How to gum up the ribosomal works

Ribosomes are the protein factories of the cell. Lined up to form veritable assembly lines, these molecular complexes link together the amino-acid building blocks that make up each of the thousands of proteins found in every cell. The heart of the ribosome is the so-called peptidyl transferase center, where the genetic information in the messenger RNA molecule to which the ribosome is attached is translated into the specific sequence of amino acids that forms the corresponding protein. Some mRNA sequences contain signals that cause the process to stall at defined positions. These interruptions play a role in regulating both protein biosynthesis and gene activity. Biochemists led by Dr. Daniel Wilson and Professor Roland Beckmann, both of whom work at LMU's Gene Center and are members of the Excellence Cluster "Center for Integrated Protein Science Munich" (CiPSM), have now elucidated the mechanism that underlies this mode of regulation. "Our findings may some day make it possible to exploit the effect for targeted control of gene activity", says Wilson, "but our immediate priority is to study the termination process in other peptides and other organisms." (Molecular Cell online, 8 October 2010)

Proteins are the workhorses of the cell, serving as structural materials and chemical catalysts. In the cells of multicellular organisms - plants, animals and fungi - which are classified as eukaryotes, the genetic material that specifies the structures of proteins resides in a specialized compartment called the nucleus. The ribosomes that produce proteins, however, are located in the surrounding cytoplasm. So the instructions for each protein must be exported, as mRNAs, from the nucleus to the ribosome, which translates each blueprint into a specific sequence of amino acids. "Although we know a lot about the biochemistry of protein biosynthesis, many of the steps involved remain poorly understood", says Dr. Daniel Wilson, a biochemist at LMU's Genzentrum.

One of the black boxes in the process is the regulatory mechanism that leads to termination of translation. To gain insight into this step, a team of researchers led by Wilson and Professor Roland Beckmann has taken a closer look at stalled eukaryotic ribosomes. Using a newly developed protocol, they prepared ribosomes that were programmed to synthesize two different protein fragments that induce stalling. "In both cases, the fragments produced were very short peptides that are known to have regulatory functions", says Beckmann.

Normally, as synthesis proceeds, the addition of new amino acids at one end progressively pushes the growing protein chain into the so-called ribosomal tunnel. The short regulatory peptides are distinguished by the fact that they interact with this tunnel during the elongation process. Electron microscopic examination of the stalled ribosomes reveals how the interaction occurs. One of the protein fragments studied by Wilson and his colleagues, the fungal Arginine Attenuator Peptide (AAP), appears to adopt a compact conformation close to the peptidyltransferase center (PTC) that links the amino acids together. Blockage of the PTC probably accounts for the cessation of translation. In the case of the AAP and the other peptide investigated, which was derived from the virus hCMV (human cytomegalovirus), the blockade also stabilizes a segment of the amino-acid chain that has reached a constriction in the tunnel.

"The stabilization may itself have a significant effect on the folding of the protein", says Wilson. The linear sequence of amino acids determines the three-dimensional structure of the finished protein, which in turn is crucial for its biological function. In fact, errors in protein folding can result in disease. "These results provide our first glimpse of the mechanisms that terminate the translation process", says Beckmann. "A detailed understanding of this step might allow us to activate and inactivate therapeutic proteins at will, but, of course, this goal is still a long way off." The team now intends to study translation termination further using other peptides from other organisms. (CA/suwe)

Publication:
Structural basis for translational stalling by human cytomegalovirus (hCMV) and fungal arginine attenuator peptide (AAP)
Shashi Bhushan, Helge Meyer, Agata L. Starosta, Thomas Becker, Thorsten Mielke, Otto Berninghausen

Source: LMU Munich