Wieslaw Kudlicki

Ribosomes and ribosomal RNA as chaperones for folding of proteins

BACKGROUND Provocative recent reports indicate that the large subunits of either prokaryotic or eukaryotic ribosomes have the capacity to promote refolding of denatured enzymes. RESULTS Salt-washed Escherichia coli ribosomes are shown to promote refolding of denatured rhodanese. The ability of the ribosomes to carry out renaturation is a property of the 50S ribosomal subunit, specifically the 23S rRNA. Refolding and release of enzymatically active rhodanese leaves the ribosomes in an inactive state or conformation for subsequent rounds refolding. Inactive ribosomes can be activated by elongation factor G (EF-G) plus GTP or by cleavage of their 23S rRNA by alpha-sarcin. Activation by either mechanism is strongly inhibited by the EF-G.GDP.fusidic acid complex. CONCLUSIONS Large subunits of E. coli ribosomes, specifically 23S rRNA, have the capacity to mediate refolding of denatured rhodanese. Refolding activity is related to the state or conformation of ribosomes that is promoted by EF-G. Activation by either mechanism is strongly inhibited by the EF-G.GDP.fusidic acid complex.

Renaturation of Rhodanese by Translational Elongation Factor (EF) Tu PROTEIN REFOLDING BY EF-Tu FLEXING

The translation elongation factor (EF) Tu has chaperone-like capacity to promote renaturation of denatured rhodanese. This renaturation activity is greatly increased under conditions in which the factor can oscillate between the open and closed conformations that are induced by GDP and GTP, respectively. Oscillation occurs during GTP hydrolysis and subsequent replacement of GDP by EF-Ts which is then displaced by GTP. Renaturation of rhodanese and GTP hydrolysis by EF-Tu are greatly enhanced by the guanine nucleotide exchange factor EF-Ts. However, renaturation is reduced under conditions that stabilize EF-Tu in either the open or closed conformation. Both GDP and the nonhydrolyzable analog of GTP, GMP-PCP, inhibit renaturation. Kirromycin and pulvomycin, antibiotics that specifically bind to EF-Tu and inhibit its activity in peptide elongation, also strongly inhibit EF-Tu-mediated renaturation of denatured rhodanese to levels near those observed for spontaneous, unassisted refolding. Kirromycin locks EF-Tu in the open conformation in the presence of either GTP or GDP, whereas pulvomycin locks the factor in the closed conformation. The results lead to the conclusion that flexing of EF-Tu, especially as occurs between its open and closed conformations, is a major factor in its chaperone-like refolding activity.

Evidence for a second phosphorylation site on eIF-2α from rabbit reticulocytes

Ser 51 in the NH2‐terminal sequence of the α‐subunit of eukaryotic peptide initiation factor 2 (eIF‐2) has been identified as a second phosphorylation site for the heme‐controlled eIF‐2α kinase from rabbit reticulocytes. Increased phosphorylation of this serine relative to the previously described phosphorylation site (Ser 48) is observed when the kinase reaction is carried out in the presence of the α‐subunit of spectrin. A synthetic peptide corresponding to eIF‐2α(41–54) is phosphorylated only in Ser 51 by the eIF‐2α kinase.

BINDING OF AN N-TERMINAL RHODANESE PEPTIDE TO DNAJ AND TO RIBOSOMES

A peptide corresponding to the N-terminal 17 amino acids of bovine rhodanese was fluorescently labeled with a coumarin derivative at its primary amino group(s) and then purified by high performance liquid chromatography. This peptide interacted with the molecular chaperone DnaJ in the absence of other chaperones and ATP. In the presence of ATP, the molecular chaperone DnaK bound to the DnaJ-peptide complex, but not to the peptide alone. The chaperone GrpE appeared to cause the release of the peptide bound to the ternary complex in the presence of ATP but not in the presence of ADP. This nucleotide apparently stabilized the complex. The peptide also bound to salt-washed Escherichia coli 70 S ribosomes, specifically to 50 S ribosomal subunits, not to 30 S subunits. DnaJ plus DnaK interacted with the peptide on the ribosome. GrpE caused dissociation of the peptide from the ribosome; ATP was required for this reaction. It was inhibited by ADP. A comparable series of chaperone-mediated reactions is assumed to occur with the N-terminal segment of the nascent polypeptide to facilitate its folding on ribosomes.

N-terminal and C-terminal modifications affect folding, release from the ribosomes and stability of in vitro synthesized proteins.

Important aspects of translation are release and folding of the synthesized protein into its three-dimensional structure. Studies from our group indicated that during in vitro protein synthesis a large portion of full-length polypeptides apparently accumulated as peptidyl-tRNA on ribosomes. We have also shown that some proteins though released in biologically active form may be inactivated without being degraded. These experiments were carried out by coupled transcription/translation using an Escherichia coli extract in which eukaryotic or prokaryotic test proteins were synthesized from their coding sequence inserted into specific plasmids. Experiments described here were designed to analyze the effects of N-terminal and C-terminal modifications of the coding sequence on the ribosomal release/termination process and on the stability of the newly synthesized protein. Elimination of the leader sequence in two proteins tested, mitichondrial rhodanese and bacterial beta-lactamase, caused an increase in the percentage of polypeptides released from the ribosomes relative to total synthesis. Conversely, an N-terminal extension such as a histidine-lag impaired the ribosomal release process. Also, a hydrophobic N-terminal modification of the synthesized protein reduced release of newly formed protein from the ribosomes. A C-terminal extension of the coding sequence for rhodanese by one amino acid decreased the percentage released polypeptide and furthermore affected the stability of the in vitro formed protein. We propose that a regulatory mechanism exists by which N-terminal and C-terminal sequences of a newly synthesized protein have feed-back effects on the termination factor-mediated release and on the stability of the native three-dimensional structure.

Inhibition of protein synthesis by the β-subunit of spectrin

The 220 kDa β‐subunit of erythroid cell spectrin is a potent inhibitor of protein synthesis in lysates from rabbit reticulocytes. On the basis of weight of protein added to a lysate reaction mixture, it has about half the inhibitory activity of highly purified heme‐regulated eIF‐2α kinase. Inhibition appears to be at the level of peptide initiation but does not involve a kinase that phosphorylates eIF‐2 on its α‐subunit.

The Conformation and Path of Nascent Proteins in Ribosomes

Publisher Summary Nascent proteins are extended into a tunnel or cavity within the large ribosomal subunit as they are formed by the successive addition of amino acids to their N-terminus. This process appears to be associated with the acquisition of secondary and tertiary structure that is important for folding into the native conformation or transport of the newly formed protein into membranes or other subcellular structures. This chapter discusses the aspects of the structure and function of ribosomes that contribute to these processes. The synthesis of proteins in all living cells is carried out by ribosomes that are composed of two structurally different subunits that associate upon initiation of protein biosynthesis. Ribosomes are massive entities comparable in size to large multienzyme complexes. They have unique structures composed of RNA and proteins of specific primary sequence. The nascent peptide provides the basis for the intimate relationship between active ribosomes and membranes in bacteria.

Regulation of Reticulocyte eIF-2α Kinases by Phosphorylation

Rabbit reticulocytes have served as one of the model systems for studies on eukaryotic protein synthesis for more than three decades. Isolation and characterization of mammalian initiation factors are based to a major extent on results obtained with these cells. In 1975 it was found by Richard Jackson, Tim Hunt, and co-workers that reticulocyte initiation factor 2 (eIF-2) can be phosphorylated in its a subunit. Initial characterization of an apparently unique protein kinase followed.1–4 This enzyme is active in reticulocytes under heme deficiency and leads to cessation of protein synthesis. Without knowing its identity, it had been described several years earlier as the causing agent for inhibition of protein synthesis; it was activated when the postribosomal supernatant was incubated without hemin. The name “heme-controlled repressor” (HCR) had been given to this agent.5 A second protein kinase phosphorylating eIF-2α was found in reticulocytes; it is associated with polysomes and activated by incubation with low concentrations of double-stranded (ds) RNA and ATP.1,6,7 Its relationship to the interferon-induced mammalian eIF-2α kinase described in other chapters of this book is obvious.

Association of the Heme-Controlled eIF-2α Kinase with Spectrin-Derived Peptides

Translational control of mammalian protein synthesis frequently occurs at the level of peptide initiation. One control system in particular has been intensively studied during the last decade. It involves phosphorylation and dephosphorylation of the smallest subunit (α-subunit) of initiation factor 2, eIF-2. Two different substrate-specific protein kinases are recognized that can carry out this phosphorylation and thereby cause inhibition of protein synthesis. Both occur in inactive form in mammalian cells [1,2]. One of the kinases is induced by interferon and activated by double-stranded RNA. The other is activated under conditions of heme deficiency and is known as the heme-controlled repressor (HCR) kinase.