Gisela Kramer

Structure, function, and genetics of ribosomes

The papers in this book cover the Ribosome conference held at the University of Texas Marine Science Institute. The topics covered include: Structure of ribosomes; Self-organization of ribosomal RNA; Structural dynamics of the translating ribosome; and Mechanism of Ribosome Translocation.

Folding of a nascent peptide on the ribosome

Even though very significant progress has been made recently in elucidating the structure of the bacterial ribosome and topological assignments of its functional parts, the molecular mechanism of how a peptide is formed and how the nascent peptides is folded on the ribosomes remains uncertain. Here, the current progress and remaining problems are considered from the standpoint of the authors. Topics considered include formation of peptide bonds and models that represent this process, the vicinity of RNA to the nascent peptide, the cotranslational folding hypothesis, evidence that some but not all nascent peptides pass through a region within the 50S ribosomal subunit, presumably the tunnel, in which they are folded and sheltered, pause-site peptides, and the involvement of chaperones in folding of nascent proteins on ribosomes. The chaperone-like activity of the large ribosomal subunit in renaturation of denatured proteins is reviewed. It is concluded that cotranslational folding of some but not all nascent peptides occurs in the large ribosomal subunit. It is suggested that this folding is facilitated by changes in the conformation of the ribosome that are related to the reaction cycle of peptide elongation.

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.

Polyamines are necessary for maximum in vitro synthesis of globin peptides and play a role in chain initiation

Abstract The salt wash fraction removed from rabbit reticulocyte ribosomes with 0.5 m KCl contains dialyzable components required for maximum in vitro synthesis of globin peptides. The active substances were identified as spermidine and spermine. Rabbit reticulocyte ribosomes contain spermine and spermidine in a 1:3 ratio of which about 75% is removed in the 0.5 m KCl wash fraction. Dialyzed salt wash can be reactivated for in vitro protein synthesis by addition of either spermine, spermidine, or Mg 2+ ion. A twofold higher leucine incorporation into protein was obtained with the optimum concentration of either polyamine than with Mg 2+ . Spermidine is effective in lowering the Mg 2+ requirement for initiation of phenylalanine peptides in the poly(U)-directed system, apparently by formation of an initiation complex. Also, spermidine competitively interferes with edeine inhibition of globin chain initiation. These results indicate that spermidine may play a special role in peptide initiation.

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.

Co-translational folding.

Nascent proteins appear to fold co-translationally. The ribosome itself may function as a chaperone, providing a sheltered environment in which the nascent peptide is protected from aggregation and degradation, and in which folding into the tertiary structure is facilitated by interactions both with ribosomal proteins and with specific segments of the ribosomal RNA.

Cotranslational folding--omnia mea mecum porto?

Evidence for cotranslational folding on both prokaryotic and eukaryotic ribosomes is reviewed. Molecular chaperones appear to assist only a small fraction of newly synthesized proteins in folding into their native conformation. The recently published crystal structure of the large ribosomal subunit at 2.5 A resolution has provided the basis for understanding where and how peptide synthesis takes place on the ribosome. The nascent peptide is concluded to pass through a tunnel that extends about 100 A between the peptidyl transferase center and its exit site. The minimum diameter of the tunnel and the apparent physical and chemical properties of its walls appear to preclude complex folding of the nascent peptide within most of the length of the tunnel. However, results indicate that nascent peptides that are protected within the ribosomes vary in length from about 30 to 72 amino acid residues. This suggests that nascent peptides have different conformations. It is hypothesized that folding of the nascent polypeptide into its native conformation starts in the distal portion of the tunnel, and proceeds at the surface of the ribosomal subunit in a depression or bay near the exit opening of the tunnel.

THREE PHOSPHORYLATION SITES IN ELONGATION FACTOR 2

Elongation factor 2 (EF‐2) of rabbit reticulocytes was phosphorylated in vitro by incubation with partially purified EF‐2 kinase and (γ32P)ATP. After exhaustive tryptic hydrolysis 4 phosphopeptides were revealed by two‐dimensional peptide mapping. The phosphopeptides were isolated by high performance liquid chromatography and sequenced. A comparison of the primary structure of the phosphopeptides with that of EF‐2 showed that all 4 phosphopeptides originated from one region of EF‐2 located near the N‐terminus that contains 3 threonine residues: Thr‐53, Thr‐56, Thr‐58. A direct estimation of localization of radioactive phosphate in the phosphopeptides demonstrated that all the enumerated threonine residues in EF‐2 can be phosphorylated in vitro.

Yeast Mitochondrial Initiator tRNA Is Methylated at Guanosine 37 by the Trm5-encoded tRNA (Guanine-N1-)-methyltransferase

The TRM5 gene encodes a tRNA (guanine-N1-)-methyltransferase (Trm5p) that methylates guanosine at position 37 (m1G37) in cytoplasmic tRNAs in Saccharomyces cerevisiae. Here we show that Trm5p is also responsible for m1G37 methylation of mitochondrial tRNAs. The TRM5 open reading frame encodes 499 amino acids containing four potential initiator codons within the first 48 codons. Full-length Trm5p, purified as a fusion protein with maltose-binding protein, exhibited robust methyltransferase activity with tRNA isolated from a Δtrm5 mutant strain, as well as with a synthetic mitochondrial initiator tRNA (tRNAMetf). Primer extension demonstrated that the site of methylation was guanosine 37 in both mitochondrial tRNAMetf and tRNAPhe. High pressure liquid chromatography analysis showed the methylated product to be m1G. Subcellular fractionation and immunoblotting of a strain expressing a green fluorescent protein-tagged version of the TRM5 gene revealed that the enzyme was localized to both cytoplasm and mitochondria. The slightly larger mitochondrial form was protected from protease digestion, indicating a matrix localization. Analysis of N-terminal truncation mutants revealed that a Trm5p active in the cytoplasm could be obtained with a construct lacking amino acids 1–33 (Δ1–33), whereas production of a Trm5p active in the mitochondria required these first 33 amino acids. Yeast expressing the Δ1–33 construct exhibited a significantly lower rate of oxygen consumption, indicating that efficiency or accuracy of mitochondrial protein synthesis is decreased in cells lacking m1G37 methylation of mitochondrial tRNAs. These data suggest that this tRNA modification plays an important role in reading frame maintenance in mitochondrial protein synthesis.

Fluorophores at the N Terminus of Nascent Chloramphenicol Acetyltransferase Peptides Affect Translation and Movement through the Ribosome

Structurally different fluorescent probes were covalently attached to methionyl-tRNAf and tested for their incorporation into nascent peptides and full-length protein using an Escherichia coli cell-free coupled transcription/translation system. Bovine rhodanese and bacterial chloramphenicol acetyltransferase (CAT) were synthesized using derivatives of cascade yellow, eosin, pyrene, or coumarin attached to [35S]Met-tRNAf. All of the probes tested were incorporated into polypeptides, although less efficiently when compared with formyl-methionine. Eosin, the largest of the fluorophores used with estimated dimensions of 20 × 11 Å, caused the largest reduction in product formed. The rate of initiation was reduced with the fluorophore-Met-tRNAf compared with fMet-tRNAf with pyrene having the least and eosin the biggest effect. Analysis of the nascent polypeptides showed that the modifications at the N terminus affected the rate at which nascent CAT peptides were elongated causing accumulation of peptides of about 4 kDa, possibly by steric hindrance inside the tunnel within the 50 S ribosomal subunit. Fluorescence measurements indicate that the probe at the N terminus of nascent pyrene-CAT peptides is in a relatively hydrophilic environment. This finding is in agreement with recent data showing cross-linking of the N terminus of nascent peptides to nucleotides of the 23 S ribosomal RNA.