In all living organisms, ribosomes translating membrane proteins are targeted to membrane translocons early in translation, by the ubiquitous signal recognition particle (SRP) system. In eukaryotes, the SRP Alu domain arrests translation elongation of membrane proteins until targeting is complete. Curiously, however, the Alu domain is lacking in most eubacteria. In this study, by analyzing genome-wide data on translation rates, we identified a potential compensatory mechanism in E. coli that serves to slow down the translation during membrane protein targeting. The underlying mechanism is likely programmed into the coding sequence, where Shine–Dalgarno-like elements trigger elongation pauses at strategic positions during the early stages of translation. We provide experimental evidence that slow translation during targeting and improves membrane protein production fidelity, as it correlates with better folding of overexpressed membrane proteins. Thus, slow elongation is important for membrane protein targeting in E. coli, which utilizes mechanisms different from the eukaryotic one to control the translation speed.
Proteins are built as long chain-like molecules. First, a length of DNA is copied to make a messenger RNA (or mRNA) molecule, which then binds to a large molecular complex called a ribosome. The ribosome reads and translates the code in the mRNA sequence to build a protein chain, which then folds into a specific three-dimensional shape to allow the protein to perform its function.
Many proteins also need to be targeted to the right location within the cell in order to carry out their role. Some proteins have to be inserted into the membranes of cells and these proteins are directed, as they are being built, to the membrane by another molecular complex called the signal recognition particle (or SRP for short). The SRP binds to the new protein as it emerges from the ribosome and helps to direct it to the membrane. To make sure that membrane proteins fold correctly, their translation is paused whilst the protein is being targeted to the membrane. Plants, animals, and other eukaryotes do this via a unique part of the SRP complex that only allows the translation to continue once the translating ribosome has been brought close to the membrane. However, most bacteria lack this part of the SRP complex, and yet they are still able to accurately insert new, correctly folded, proteins into their membranes. This suggests that an alternative mechanism must exist in bacterial cells.
Fluman et al. looked at an existing data set that had measured how many ribosomes are found at different points along the length of mRNA molecules at any given time in the bacterium E. coli. If ribosomes are consistently found at specific sites in given mRNA molecules, it suggests that these are the sites where a pause in translation occurs. Specific short mRNA sequences—that bind to a ribosome and hold it in place—are often found in these pause sites. These sequences are similar to another sequence, called the Shine–Dalgarno sequence that is often also found at the very start of an mRNA molecule, where it functions to recruit a ribosome and begin the translation process.
Fluman et al. reveal that mRNAs of membrane proteins contained these similar sequences early on in their coding region. Some looked likely to pause the translation before the newly formed protein chain emerged from the ribosome, which could give the ribosome time to be targeted to the membrane. Other Shine–Dalgarno-like sequences were found slightly later on in the mRNA molecules for protein chains that span back-and-forth through the membrane several times.
Fluman et al. show that slowing translation in this manner—which is different to that used by eukaryotes—helps to ensure that membrane proteins are folded correctly in E. coli. Although these pauses occur frequently, mainly in the early stages of the translation of membrane proteins, there are many other translation pause sites that are known to exist in other mRNAs. The next challenge is to understand the function of these other pause sites, and how they work together with other mechanisms to regulate translating ribosomes inside cells.