Peter Gegenheimer

Novel mechanisms for maturation of chloroplast transfer RNA precursors.

Despite the prokaryotic origins of chloroplasts, a plant chloroplast tRNA precursor is processed in a homologous in vitro system by a pathway distinct from that observed in Escherichia coli, but identical to that utilized for maturation of nuclear pre‐tRNAs. The mature tRNA 5′ terminus is generated by the site‐specific endonucleolytic cleavage of an RNase P (or P‐type) activity. The 3′ end is likewise produced by a single precise endonucleolytic cut at the 3′ terminus of the encoded tRNA domain. This is the first complete structural characterization of an organellar tRNA processing system using a homologous substrate. In contrast to eubacterial RNase P, chloroplast RNase P does not appear to contain an RNA subunit. The chloroplast activity bands with bulk protein at 1.28 g/ml in CsCI density gradients, whereas E.coli RNase P bands as ribonucleoprotein at 1.73 g/ml. Chloroplast RNase P activity survives treatment with micrococcal nuclease (MN) at levels 10‐ to 100‐fold higher than those required to totally inactivate the E.coli enzyme. The chloroplast system is sensitive to a suppression of tRNA processing, caused by binding of inactive MN to pre‐tRNA substrate, which is readily overcome by addition of carrier RNA to the assay.

Structural Analysis of the Regulatory Dithiol-containing Domain of the Chloroplast ATP Synthase γ Subunit

The γ subunit of the F1 portion of the chloroplast ATP synthase contains a critically placed dithiol that provides a redox switch converting the enzyme from a latent to an active ATPase. The switch prevents depletion of intracellular ATP pools in the dark when photophosphorylation is inactive. The dithiol is located in a special regulatory segment of about 40 amino acids that is absent from the γ subunits of the eubacterial and mitochondrial enzymes. Site-directed mutagenesis was used to probe the relationship between the structure of the γ regulatory segment and its function in ATPase regulation via its interaction with the inhibitory ϵ subunit. Mutations were designed using a homology model of the chloroplast γ subunit based on the analogous structures of the bacterial and mitochondrial homologues. The mutations included (a) substituting both of the disulfide-forming cysteines (Cys199 and Cys205) for alanines, (b) deleting nine residues containing the dithiol, (c) deleting the region distal to the dithiol (residues 224-240), and (d) deleting the entire segment between residues 196 and 241 with the exception of a small spacer element, and (e) deleting pieces from a small loop segment predicted by the model to interact with the dithiol domain. Deletions within the dithiol domain and within parts of the loop segment resulted in loss of redox control of the ATPase activity of the F1 enzyme. Deleting the distal segment, the whole regulatory domain, or parts of the loop segment had the additional effect of reducing the maximum extent of inhibition obtained upon adding the ϵ subunit but did not abolish ϵ binding. The results suggest a mechanism by which the γ and ϵ subunits interact with each other to induce the latent state of the enzyme.

The 20 C-terminal amino acid residues of the chloroplast ATP synthase gamma subunit are not essential for activity.

It has been suggested that the last seven to nine amino acid residues at the C terminus of the γ subunit of the ATP synthase act as a spindle for rotation of the γ subunit with respect to the αβ subunits during catalysis (Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994)Nature 370, 621–628). To test this hypothesis we selectively deleted C-terminal residues from the chloroplast γ subunit, two at a time starting at the sixth residue from the end and finishing at the 20th residue from the end. The mutant γ genes were overexpressed in Escherichia coli and assembled with a native α3β3 complex. All the mutant forms of γ assembled as effectively as the wild-type γ. Deletion of the terminal 6 residues of γ resulted in a significant increase (>50%) in the Ca-dependent ATPase activity when compared with the wild-type assembly. The increased activity persisted even after deletion of the C-terminal 14 residues, well beyond the seven residues proposed to form the spindle. Further deletions resulted in a decreased activity to ∼19% of that of the wild-type enzyme after deleting all 20 C-terminal residues. The results indicate that the tip of the γC terminus is not essential for catalysis and raise questions about the role of the C terminus as a spindle for rotation.

Structure, mechanism and evolution of chloroplast transfer RNA processing systems.

Chloroplasts of land plants have an active transfer RNA processing system, consisting of an RNase P-like 5′ endonuclease, a 3′ endonuclease, and a tRNA:CCA nucleotidyltransferase. The specificity of these enzymes resembles more that of their eukaryotic counterparts than that of their cyanobacterial predecessors. Most strikingly, chloroplast RNase P activity almost certainly resides in a protein, rather than in an RNA protein complex as in Bacteria, Archaea, and Eukarya. The chloroplast enzyme may have evolved from a preexisting chloroplast NADP-binding protein. Chloroplast RNase P cleaves pre-tRNA by a reaction mechanism in which at least one of the Mg2+ ions utilized by the bacterial ribozyme RNase P is replaced by an amino acid side chain.

PPR proteins shed a new light on RNase P biology

A fast growing number of studies identify pentatricopeptide repeat (PPR) proteins as major players in gene expression processes. Among them, a subset of PPR proteins called PRORP possesses RNase P activity in several eukaryotes, both in nuclei and organelles. RNase P is the endonucleolytic activity that removes 5′ leader sequences from tRNA precursors and is thus essential for translation. Before the characterization of PRORP, RNase P enzymes were thought to occur universally as ribonucleoproteins, although some evidence implied that some eukaryotes or cellular compartments did not use RNA for RNase P activity. The characterization of PRORP reveals a two-domain enzyme, with an N-terminal domain containing multiple PPR motifs and assumed to achieve target specificity and a C-terminal domain holding catalytic activity. The nature of PRORP interactions with tRNAs suggests that ribonucleoprotein and protein-only RNase P enzymes share a similar substrate binding process.

Over-expression and refolding of β-subunit from the chloroplast ATP synthase

We established a bacterial system for high‐level over‐expression of the spinach chloroplast atpB gene which encodes the ATP Synthase β subunit. Upon induction, atpB was expressed as at least 50% to 70% of total cell protein. Although the over‐expressed β polypeptide formed insoluble inclusion bodies, more than fifty percent of it was restored to a functional form by solubilizing the inclusion bodies with 4 M urea and slowly removing the urea by stepwise dialysis. The resulting β subunit exhibited specific and selective nucleotide binding properties identical to those of the native β subunit.

Enzyme nomenclature: Functional or structural?

Altman and colleagues (this issue) call attention to the inability of current standardized enzyme nomenclature to distinguish between enzymatic activities that reside in nonhomologous macromolecules+ This issue is highlighted by the fact that the pre-tRNA 59-maturation activities of bacteria and plant chloroplasts present the first instance (of which I am aware) of two naturally occurring enzymes that cannot be evolutionarily related, but which catalyze an identical reaction+ (In the classic example of convergent evolution between the trypsin family and subtilisin, the enzymes do not have an identical substrate specificity+) Altman and colleagues propose that a single trivial name be used only for members of a family of homologous macromolecules; in other words, that different trivial names be given to enzymes that catalyze the same precursor–product conversion but do so with different catalytic mechanisms, or which are not members of a single family of homologous macromolecules+ I am not convinced that there is a problem needing solution+ The current proposal seems to run counter to the rationale behind current EC nomenclature, and could create more confusion than it would alleviate+ One can distinguish between a function-based nomenclature based on the biochemical reaction catalyzed—the substrate–product conversion—and a structure-based nomenclature based on the physical nature of the catalyst+ For a classical enzymologist, the reaction type being catalyzed is paramount: It is the reaction that one uses to purify the enzyme+ One identifies the enzyme based on its activity,whereas its physical structure may initially be of secondary importance+ The value of function-based nomenclature is precisely that it allows the biochemical reaction (the substrate– product conversion) to be described, specified, and studied concomitant with continuing purification and analysis of the corresponding enzyme+ Further, as more is learned about the enzyme’s structure and catalytic mechanism, it is not necessary to rename it+ Indeed, the utility of function-based nomenclature is exemplified by the history of bacterial RNase P purification and characterization+

Processing of rRNA and tRNA in Escherichia coli: Cooperation between Processing Enzymes

rRNAs of Escherichia coli are transcribed from a number of polycistronic transcription units, each of which codes for 16S, 23S, and 5S rRNA (for a review, see Pace 1973). Genes for some tRNAs are located within rRNA transcription units, either in the central spacer or in the 3′ trailer region (Lund et al. 1976; Lund and Dahlberg 1977; Morgan et al. 1977, 1978; Gegenheimer and Apirion 1978). Processing of rRNA transcripts, then, must include tRNA processing steps. Inasmuch as intact polycistronic transcripts of rRNA are not detectable in wild-type bacterial cells, the first processing cleavage events must take place while the polycistronic precursor is still being transcribed (see Pace 1973; Gegenheimer and Apirion 1975; Gegenheimer et al. 1977; Hoffman and Miller 1977). Mutant strains of E. coli that are defective in rRNA or in tRNA processing enzymes have been isolated. These include the mutations rnc-105, rne-3071, and rnp-49, which affect the enzymes RNase III, RNase E, and RNase P, respectively (Kindler et al. 1973; Apirion and Watson 1975; Apirion 1978; Apirion and Lassar 1978; Schedl and Primakoff 1973). This discussion describes the structural analysis and in vitro processing of rRNA processing intermediates accumulated in mutant strains defective in the enzymes RNase III, RNase E, and RNase P, singly or in combinations, and demonstrates that all of these enzymes participate in production of mature cellular rRNA and tRNAs, including tRNA species not cotranscribed with rRNA. These studies also demonstrate the existence of another RNA processing enzyme, RNase F. These four enzymes...

Role of the Gamma Subunit in Regulating the Activity of the Chloroplast ATP Synthase

The ATP synthase enzymes of the inner membranes of mitochondria, chloroplasts and of the bacterial cytoplasmic membrane, couple the energy of a transmembrane electrochemical proton gradient to the synthesis of ATP from ADP and inorganic phosphate. The general structural features of the enzyme are highly conserved from one organism to another. It is comprised of an integral membrane-spanning H+- translocating segment (F0 or factor O) and a peripheral membrane segment (F1 or factor 1) which contains the catalytic sites for ATP synthesis and hydrolysis. The F1 segment is comprised of five different polypeptide subunits designated α to e in order of decreasing molecular weight. The subunit stoichiometry is α3β3γ1δ1 and e1 (1).