(ORDO NEWS) — Skoltech researchers and their colleagues studied more than 30 thousand variants of genetic sequences encoding two fluorescent proteins in order to understand what characteristics of mRNA and the first ten codons in it can increase the efficiency of the translation process. Among other things, they found that the rare codons at the beginning of the sequence did not appear to increase translation efficiency, as previously suggested.
The study was published in the journal Nucleic Acids Research. Translation is one of the fundamental processes in the cell: on the basis of messenger RNA (obtained from DNA during transcription), the ribosome builds a chain of amino acids, which then folds into a protein that sets off to perform various vital functions.
Each amino acid is encoded by a codon, a triple of nucleotides in the mRNA chain. Only 20 amino acids that the ribosome connects have 61 codons , which means that some codons are synonymous – they encode the same amino acid.
After decades of research, scientists are still not completely sure what makes the cellular “protein plant” more or less effective. For example, there is evidence that some secondary structures of mRNA — how it is laid out in space at its beginning — may interfere with the ribosome binding to it and doing its job.
Another possible factor is the very synonymous codons: previous studies have indicated that perhaps statistically more rarely used codons can increase translation efficiency if they are at the beginning of an open reading frame. These codons slow down the movement of the ribosome along the mRNA at its beginning so that there are no further “queues” from the ribosomes.
This is not an idle search: the study of translation efficiency will help to better understand gene expression and increase the efficiency of biotechnological workhorse bacteria that produce the desired proteins. Therefore, Ilya Osterman and Zoya Chervontseva from the groups of Peter Sergiev, Olga Dontsova and Mikhail Gelfand at Skoltech and Moscow State University named after M.V. Lomonosov and their colleagues decided to hold a kind of competition: they tested more than 30 thousand variants of mRNA encoding the same proteins to Understand which options will give a more efficient translation.
Researchers were interested in codons with numbers from 2 to 11 (the first codon is always the ATG start codon, just as the first lines of any program in some programming languages tell the machine that the program code will follow).
Scientists used Escherichia coli Escherichia coli and plasmids – circular DNAs encoding the so-called double fluorescent reporter (the “duet” in this case is two fluorescent proteins, red RFP and blue CER).
Randomized sequences of 30 nucleotides were inserted immediately after the start codon so that they became second to eleventh codons in the mRNA. Having grown the bacteria and sorted them by how efficiently they managed to produce CER and RFP, the scientists used the flowseq method to figure out which sequences provided the most efficient protein production.
“Flowseq is a combination of flow cytometry (a technique in which the physical and chemical characteristics of cells are measured by scattering a laser beam) and sequencing of separated fractions. This method allows you to evaluate the effectiveness of protein synthesis for thousands of options at a time, ”says Ilya Osterman.
It turned out that the secondary structure of mRNA can indeed interfere with translation, but scientists were not able to show that rare codons at the beginning of a protein coding sequence positively affect translation. However, they found that additional start codons contribute to efficient translation, and additional Shine-Dalgarno sequences that “call” the ribosome to mRNA, on the contrary, interfere with it.
Researchers believe their findings will help develop more efficient artificial gene constructs that can be used to turn ordinary bacteria like E. coli into powerful biotechnological tools.
Other organizations involved in the study include the Institute of Bioorganic Chemistry named after Academicians M.M. Shemyakin and Yu.A. Ovchinnikov, the Institute of Chemical Biology and Fundamental Medicine of the SB RAS and the Institute for Information Transmission Problems named after A. A. Kharkevich.
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