Chapter 24 : Folding of Nascent Peptides on Ribosomes

Translational pause sites differ for different proteins. Six different proteins were synthesized by coupled transcription-translation in the cell-free E. coli system with a small amount of the A19 S30 fraction (cf. Tsalkova et al., 1999 ). The coding sequences were in plasmids under the control of the T7 promoter. [14C]leucine was the radioactive precursor. After coupled transcription- translation, an aliquot (15 µl) was withdrawn to determine the amount of polypeptides formed, another aliquot (15 µl) of the reaction mixtures was analyzed by polyacrylamide gel electrophoresis according to the method of Schägger and von Jagow (1987) , and the radioactive bands were visualized by phosphorimaging. Lane 1, CAT (M r, ˜25,600; 13 leucines; 207 pmol of leucine incorporated); lane 2, hamster rhodanese (M r, ˜33,000; 28 leucines; 103 pmol of leucine incorporated); lane 3, bovine rhodanese (M r, ˜33,000; 25 leucines; 115 pmol of leucine incorporated); lane 4, trigger factor (M r, ˜58,000; 31 leucines; 73 pmol of leucine incorporated); lane 5, release factor 1 (M r, ˜40,500; 32 leucines; 50 pmol of leucine incorporated); lane 6, release factor 2 (M r, ˜41,200; 29 leucines; 61 pmol of leucine incorporated).

Puromycin reactivity of nascent peptides. Hamster (H) and bovine (B) rhodanese were synthesized as described in the legend to Fig. 1 except that 5 µl of S30 (E. coli MRE 600) and nonradioactive amino acids were used. After coupled transcription-translation, 32P-labeled C-puro was added to give 8 µM, and the incubation continued for 10 min. Then one aliquot was used to determine the incorporation of C-puro into nascent peptides, and another aliquot was processed for polyacrylamide gel electrophoresis and phosphorimaging. The result is shown. Lane 1, no plasmid added; lane 2, hamster rhodanese; lane 3, bovine rhodanese. About 4.5 and 5 pmol of C-puro was incorporated into hamster and bovine polypeptides, respectively. The numbers on the left side of the gel indicate the numbers of amino acids (a a #) corresponding to the puromycin-labeled band in either lane 2 (H) or lane 3 (B). On the right side of the gel, amino acid positions in which a change in amino acid composition from H to B occurred are given.

Structures of different fluorophore–Met-tRNAf species that were synthesized and used in coupled transcription-translation.

Time course of CAT synthesis with fluorophore–Met-tRNAf. CAT was synthesized by coupled transcription-translation with a reduced amount of the S30 fraction (2.5 µl / 30-µl assay). Protein synthesis was initiated with either f[35S]Met-tRNAf, cascade yellow–[35S]Met-tRNAf, pyrene–[35S]Met-tRNAf, coumarin–[35S]Met-tRNAf, or eosin–[35S]Met-tRNAf. The reaction mixtures were incubated, and at the indicated times aliquots were withdrawn and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and phosphorimaging. The results are shown.

Fusidic acid keeps short CAT peptides in the ribosomal P site. CAT was synthesized for either 5 min (lanes 1 and 2) or 30 min (lanes 3 and 4) by coupled transcription-translation with an S30 fraction from MRE 600 and unlabeled amino acids. Then, either H2O (lanes 1 and 3) or fusidic acid (lanes 2 and 4) was added and the samples were kept on ice for 3 min before the addition of 32P-labeled C-puro. The samples were incubated and processed as described in the legend to Fig. 2. The numbers on the left refer to positions of molecular weight markers as follows: 31, carbonic anhydrase; 20, soybean trypsin inhibitor; 14, lysozyme; 6, aprotinin; 3.5, insulin β chain. +, present; −, absent.

Interaction of eosin–Met-tRNAf with IF2 and its binding to salt-washed ribosomes. (A) Binding of f[35S]Met-tRNAf and eosin–[35S]Met-tRNAf to IF2 was determined by the Millipore filter binding assay in the absence of Mg2+ ( Sundari et al., 1976 ). IF2 was isolated according to the method of Mortensen et al. (1991) from an E. coli strain transformed by a plasmid containing the IF2 sequence. (We thank U. RajBhandari for providing the plasmid.) (B) Binding of f[35S]Met-tRNAf and eosin–[35S]Met-tRNAf to salt-washed ribosomes was carried out in the presence of 18 mM Mg2+ in the absence (w/o) or presence (w/ ) of an excess of IF2.

Increased synthesis of full-length protein in the S30 fraction from MRE 600. Both RHO and CAT were synthesized for 20 min in the presence of [14C]Leu. The samples shown in lanes 2 to 4 received increasing amounts of a fraction derived from a different S30 before protein synthesis was started. This fraction was isolated by S300 chromatography and eluted well behind ribosomes but before ˜100-kDa proteins. The arrows indicate the positions of the full-length proteins.