Fluorescence Studies Of Subunit Exchange In 'Thermotoga Maritima' Ribosomal Stalk Protein L12

Abstract

The "stalk" of the prokaryotic 50S ribosomal subunit contains four copies of the protein L7/L12. In 'E. Coli', L7/L12 is a dimeric protein, which is able to undergo rapid subunit exchange (Hamman et al., 1996 Biochemistry 35: 16680-6). Using fluorescence polarization we investigated subunit exchange between labeled cysteine mutants of 'T. maritima' L12 and wild type proteins. In these systems, polarization increases after subunit exchange due to relief of depolarization caused by homo-energy transfer. We demonstrated that the rate of subunit exchange in 'T. maritima' L12 is significantly slower at 25C than in the 'E. coli' system. We also show that in 'T. maritima' the exchange rate increases with increasing temperature. Possible factors responsible for the difference between these two systems are discussed. We also investigated the mode of interactions between L12 subunits. The crystal structure suggested a tetrameric arrangement of the L12 proteins from 'T. maritima' (Wahl, et al., 2000 Embo J 19, 174-86). In our solution studies, however, data from Forster Resonance Energy Transfer experiments supported a dimer model for L12. From the X-ray data, 2 possible dimerization modes were proposed. In the first mode, the monomers form a tight symmetric and parallel dimer. In the second mode, the monomers bind through their N-terminal region in an anti-parallel configuration with one monomer in a tight conformation and the other monomer adopting an elongated shape. Our results support the second mode. These studies were supported by NSF grant MCB9808427 (DMJ).