Antibiotic-Resistant Ribosome Sheds Light on Protein Synthesis

Escherichia coli

E. coli

Ribosomes bring together two different forms of RNA—messenger RNA (mRNA) that carries genetic information from the DNA in a cell's nucleus, and transfer RNA (tRNA) that carries the amino acids in a cell's cytoplasm. A chain of amino acids is then joined together by the ribosome to make proteins in accordance with the genetic blueprints provided in the mRNA.

Protein assembly on the ribosome depends upon an accurate decoding of mRNA's genetic information for tRNA. However, even though ribosomes have been studied for more than 40 years, the way this process actually works is only now being unraveled. A key to understanding protein synthesis is the ability to study the structure of the ribosome, which is an ancient organelle so highly conserved by evolution that its core components are pretty much the same for all forms of life. The researchers have been looking into the ribosome of the common E. coli bacterium, which has long been a valuable scientific subject for protein synthesis research. Many decades have been spent developing the biochemical and genetic tools necessary to study protein synthesis in E. coli. Ribosomes from this organism should provide the best means for determining the atomic-resolution structure of the ribosome in all stages of protein synthesis.

The High-Fidelity Ribosome

What do consumer electronics have in common with bacterial ribosomes? Computer files, video DVDs, music CDs—all store encoded information that must be decoded with a high degree of accuracy. To do this, the read/write technologies for these media employ error-correction protocols; without them you end up with too many corrupted files, unplayable DVDs, and CD coasters. A strand of RNA similarly contains encoded (genetic) data that must be accurately "read" by a ribosome to produce the proteins needed by the host organism. Antibiotics such as streptomycin work by binding to bacterial ribosomes and causing an increase in "read errors." However, a particular mutation in the E. coli ribosome has been found to endow the bacteria with resistance to streptomycin by counteracting the antibiotic's error-inducing effects. Ribosomes of this so-called "hyper-accurate" strain of E. coli provide an excellent opportunity to gain valuable insight into which ribosomal mechanisms play a role in deciphering the genetic code.

In this study, the researchers specifically compared the x-ray crystal structures of the wild type (WT) and hyper-accurate forms of the 70S ribosome. The hyper-accurate form is found in a mutant strain of E. coli that is extremely resistant to the antibiotic streptomycin. Streptomycin binds to ribosomes and leads to error-prone protein synthesis, but streptomycin resistance mutations in the ribosome are able to counteract the error-inducing effects of the antibiotic. The researchers were looking for structural differences between ribosomes resistant and sensitive to streptomycin that might have an effect on mRNA decoding.

ALS Beamline 8.3.1 was used to image the 70S ribosome of E. coli samples at a resolution of 10 Å for the WT form, and 9 Å for the hyper-accurate form. The research team focused on two conformational changes known to take place on the 30S subunit, the smaller of two asymmetric subunits that make up the 70S ribosome. One change is the closing of the 30S head around tRNA when this subunit is joined with the larger 50S subunit to create an intact ribosome. The second change is an RNA helical switch near the mRNA decoding site on the 30S subunit. Although both have been proposed as sites where mRNA decoding takes place, the images obtained by the researchers indicate otherwise.

While they did see the 30S head clamping down on tRNA much like a tape head clamps down to read data on a magnetic tape, the images suggest the clamping is independent of mRNA decoding and probably has more to do with forming the intact ribosome. They saw no evidence in the images nor in subsequent biochemical tests that the RNA helical switch played any role in mRNA decoding.

The "head" (H) of the ribosome's 30S subunit (purple) tilts 5–8 Å toward the 50S subunit (gray) when the intact ribosome is formed. The images obtained suggest that the clamping is independent of mRNA decoding. Also shown for reference are bound mRNA and tRNA (green and blue).

Research conducted by A. Vila-Sanjurjo, W.K. Ridgeway, V. Seymaner, and W. Zhang (Univ. of California, Berkeley); J.H. Doudna Cate (Univ. of California, Berkeley, and Berkeley Lab); and S. Santoso and K. Yu (Massachusetts Institute of Technology).

Research funding: The Whitehead Institute for Biomedical Research, the Massachusetts Institute of Technology, the Searle Scholars Program, and the National Institutes of Health. Operation of the ALS is supported by the U.S. Department of Energy, Office of Basic Energy Sciences (BES).

Publication about this research: A. Vila-Sanjurjo, W.K. Ridgeway, V. Seymaner, W. Zhang, S. Santoso, K. Yu, and J.H. Doudna Cate, "X-ray crystal structures of the WT and a hyper-accurate ribosome from Escherichia coli," Proc. Natl. Acad. Sci. 100, 8682 (2003).

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