The ribosome is the organelle that translates the genetic code into proteins in all types of living cells. The ribosome has two subunits, a small subunit that is responsible for decoding the genetic information, and a large subunit where coupling of amino acids into a protein takes place. Newly synthesized proteins exit the ribosome through a long, narrow tunnel in the large ribosomal subunit. Under some circumstances, growth of specific proteins is interrupted for short or long periods of time. This project addresses the mechanisms that control which proteins are subject to such arrest of synthesis. Current evidence indicates that the stoppage involves interactions between the portion of the protein that was synthesized immediately before the polymerization arrest and RNA and/or protein components of the tunnel. The project focuses on a very short protein (CrbCmlA; only 9 amino acids long), which is involved in the regulation of another protein (CmlA) that makes cells resistant to the ribosome-targeting antibiotic chloramphenicol. A low concentration of chloramphenicol (insufficient to inhibit ribosomes synthesizing other proteins) is required to arrest the polymerization of CrbCmlA, but it is not known how or why chloramphenicol specifically inhibits synthesis of this protein. The hypothesis is that the growing protein and chloramphenicol both interact with ribosomal RNA and proteins to bring about the conditions that stop synthesis. The experiments in this project are designed to define which features of each of the three interacting components (CrbCmlA, the ribosome and chloramphenicol) are required for this tri-component interaction and how these interactions establish translation arrest. The bacterium Escherichia coli is chosen as the experimental organism because of the ease of its genetic manipulation and the availability of procedures for biochemical analysis of its ribosomes. Specifically, the study will employ a mixture of genetic, biochemical, and physiological methods to (1) identify critical features of the CrbCmlA pausing peptide (length, sequence, and electrostatic properties), the ribosome, and the co-inducer necessary for pausing, (2) characterize the interactions between CrbCmlA, ribosomal components, and chloramphenicol, and (3) probe the effect(s) of CrbCmlA on the interaction of chloramphenicol with the ribosome. For context, pausing of Crb will be compared with translational arrest of another protein (SecM), which does not require binding of a separate molecule to the ribosome. The collective results of these experiments will provide model(s) for the molecular mechanism of translation arrest and comparison of the results for experiments with the two proteins will reveal whether peptide synthesis pausing can be explained by a single mechanism or if there are different pausing pathways in the ribosome. This research will have impact in two areas. First, it will generate a more comprehensive understanding of the function of the ribosome, an organelle that is universally required for the survival and growth of all types of biological cells. Second, it will provide training opportunities for both graduate and undergraduate students in biological research that integrate molecular genetics with advanced biochemical probing of interactions in a major multi-component complex.
Ribosomes are found in all forms of life. They translate the genetic code into protein, the entities responsible for most biochemical function and structures in biological cells. Ribosomes are therefore essential to all life. Ribsomes are complex structures that contain several RNA and many protein molecules. We investigate the formation from their many components and function of ribosomes. Major findings resulting from work funded by this NSF grant are: (i) Mutations in portions of two specific ribosomal proteins (L4 and L22) reduce the speed of coupling of amino acids into protein chains ribosome the catalytic center. This suggests that protein L4 and L22 are part of a mechanism that couples the rate of growth of protein chains with the transport of new proteins out of the ribosome. (ii) The same segments of protein L4 and L22 are necessary for normal formation of ribosomes, probably because they help organize the ribosomal RNA during assembly of ribosomes from its constituent parts. Nevertheless, protein L4 is not required in one bacterial species, but cells without L4 are extremely sick and growl at rates about 4-5 times lower than wildtype cells (iii) In the yeast, a simple model organism for investigation of eukaryotes, ribosome formation is required for initiating the cycle that generates new daughter cells. Our experiments have pinpointed essential features of the mechanism for this coordination.
