for a close similarity in the quaternary arrangement of a subset of archaeal and eukaryotic RNA polymerase subunits and the conservation of the protein-protein contacts formed between them.     

Early steps in the path of nascent ribonucleic acid across the surface of ribonucleic acid polymerase, determined by photoaffinity labeling

The photoaffinity probes beta-(4-azidophenyl) adenosine 5'-diphosphate (N3PhppA) and beta-(4-azidophenyl) adenylyl-(3'--5')-uridine 5'-diphosphate (N3PhppApU) were used to determine the RNA polymerase subunit contacts made by the 5' ends of three nascent RNA chains. Ternary enzyme-poly[d(A-T)].oligonucleotide complexes were prepared in which the nascent oligonucleotide contained a photoaffinity label at the 5' end and a 32P radiolabel only at the 3' end. The length of the RNA was fixed at two, three, or four nucleotides. Photolysis of the ternary complexes was followed by dissociation, polyacrylamide gel electrophoresis, autoradiography, and scintillation counting. With a dinucleotide probe, the enzyme subunits labeled were beta' (71%) and sigma (21%). Photolysis of the ternary complex containing trinucleotide RNA also resulted in labeling of the beta' (64%) and sigma (35%) subunits. With a tetranucleotide, the beta' subunit was very heavily labeled (88%), and a small amount of labeling of the beta (7%) and sigma (4%) subunits was observed. The alpha subunit was not labeled with any of the probes. These results imply that a conformational change, possibly involving dissociation of the sigma subunit, occurs in the enzyme as the ribonucleotide is elongated from a tri- to a tetranucleotide.     

Structural and functional homology between the RNAP(I) subunits A14/A43 and the archaeal RNAP subunits E/F

In the archaeal RNA polymerase and the eukaryotic RNA polymerase II, two subunits (E/F and RPB4/RPB7, respectively) form a heterodimer that reversibly associates with the core of the enzyme. Recently it has emerged that this heterodimer also has a counterpart in the other eukaryotic RNA polymerases: in particular two subunits of RNA polymerase I (A14 and A43) display genetic and biochemical characteristics that are similar to those of the RPB4 and RPB7 subunits, despite the fact that only A43 shows some sequence homology to RPB7. We demonstrate that the sequence of A14 strongly suggests the presence of a HRDC domain, a motif that is found at the C-terminus of a number of helicases and RNases. The same motif is also seen in the structure of the F subunit, suggesting a structural link between A14 and the RPB4/C17/subunit F family, even in the absence of direct sequence homology. We show that it is possible to co-express and co-purify large amounts of the recombinant A14/A43 heterodimer, indicating a tight and specific interaction between the two subunits. To shed light on the function of the heterodimer, we performed gel mobility shift assays and showed that the A14/A43 heterodimer binds single-stranded RNA in a similar way to the archaeal E/F complex.     

Monoclonal antibodies as probes of the topological arrangement of the alpha subunits of Escherichia coli RNA polymerase

Three monoclonal anti-alpha antibodies were used to study the properties of the alpha subunit of Escherichia coli RNA polymerase. None of the monoclonal antibodies inhibited the d(A-T)n-directed synthesis of r(A-U)n. Reassembly of the RNA polymerase core was blocked by mAb 129C4 or mAb 126C6 while no effect was observed with mAb 124D1. The conversion of premature to mature core was partially inhibited by mAb 129C4 and almost totally inhibited by mAb 126C6. The data suggest that during the course of core assembly at least one of the alpha subunits undergoes conformational changes. The increase in affinity of mAb 126C6 for assembled alpha compared with free alpha also implies that alpha undergoes conformational changes during RNA polymerase assembly. Double antibody binding studies showed that the epitopes for mAb 124D1 and mAb 129C4 are available on only one of the alpha subunits in RNA polymerase. It would appear that the relevant domain on one of the alpha subunits in RNA polymerase is well exposed whereas this domain on the second alpha subunit is shielded by interaction with regions of the large beta and beta' subunits. The alpha domain in which the epitope for mAb 126C6 resides is not impeded by subunit interactions in the RNA polymerase. The data obtained also suggest that in the holoenzyme the sigma subunit may be positioned close to one of the alpha subunits, probably to the more exposed alpha. The alpha beta complex is the minimal stable subassembly since one of the alpha subunits dissociates from the alpha 2 beta complex following binding of any of the monoclonal antibodies studied.(ABSTRACT TRUNCATED AT 250 WORDS)     

Proteomic characterization of archaeal ribosomes reveals the presence of novel archaeal-specific ribosomal proteins

Protein synthesis occurs in macromolecular particles called ribosomes. All ribosomes are composed of RNA and proteins. While the protein composition of bacterial and eukaryotic ribosomes has been well-characterized, a systematic analysis of archaeal ribosomes has been lacking. Here we report the first comprehensive two-dimensional PAGE and mass spectrometry analysis of archaeal ribosomes isolated from the thermophilic Pyrobaculum aerophilum and the thermoacidophilic Sulfolobus acidocaldarius Crenarchaeota. Our analysis identified all 66 ribosomal proteins (r-proteins) of the P. aerophilum small and large subunits, as well as all but two (62 of 64; 97%) r-proteins of the S. acidocaldarius small and large subunits that are predicted genomically. Some r-proteins were identified with one or two lysine methylations and N-terminal acetylations. In addition, we identify three hypothetical proteins that appear to be bona fide r-proteins of the S. acidocaldarius large subunit. Dissociation of r-proteins from the S. acidocaldarius large subunit indicates that the novel r-proteins establish tighter interactions with the large subunit than some integral r-proteins. Furthermore, cryo electron microscopy reconstructions of the S. acidocaldarius and P. aerophilum 50S subunits allow for a tentative localization of the binding site of the novel r-proteins. This study illustrates not only the potential diversity of the archaeal ribosomes but also the necessity to experimentally analyze the archaeal ribosomes to ascertain their protein composition. The discovery of novel archaeal r-proteins and factors may be the first step to understanding how archaeal ribosomes cope with extreme environmental conditions.     

Role of second-largest RNA polymerase I subunit Zn-binding domain in enzyme assembly

The second-largest subunits of eukaryal RNA polymerases are similar to the β subunits of prokaryal RNA polymerases throughout much of their lengths. The second-largest subunits from eukaryal RNA polymerases contain a four-cysteine Zn-binding domain at their C termini. The domain is also present in archaeal homologs but is absent from prokaryal homologs. Here, we investigated the role of the C-terminal Zn-binding domain of Rpa135, the second-largest subunit of yeast RNA polymerase I. Analysis of nonfunctional Rpa135 mutants indicated that the Zn-binding domain is required for recruitment of the largest subunit, Rpa190, into the RNA polymerase I complex. Curiously, the essential function of the Rpa135 Zn-binding domain is not related to Zn²⁺ binding per se, since replacement of only one of the four cysteine residues with alanine led to the loss of Rpa135 function. Even more strikingly, replacement of all four cysteines with alanines resulted in functional Rpa135.