The f luorescence decay functions of individual, specifically labeled tRNA Phe molecules exhibit nonexponential character as a result of conformational dynamics occurring during the measurement on a single molecule. tRNA Phe conformational states that interchange much more slowly are evidenced by the distribution of lifetimes observed for many individual molecules. A structural model for the nonexponential decay indicates that the tRNA Phe -probe adduct f luctuates between two states, one of which provides conditions that quench the probe f luorescence.

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The ribosome is universally responsible for synthesizing proteins by translating the genetic code transcribed in mRNA into an amino acid sequence. Ribosomes use cellular accessory proteins, soluble transfer RNAs, and metabolic energy to accomplish the initiation, elongation, and termination of peptide synthesis. In translocating processively along the mRNA template during the elongation cycle, ribosomes act as supramolecular motors. Here we demonstrate that ribosomes adsorbed on a surface, as for mechanical or spectroscopic studies, are capable of polypeptide synthesis and that tethered particle analysis of fluorescent beads connected to ribosomes via polyuridylic acid can be used to estimate the rate of polyphenylalanine synthesis by individual ribosomes. This work opens the way for application of biophysical techniques, originally developed for the classical motor proteins, to the understanding of protein biosynthesis.

Reverse-phase HPLC was used to fractionate 40s ribosomal proteins from human placenta. Application of a C, reverse-phase column allowed us to obtain 27 well-resolved peaks. The protein composition of each chromatographic fraction was established by two-dimensional polyacrylamide gel electrophoresis and N-terminal sequencing. N-terminally blocked proteins were cleaved with endoproteinase Lys-C, and suitable peptides were sequenced. All sequences were compared with those of ribosomal proteins available from data bases. This allowed us to identify all proteins from the 40s human ribosomal subunit in the HPLC elution profile. By matrix-assisted laser-desorption ionization mass spectrometry the masses of the 40s proteins were determined and checked for the presence of post-translational modifications. For several proteins differences to the deduced sequences and the calculated masses were found to be due to post-translational modifications.Keywords: human ribosomal proteins ; HPLC ; N-terminal and internal amino acid sequencing ; matrixassisted-laser-desorption ionization MS ; post-translational modifications.Human ribosornd proteins are the main components of the fundamental processes which translate genetic information into proteins at the ribosome. Some ribosomal proteins (rp) are further involved in non-ribosomal functions. Recently, rp L7 was shown to contain the basic-region-leucine-zipper sequence which mediates the stable binding of this protein to DNA [I]. Moreover, rp S3 was found to act as an endonuclease in the DNA-repairing system [2]. Finally, some data indicate that tumor diseases may be accompanied by overexpression of rp genes 13, 4) and by chromosomal rearrangements in regions of rp genes [ S ] . Therefore, structural analysis of these proteins is necessary for complete understanding of their fundamental role in the cell. The task of structure determination of rat rp has been almost fully resolved by the application of recombinant-DNA technology ; details are reviewed in [6]. However, knowledge of the cDNA sequences does not provide information concerning post-translational modifications of the proteins after their biosynthesis and assembly into the ribosomal subunits. Several modifications of rp have been determined in the eubacterial ribosome [7, 81, and the eukaryotic rprotein S6 is known to be phosphorylated 191. This phosphorylation was shown to be connected with cell growth [I 01 and cell cycle control [I I]. At least seven 40s proteins from HeLa cells were found to be methylated, and the level of their methylation was shown to be altered in the Abbreviations. MALDI, matrix-assisted laser-desorption ionization ; Lys-C, endoproteinase Lys-C; RP, reverse-phase; rp, ribosomal protein ; S-proteins, proteins of the small ribosomal subunit; 40S, small ribosomal subunit; TP40, total protein mixture of the small ribosomal subunit; TOF, time-of-flight ; Pth-Xaa, phenylthiohydantoin -amino-acid.course of the cell cycle [12]. It is also known that ribosomal proteins may be acetylated 17, 13, 141. However...