Making Proteins: Where to Begin?
By Tegan Armarego-Marriott
Let’s start at the very beginning…
Whether singing a song, baking a cake or reading a book, I’ve heard that the beginning is a (very) good place to start. The same holds true if you happen to be a ribosome, creating proteins from messanger RNA templates inside plant or animal cells. Unfortunately, finding the beginning of a protein is not as simple as opening a book to chapter one: working out the place to start is, in itself, half of the battle.
Recent work by Scharff and colleagues demonstrates some of the tricks of the trade that ribosomes use to find the “true start”, when making proteins in the chloroplasts of plants.
When you read you begin with ABC…
Unless you’re a ribosome. In which case, you begin with AUG.
RNA, like DNA, is a polymer of nucleotides strung together in a chain. The four nucleotides that make up RNA are adenine (A), guanine (G), cytosine (C), and uracil (U). The function of the RNA is to carry a coded message, consisting series of three-letter nucleotide ‘words’ that can be translated into a string of amino acids. For example, the RNA triplet UGG codes for the amino acid tryptophan, while UCU can be translated into serine. Put enough of these amino acids together, and you’ve got yourself a protein. Ribosomes do this by providing a platform and the enzymatic machinery necessary for binding amino acids together, in the sequence dictated by the mRNA.
Unfortunately, while translating the code itself is pretty straight forward, finding the correct place to start reading is a bit more complex. Just as movies in the cinema are preceded by ads and trailers, and are followed by credits of varying length, so too is the RNA message that codes for a protein surrounded by hundreds of nucleotides that never get translated. So, for each protein, the ribosome that translates the message must first find the proper place to start.
As it happens, most proteins begin with the amino acid methionine, which is coded by the RNA letters AUG. So ribosomes recognize that they must start with AUG. Unfortunately, AUGs are not only found at the beginning of proteins, but also within them (methionine is a common amino acid), and are also found in the untranslated regions before and after the protein coding RNA. In searching a single long mRNA, which may encode multiple proteins, the ribosome might therefore encounter a bunch of AUGs in the untranslated region of an mRNA, followed at some point by the all-important “start” AUG, then followed by a whole lot of internal protein AUGs in amongst other code, then a triplet signaling to stop making the first protein, followed by another AUG to start making the second protein. And so on.
Finding the “true” AUG starting point for each protein is critical, as it represents the difference between making protein that functions properly in the cell, and making the wrong protein, which can waste energy or might even be harmful.
Shining a guiding light: the Shine-Dalgarno sequence
In order to find the start AUG, bacterial-type ribosomes, including those found in chloroplasts, rely on the laws of attraction. In the world of nucleotides, C is attracted to G while U is attracted to A. These attractive forces are very weak for single nucleotides, but in a string of RNA, they become strong enough to hold complementary sequences together in a double strand. The ribosome itself contains a conserved short sequence of nucleotides, called the anti-Shine-Dalgarno sequence. This sequence, which reads simply CCUCCU, is attracted to its complementary sequence — the Shine-Dalgarno sequence (GGAGGA) — found within many mRNA molecules, adjacent to and just a few base pairs upstream of the start AUG. The attraction between the anti-Shine-Dalgarno (aSD) and the mRNA’s Shine-Dalgarno (SD) helps to line up the ribosome with the start AUG, indicating that this particular AUG is the right place to start.
The problem is that not all chloroplast mRNA have a Shine-Dalgarno sequence, and yet these SD-less mRNAs are nonetheless translated. Which has led to a long-held debate within the scientific community over how important the Shine-Dalgarno sequence, and the SD-aSD attraction really are.
The importance of attraction
In order to investigate the importance of the SD-aSD attraction, Scharff and colleagues created plants containing ribosomes with modified aSD sequences. They created a number of modifications specifically designed to weaken the interaction between the aSD and the SD by varying amounts, or to abolish the attraction completely.
Introducing even a weak mutation (CCUCCU to UCUCCU), designed to cause only a slight disruption in the interaction, resulted in pale green plants. Furthermore, a genome-wide analysis of translation in these mutant plants revealed a link between the weakened aSD-SD attraction and translational efficiency. Introduction of a slightly stronger mutation (CCUCCUàCCCCCU) gave rise to slow growing, extremely sick plants, while plants with even stronger mutations could not be obtained, suggesting that those changes were lethal.
These results suggest that not only is the SD-aSD attraction important for plant health — it is absolutely essential for plant survival!
Structure matters
Given that some chloroplast mRNA do not have any recognizable SD sequence, it’s clear that not all mRNA rely on the aSD attraction to be translated, suggesting that there is another factor that can influence the efficiency of translation. Previous research suggested that the shape of the mRNA molecule itself might come into play (Scharff et al., 2011).
Although mRNA message looks like a straight line of letters on the page, in its true form inside the cell it twists and turns back on itself, forming a complex secondary structure. It has been suggested that mRNA wrapped into three dimensional form might be harder to reach for ribosomes, and might also make it harder for the ribosomes ‘see’ that start AUG. In line with this idea, the authors found that mRNA with more complicated secondary structure around the AUG generally depended on the aSD-SD for help: translation from these mRNAs was more likely to be reduced in the aSD mutants with weakened aSD-SD attraction. By contrast, mRNA without all the mess around their AUG seemed much less reliant on the attraction, and could therefore be translated efficiently even in the mutant.
Although the work by Scharff and colleagues has revealed a great deal about the SD-aSD attraction and its impact on translation efficiency in chloroplasts, we still are not at the point where we can predicted the way in which any single mRNA is translated based on its individual aSD sequences. This is likely because chloroplast translation relies on more than just the ribosome and the mRNA: a whole cohort of regulatory factors influences translational efficiency, and allows translation to change with external environment and the needs of the plant. Which means that, as always, there’s room for further research!
Tegan Armarego-Marriott
Max Planck Institute of Molecular Plant Physiology
Potsdam, Germany
Armarego@mpimp-golm.mpg.de
ORCID: 0000–0002–8745–9468
The ‘it’s a little more complicated’ section:
Although we’ve stated that the Shine-Dalgarno sequence is ‘GGAGGA’, there’s actually a bit of flexibility. We’ve also not really discussed here the fact that there’s bit of wiggle room when it comes to the attractions between the nucleic acids, for example, Gs can also be attracted to Us. Also important in this equation is not just the letters themselves, but the spacing of these letters. So in the case of the SD-aSD interaction, the SD is often less than a perfect ‘GGAGGA’. This makes them harder to find in the genome sequence, but might have interesting consequences: different types of sequence might lead to different efficiencies of translation!
Read the research article upon which this story is based:
Scharff, L.B., Ehrnthaler, M., Janowski, M., Childs, L.H., Hasse, C., Gremmels, J., Ruf, S., Zoschke, R., and Bock, R. (2017). Plant Cell 29: 3085–3101; DOI: https://doi.org/10.1105/tpc.17.00524
Other cited references:
Scharff, L.B., Childs, L., Walther, D. and Bock, R. (2011). Local absence of secondary structure permits translation of mRNAs that lack ribosome-binding sites. PLoS Genet. 7, e1002155.
