Purification of the large ribosomal subunit via its association with the small subunit

We have developed an affinity purification of the large ribosomal subunit from Deinococcus radiodurans that exploits its association with FLAG-tagged 30S subunits. Thus, capture is indirect so that no modification of the 50S is required and elution is achieved under mild conditions (low magnesium) that disrupt the association, avoiding the addition of competitor ligands or coelution of common contaminants. Efficient purification of highly pure 50S is achieved, and the chromatography simultaneously sorts the 50S into three classes according to their association status (unassociated, loosely associated, or tightly associated), improving homogeneity.     

Mutations in the small subunit of ribulosebisphosphate carboxylase affect subunit binding and catalysis

Fully functional Synechococcus PCC 6301 ribulose 1,5-bisphosphate carboxylase-oxygenase (kcat = 11.8 s-1) was assembled in vitro following separate expression of the large- and small-subunit genes in different Escherichia coli cultures. The small subunits were expressed predominantly as monomers, in contrast to the large subunits which have been shown to be largely octameric when expressed separately [Andrews, T. J. (1988) J. Biol. Chem. 263, 12213-12219]. This separate expression system was applied to the study of mutations in the amino-terminal arm of the small subunit, which is one of the major sites of contact with the large subunit in the assembled hexadecamer. It enabled the effects of a mutation on the tightness of binding of the small subunit to the large-subunit octamer to be distinguished from the effects of the same mutation on catalysis carried out by the assembled complex when fully saturated with mutant small subunits. This important distinction cannot be made when both subunits are expressed together in the same cell. Substitutions of conserved amino acid residues at positions 14 (Ala, Val, Gly, or Asp instead of Thr) and 17 (Cys instead of Tyr), which make important contacts with conserved large-subunit residues, were introduced by site-directed mutagenesis. All mutant small subunits were able to bind to large subunits and form active enzymes. A potential intersubunit hydrogen bond involving the Thr-14 hydroxyl group is shown to be unimportant. However, the binding of Gly-14, Asp-14, and Cys-17 mutant small subunits was weaker, and the resultant mutant enzymes had reduced catalytic rates compared to the wild type.(ABSTRACT TRUNCATED AT 250 WORDS)     

Internal structure of the chromatin subunit

The digestion of chromatin in situ with DNase I reveals, after denaturation, a regular series of single stranded DNA fragments the lengths of which represent multiples of 10 bases. These experiments are compatible with the DNA being on the outside of the chromatin subunit and suggest that the subunit structure itself contains repetitive structural elements. Possible models are discussed.     

p-Cresol methylhydroxylase: alteration of the structure of the flavoprotein subunit upon its binding to the cytochrome subunit

The structures of two forms of a recombinant flavoprotein have been determined at high resolution and compared. These proteins are (1) the flavocytochrome c p-cresol methylhydroxylase (rPCMH, 1.85 A resolution) and (2) the cytochrome-free flavoprotein subunit of rPCMH (PchF, 1.30 A resolution). A significant conformational difference is observed in a protein segment that is in contact with the re face of the isoalloxazine ring of FAD when the structure of PchF is compared to the subunit in the intact flavocytochrome. This structural change is important for optimum catalytic function of the flavoprotein, which has been shown to be dependent on the presence of the cytochrome subunit. This change results in different protein-flavin and apparently different protein-substrate interactions that have a "tuning effect" on the electronic and redox properties of bound p-cresol and the covalently bound FAD. The conformational change in the segment in the cofactor-binding site is induced by a small rearrangement in the flavoprotein-cytochrome interface region of the flavoprotein.     

Amino-terminal truncations of the ribulose-bisphosphate carboxylase small subunit influence catalysis and subunit interactions.

The first 20 residues at the amino terminus of the small subunit of spinach ribulose-1,5-bisphosphate carboxylase form an irregular arm that makes extensive contacts with the large subunit and also with another small subunit (S. Knight, I. Andersson, and C.-I. Brändén [1990] J Mol Biol 215: 113-160). The influence of these contacts on subunit binding and, indirectly, on catalysis was investigated by constructing truncations from the amino terminus of the small subunit of the highly homologous enzyme from Synechococcus PCC 6301 expressed in Escherichia coli. Removal of the first six residues (and thus the region of contact with a neighboring small subunit) affected neither the affinity with which the small subunits bound to the large subunits nor the catalytic properties of the assembled holoenzyme. Extending the truncation to include the first 12 residues (which encroaches into a highly conserved region that interacts with the large subunit) also did not weaken intersubunit binding appreciably, but it reduced the catalytic activity of the holoenzyme nearly 5-fold. Removal of an additional single residue (i.e. removal of a total of 13 residues) weakened intersubunit binding approximately 80-fold. Paradoxically, this partially restored catalytic activity to approximately 40% of that of the wild-type holoenzyme. None of these truncations materially affected the Km values for ribulose-1,5-bisphosphate or CO2. Removal of all 20 residues of the irregular arm (thereby deleting the conserved region of contact with large subunits) totally abolished the small subunit's ability to bind to large subunits to form a stable holoenzyme. However, this truncated small subunit was still synthesized by the E. coli cells. These data are interpreted in terms of the role of the amino-terminal arm of the small subunit in maintaining the structure of the holoenzyme.     

Structure of the theta subunit of Escherichia coli DNA polymerase III in complex with the epsilon subunit

The catalytic core of Escherichia coli DNA polymerase III contains three tightly associated subunits, the alpha, epsilon, and theta subunits. The theta subunit is the smallest and least understood subunit. The three-dimensional structure of theta in a complex with the unlabeled N-terminal domain of the epsilon subunit, epsilon186, was determined by multidimensional nuclear magnetic resonance spectroscopy. The structure was refined using pseudocontact shifts that resulted from inserting a lanthanide ion (Dy3+, Er3+, or Ho3+) at the active site of epsilon186. The structure determination revealed a three-helix bundle fold that is similar to the solution structures of theta in a methanol-water buffer and of the bacteriophage P1 homolog, HOT, in aqueous buffer. Conserved nuclear Overhauser enhancement (NOE) patterns obtained for free and complexed theta show that most of the structure changes little upon complex formation. Discrepancies with respect to a previously published structure of free theta (Keniry et al., Protein Sci. 9:721-733, 2000) were attributed to errors in the latter structure. The present structure satisfies the pseudocontact shifts better than either the structure of theta in methanol-water buffer or the structure of HOT. satisfies these shifts. The epitope of epsilon186 on theta was mapped by NOE difference spectroscopy and was found to involve helix 1 and the C-terminal part of helix 3. The pseudocontact shifts indicated that the helices of theta are located about 15 A or farther from the lanthanide ion in the active site of epsilon186, in agreement with the extensive biochemical data for the theta-epsilon system.     

Changed properties of the A subunit in DNA gyrase with a B subunit mutation

Summary To investigate the interaction of subunits A and B of DNA gyrase during DNA supercoiling, a Cou^r mutant of Escherichia coli was obtained and the effect of nalidixic acid on the supercoiling of DNA by wild-type and mutant enzymes was assayed. The enzyme of the Cou^r strain proved to be more sensitive to nalidixic acid than the wild-type DNA gyrase. Hence the mutation affecting the B subunit can also change the properties of the A subunit, which fact suggests that the two subunits of DNA gyrase are in contact during DNA supercoiling.     

Identification of a subunit assembly domain in the alpha subunit of Escherichia coli RNA polymerase

The alpha subunit of Escherichia coli DNA-dependent RNA polymerase is encoded by the rpoA gene and plays a major role in enzyme assembly. A set of C-terminal deletion mutations of the rpoA gene was constructed. The results of mixed reconstitution experiments in vitro, using the truncated alpha polypeptides encoded by the rpoA deletion mutants, suggest that the amino-terminal two-thirds of alpha subunit is sufficient for the formation of pseudo-core complexes containing both beta and beta' subunits.     

Tissue specificity of subunit composition and EGF-dependent changes in the subunit pattern of 20S-proteasomes

The subunit pattern of 20S proteasomes from rat kidney, rat liver, human A-431 cells, human K-562 cells and mouse NIH 3T3 cells were studied. Proteasomes in cells of a common tissue origin appeared to be similar, independently of the intensity of cell proliferation. Unlike, proteasomes in cells of various types of tissue specificity differed from each other. Besides, EGF was shown to induce changes in the subunit pattern of proteasomes in A-431 cells.     

Determination of the native subunit pattern of gizzard tropomyosin using antibodies specific to each subunit

The gizzard tropomyosin molecule is composed of two subunits at 1:1 molar ratio. Possible composites of the tropomyosin molecule are two kinds of homodimer (one for each subunit), a heterodimer of two subunits, or a mixture of heterodimer and homodimer(s). We tried to evaluate the native subunit composition of gizzard tropomyosin by cross-linking experiments and immunological methods using specific antibodies to each subunit. For the cross-linking experiment we used dimethyl suberimidate, an amino group-specific cross-linker, in the presence of dithiothreitol to avoid artificial oxidative intersubunit cross-linking. When gizzard tropomyosin was cross-linked, it generated several products which might correspond to dimers formed by intersubunit cross-linkage. When the reaction was carried out for a long time, non-cross-linked subunits completely disappeared and two or three major cross-linked products arose. All of these cross-linked products were recognized by both of the specific antibodies to each subunit. These results indicated that the predominant part, if not all, of gizzard tropomyosin is present as heterodimer.     

Effects of subunit V antibodies on the topology of the subunit and the activity of beef heart cytochrome-c oxidase

Redox-sensitive epitopes on subunit V of beef heart cytochrome-c oxidase were demonstrated previously using polyclonal subunit-specific antibodies raised in rabbits. The antibodies only slightly inhibited electron transfer, and the accessibility of their epitopes depended on the presence of a membrane and on the redox state of the oxidase. The present paper describes additional preparations of antibodies raised against subunit V. These antibodies have an even higher subunit specificity, they are more than three times as inhibitory against electron transfer, and their binding does not require a membrane. Moreover, the redox-sensitive nature of their binding to detergent-dispersed oxidase is sensitive to the method of its isolation. We discuss inferences that can be drawn from a detailed quantitative comparison of the interactions of the two antibody preparations with the antigen in different environments. The techniques used in the comparison can be used to examine other perturbants of the oxidase as to their effects on specific segments of the enzyme.     

The regulatory subunit monomer of cAMP-dependent protein kinase retains the salient kinetic properties of the native dimeric subunit

Monomeric regulatory subunit (R) fragments of type II cAMP-dependent protein kinase were compared with the parent dimeric R. The monomeric fragments were generated by either endogenous proteolysis of rabbit muscle R or by trypsin treatment of bovine heart R in the holoenzyme form. During isolation of pure R from rabbit muscle, carboxyl-terminal fragments of Mr = 42,000 (42 K) and Mr = 37,000 by denaturing gels are generated by endogenous proteolysis. Although the autophosphorylation site is retained, the 42 K is not dimeric (as is its native 56 K precursor) but, in contrast to the monomeric 37 K product, actively reassociates with purified catalytic subunit (C). Several lines of evidence indicate a type II R origin of the 42 K. N-terminal sequence analysis of the 42 K shows some homology with known bovine RI, RII, and cGMP-dependent protein kinase sequences. Both cyclic nucleotide-binding sites (two/42 K or 37 K) and the site selectivity of cAMP analogs are retained in the monomeric fragments. When purified bovine heart holoenzyme, which contains a dimeric Mr = 56,000 R (denaturing gel analysis) and two C subunits, is treated with trypsin followed by separation procedures, the product is a fully recovered active enzyme with an unaltered ratio of cAMP binding to catalytic activity. From Mr considerations, the product is a dimer containing one intact C and a proteolyzed R of Mr = 48,000 on denaturing gels. This dimeric enzyme is not significantly different from the parent tetramer in cAMP concentration dependence (Hill constant = 1.63), [3H]cAMP dissociation behavior (both intrasubunit cAMP-binding sites are present), stimulation of [3H]cIMP binding by site-selective cAMP analogs, and synergism between two analogs in kinase activation. The data indicate that 1) proteolytic cleavage of the native R dimer can cause monomerization without appreciably affecting the inhibition of C and 2) essentially all of the cAMP binding cooperativity is an intrasubunit interaction.     

Interaction of Era with the 30S ribosomal subunit implications for 30S subunit assembly

Era (E. coliRas-like protein) is a highly conserved and essential GTPase in bacteria. It binds to the 16S ribosomal RNA (rRNA) of the small (30S) ribosomal subunit, and its depletion leads to accumulation of an unprocessed precursor of the 16S rRNA. We have obtained a three-dimensional cryo-electron microscopic map of the Thermus thermophilus 30S-Era complex. Era binds in the cleft between the head and platform of the 30S subunit and locks the subunit in a conformation that is not favorable for association with the large (50S) ribosomal subunit. The RNA binding KH motif present within the C-terminal domain of Era interacts with the conserved nucleotides in the 3' region of the 16S rRNA. Furthermore, Era makes contact with several assembly elements of the 30S subunit. These observations suggest a direct involvement of Era in the assembly and maturation of the 30S subunit.     

The influence of the DNA polymerase gamma accessory subunit on base excision repair by the catalytic subunit

Mammalian DNA polymerase gamma, the sole polymerase responsible for replication and repair of mitochondrial DNA, contains a large catalytic subunit and a smaller accessory subunit, pol gammaB. In addition to the polymerase domain, the large subunit contains a 3'-5' editing exonuclease domain as well as a dRP lyase activity that can remove a 5'-deoxyribosephosphate (dRP) group during base excision repair. We show that the accessory subunit enhances the ability of the catalytic subunit to function in base excision repair mainly by stimulating two subreactions in the repair process. Pol gammaB appeared to specifically enhance the rate at which pol gamma was able to locate damage in high molecular weight DNA. One pol gammaB point mutant known to have impaired ability to bind duplex DNA stimulated repair poorly, suggesting that duplex DNA binding through pol gammaB may help the catalytic subunit locate sites of DNA damage. In addition, the small subunit significantly stimulated the dRP lyase activity of pol gammaA, although it did not increase the rate at which the dRP group dissociated from the enzyme. The ability of DNA pol gamma to process a high load of damaged DNA may be compromised by the slow release of the dRP group.     

A new subunit of horse liver alcohol dehydrogenase and subunit composition of the polymorphic form.

The most cathodal (on starch-gel electrophoresis), steroid-active band of horse liver alcohol dehydrogenase, whose catalytic properties were shown to be dependent on the livers used as a starting material [Pietruszko (1974) Biochem. Biophys. Res. Commun. 60, 687-694], has been prepared from A-type and S-type horse livers by identical methods. Results presented here show that different isoenzymes are present in these preparations.