Enzyme plays important role in bacterial translation

Cells have elaborate machinery in place to produce proteins, starting from the transcription of genes that encode a protein to the ribosomes that piece the protein together. Like many systems in nature, this can result in errors from time to time. Mechanisms that protect against these errors are still being studied, and recent research has shed some light onto a component of this system in bacteria.

Using Escherichia coli cells, researchers at the Department of Molecular Biology and Genetics in the Hopkins School of Medicine have identified a more detailed role of Release Factor 3 (RF3) in screening for errors in bacterial production of proteins. In a paper published last month in the journal Cell, the researchers explained the role of RF3 in addressing errors in protein production.

Bacteria and many other types of cells rely on ribosomes to produce proteins by connecting the correct amino acids in the proper order, based on the genes encoded in DNA. To do so, an mRNA, or messanger RNA, is made based on the sequence encoded by the gene, and it is used by the ribosome as a template to sequentially bond amino acids into a chain. Once the ribosome finds the signal towards the end of the mRNA to stop making the protein, a Class I Releasing Factor comes into the ribosome, allowing the protein chain to leave the ribosome. RF3, a Class II Releasing Factor, comes in and "recycles" the Class 1 Releasing Factor, allowing it to leave the ribosome and move on to wherever it is needed.

RF3 has an additional role in another mechanism that makes sure the right amino acid is used by the ribosome for a specific portion of the protein. If the wrong amino acid comes in, or if the stop signal is misread as another amino acid, then RF3 interacts with a mechanism that acts after an amino acid has been introduced to release the protein chain immediately from the ribosome.

To hone in on these actions of RF3, the researchers used a mutant bacteria that lacked a gene encoding RF3 to see how bacteria would grow and produce proteins under such conditions. They also utilized several strains of bacteria with and without this deletion that looked at bacteria which are ultra accurate at producing proteins correctly and those that are likely to mess something up.

One of the methods the researchers used to test the importance of RF3 was to grow bacterial strains in the presence of streptomycin, an antibiotic that fights bacteria by making errors in the production of proteins by the bacteria. Without the antibiotic, the normal bacteria and those lacking RF3 were able to grow normally with no discernible difference in growth rate, but, when grown with streptomycin at levels tolerable by the normal bacteria, those lacking RF3 failed to grow while the normal bacteria was doing fine.

As for the production of proteins in the midst of errors, the researchers looked at the importance of RF3 in a bacterial strain that was prone to making mistakes in protein production. They found that with RF3, the error-prone strain would not produce full-length proteins while the mutant lacking RF3 would go on making the proteins in full, despite possible errors. In an additional step to verify that RF3's absence led to the creation of proteins in full despite possible errors, the researchers inserted a plasmid into the RF3-lacking bacteria. This plasmid, a short strand of DNA bacteria use to pick up and pass genes to neighboring bacteria, contained RF3, so its presence would "replace" the RF3 gene missing in the bacteria's main DNA. With this plasmid, the bacteria behaved much like the normal error-prone bacteria, failing to produce full-length proteins.

Other roles of RF3 identified by the researchers include a destabilization role that RF3 has on mRNA, or an increase in mRNA stability when RF3 is absent, as well as a reduction in frame shift mutations. These sort of mutations affect the way in which a ribosome reads an mRNA, having a impact on nearly all of the protein that is encoded after the frame shift mutation.

The researchers note that RF3 is only found in a subset of bacteria, and a similar protein has yet to be found in eukaryotes, more complex cells such as fungi or amoeba and those that make up complex organisms such as ourselves. In addition, the error control mechanism that RF3 is involved in does not exist in eukaryotes, or at least that appears to be the case based on the research lab's previous work. Instead, we rely on different and multiple mechanisms to ensure our ribosomes are properly making proteins, within their realm of control to say the least.