In vitro protein folding by ribosomes from Escherichia coli, wheat germ and rat liver

Ribosomes from a number of prokaryotic and eukaryotic sources (e.g., Escherichia coli, wheat germ and rat liver) can refold a number of enzymes which are denatured with guanidine/HCl prior to incubation with ribosomes. In this report, we present our observations on the refolding of denatured lactate dehydro-genase from rabbit muscle and glucose-6-phosphate dehydrogenase from baker's yeast by ribosomes from E. coli, wheat germ and rat liver, The protein-folding activity of E. coli, ribosomes was found to be present in 50s particles and in 23S rRNA. The 30S particle or 16S rRNA did not show any protein-folding activity. The protein-folding activity of 23S rRNA may depend on its tertiary conformation. Loss of tertiary structure, by incubation with low concentrations of EDTA, inhibited the protein-folding activity of 23S rRNA. This low concentration of EDTA had no effect on folding of the denatured enzymes by themselves

Role of the ribosome in protein folding

In all organisms, the ribosome synthesizes and folds full length polypeptide chains into active three-dimensional conformations. The nascent protein goes through two major interactions, first with the ribosome which synthesizes the polypeptide chain and holds it for a considerable length of time, and then with the chaperones. Some of the chaperones are found in solution as well as associated to the ribosome. A number of in vitro and in vivo experiments revealed that the nascent protein folds through specific interactions of some amino acids with the nucleotides in the peptidyl transferase center (PTC) in the large ribosomal subunit. The mechanism of this folding differs from self-folding. In this article, we highlight the folding of nascent proteins on the ribosome and the influence of chaperones etc. on protein folding

Ribosome-DnaK interactions in relation to protein folding

Bacterial ribosomes or their 50S subunit can refold many unfolded proteins. The folding activity resides in domain V of 23S RNA of the 50S subunit. Here we show that ribosomes can also refold a denatured chaperone, DnaK, in vitro, and the activity may apply in the folding of nascent DnaK polypeptides in vivo. The chaperone was unusual as the native protein associated with the 50S subunit stably with a 1:1 stoichiometry in vitro. The binding site of the native protein appears to be different from the domain V of 23S RNA, the region with which denatured proteins interact. The DnaK binding influenced the protein folding activity of domain V modestly. Conversely, denatured protein binding to domain V led to dissociation of the native chaperone from the 50S subunit. DnaK thus appears to depend on ribosomes for its own folding, and upon folding, can rebind to ribosome to modulate its general protein folding activity

Lower fidelity of RecA protein catalysed homologous pairing with a superhelical substrate

The heteroduplex joint, a splice containing paired strands from each of two DNA molecules is a key feature of homologous genetic recombination. The formation of such a lap joint ensures that heterologous chromosomes do not recombine; presumably the degree of homology determines the frequency of crossing over between related but non-identical chromosomes. On the other hand, according to the current ideas on recombination, the formation of heteroduplex joints is not a stringent process. Genetic evidence supports the view that the classical phenomena of meiotic gene conversion and aberrant meiotic segregation result in part from the inclusion of mismatched base pairs in heteroduplex joints, and the subsequent correction of some of these mismatched pairs before replication. Thus meiotic gene conversion signals one kind of departure from perfectly faithful pairing. The association of increased mutation frequencies with crossing-over is another kind of departure from fidelity in recombination. Little is known about the effect of imperfect homology either on the initial pairing of DNA molecules, or on the formation and extension of heteroduplex joints. The nucleotide sequences of phages 3ΦX174 and G4 are related, but differ by 33% of bases in the coding regions. Here we show using combinations of 3ΦX174 and G4 DNA, that Escherichia coli RecA protein catalyses the formation of joint molecules with many mismatched base pairs, but only if one of the molecules is superhelical. By contrast, in the absence of RecA protein, superhelicity does not cause ΦX174 and G4 DNA to form D-loops spontaneously at any temperature between 37 and 75°C. These observations focus attention on the role of RecA protein in unwinding DNA, and on superhelicity as a factor that can lessen the fidelity of homologous pairing

Synapsis and the formation of paranemic joints by E. coli RecA protein

E. coli RecA protein promotes the homologous pairing of a single strand with duplex DNA even when certain features of the substrates, such as circularity, prohibit the true intertwining of the newly paired strands. The formation of such nonintertwined or paranemic joints does not require superhelicity, and indeed can occur with relaxed closed circular DNA. E. coli topoisomerase I can intertwine the incoming single strand in the paranemic joint with its complement, thereby topologically linking single-stranded DNA to all of the duplex molecules in the reaction mixture. The efficiency of formation of paranemic joints, the time course, and estimates of their length, all suggest that they represent true synaptic intermediates in the pairing reaction promoted by RecA protein

23S rRNA assisted folding of cytoplasmic malate dehydrogenase is distinctly different from its self-folding

The role of the 50S particle of Escherichia coli ribosome and its 23S rRNA in the refolding and subunit association of dimeric porcine heart cytoplasmic malate dehydrogenase (s-MDH) has been investigated. The self-reconstitution of s-MDH is governed by two parallel pathways representing the folding of the inactive monomeric and the dimeric intermediates. However, in the presence of these folding modulators, only one first order kinetics was observed. To understand whether this involved the folding of the monomers or the dimers, subunit association of s-MDH was studied using fluorescein-5-isothiocyanate–rhodamine-isothiocyanate (FITC–RITC) fluorescence energy transfer and chemical cross-linking with gluteraldehyde. The observation suggests that during refolding the interaction of the unstructured monomers of s-MDH with these ribosomal folding modulators leads to very fast formation of structured monomers that immediately dimerise. These inactive dimers then fold to the native ones, which is the rate limiting step in 23S or 50S assisted refolding of s-MDH. Furthermore, the sequential action of the two fragments of domain V of 23S rRNA has been investigated in order to elucidate the mechanism. The central loop of domain V of 23S rRNA (RNA1) traps the monomeric intermediates, and when they are released by the upper stem–loop region of the domain V of 23S rRNA (RNA2) they are already structured enough to form dimeric intermediates which are directed towards the proper folding pathway

Formation of nascent heteroduplex structures by RecA protein and DNA

E. coli RecA protein promotes homologous pairing in two distinguishable phases: synapsis and strand exchange. With circular single strands (plus strand only) and linear duplex DNA, polarized or unidirectional strand exchange appeared to cause heteroduplex joints to form and grow from a unique end of the duplex DNA. However, a variety of other pairs of substrates appeared to form joint molecules without regard to the polarity of the strands involved. This paradox has been resolved by observations that show that synapsis is fast, nonpolar and sensitive to inhibition by ADP, whereas strand exchange is slow, directional and relatively insensitive to inhibition by ADP. Thus a heteroduplex joint initiated at one end of the duplex DNA grows by continued strand exchange, whereas a joint initiated at the other end dissociates and is unable to start again because accumulating ADP inhibits synapsis. RecA protein appears to form a nascent protein-DNA structure, the RecA synaptic structure, in which at least 100-300 bp in the duplex molecule are held in an unwound configuration and in which the incoming strand is aligned with its complement

The topology of homologous pairing promoted by RecA protein

In addition to catalyzing the pairing of linear single-stranded DNA with homologous duplex DNA, recA protein promotes the pairing of circular single strands with linear duplex DNA or nicked circular duplex DNA, and of gapped circular duplex DNA with superhelical DNA. RecA protein will thus produce joint molecules of DNA at a high frequency from a pair of homologous molecules if one of them is single-stranded or partially single-stranded, and if either one has a free end. The structure made from a linear single strand and duplex DNA is a D loop. The joint molecule made from circular single-stranded DNA and linear duplex DNA is a branched structure in which the circular strand has displaced a strand from one end of the duplex molecule. In these structures, the heteroduplex regions reach sizes approaching that of full-length fd DNA. When we used restriction fragments of duplex fd DNA that were approximately half-length, we found circular molecules that were half duplex and half single-stranded. Similarly, single-stranded circles displaced a strand from nicked circular duplex DNA, yielding structures related to those made with linear duplex DNA, as well as other structures. Our observations indicate that purified recA protein catalyzes a concerted strand transfer with several features of particular biological interest, including the initiation of a strand crossover (in some cases perhaps the crossing back of a strand as well) and the production of long heteroduplex joints by a kind of branch migration. While a free end permits interwinding of DNA strands and the formation of joints containing stable right-handed helices, the free end is not essential for the promotion of homologous pairing by recA protein. When we mixed phage G4 double-stranded DNA and recA protein with single-stranded circular M13 DNA containing an insert of 274 bases of G4 DNA, we observed by electron microscopy the formation of a few percent of complexes in which single-stranded circular DNA and duplex DNA were joined side by side in the region of shared sequence homology. The frequency of such complexes was twenty to thirty times greater than that observed in a control mixture of G4 duplex DNA and single-stranded circular fd DNA, molecules which do not share a region of extensive homology. We conclude that recA protein can promote homologous association of a single strand and duplex DNA without the plectonemic colling that characterizes the normal Watson-Crick structure of DNA

An unusual case of episodic quadriparesis

The natural history of untreated asymptomatic primary hyperparathyroidism (PHPT) remains incompletely understood. Increased level of parathyroid hormone produces the characteristic biochemical phenotype of hypercalcemia, hypophosphatemia and the various clinical sequelae of chronic hypercalcemia. Periodic paralysis (PP) is a group of disorders of different etiologies with episodic, short-lived and hyporeflexic skeletal muscle weakness, with or without myotonia, but without sensory deficit and without loss of consciousness. However, PHPT has rare association with episodic quadriparesis mimicking as PP