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Connect With Us. Local Sales Support Get in touch with a nearby distributor or sales representative. Find Sales Contact. Contact Us Customer Support. L in vitro translation reaction 5. Frequently Asked Questions How do I determine the apparent molecular weight of a protein? Article References Kozak, M. Cell 44 , — Kozak, M. Nucleic Acids Res. Moreover, our data clearly showed that changes not only in the Kozak sequence but also inside the upstream region markedly affect gene expression.
All point mutations except the one in k 38 resulted in a fluorescence level higher than that associated with k 1. None of these were taken into account in the reference work by Dvir et al. Effect of point mutations in the upstream region on fluorescence relative to k 1.
We carried out simulations with RNAfold to investigate possible correlations between computed mRNA secondary structures, together with their corresponding minimum free energies MFEs , and measured fluorescence levels. In contrast, no plausible justification for the effects of single point mutations on translational efficiency emerged from simulations with RNAfold.
Each sequence was nucleotides long. From preliminary simulations, we observed that a poly-A chain with a variable length of — nucleotides had no significant effect on mRNA folding. This is the highest—and the most common—within the collection of 59 sequences analyzed in this work see Additional file 1.
The two sequences contain multiple guanines in the extended Kozak sequence involved in pairing interactions with the CDS. A similar pattern is also present in k The MFE of k 26 is substantially lower than those of k 30 and k 31 because of the presence of another stem due to pairing interactions between the upstream region and the CYC1 terminator.
Nevertheless, the fluorescence levels of k 30 and k 31 are only approximately 1. As a consequence, the giant hairpin is destroyed and replaced by one or two stems that lower the MFE of the mRNA secondary structure Table 2. However, the fluorescence levels we measured did not increase proportionally to increments in the MFE. Moreover, in two cases k 32 and k 36 RNAfold predicted a giant hairpin in the mRNA structure, whereas the fluorescence levels from our experiments were significantly lower than that of k 1 Fig.
Low MFE values are associated with reduced fluorescence expression. All sequences except k 4 contain multiple point mutations with respect to k 1. A fluorescence level just above that of k 26 was registered for k 30 and k Similarly to k 26 , the first five nucleotides of the extended Kozak region of k 30 and the first six of k 31 were sequestered into a stem with the CDS.
However, differently from k 26 , the upstream regions of k 30 and k 31 were entirely free from any pairing interactions see Fig. Moreover, not all of these nucleotides have to participate in base-pairing interactions. However, this hypothesis is contradicted by k However, their mRNA secondary structures are dissimilar.
In k 27 , the extended Kozak sequence is involved in base-pairing interactions with the CYC1 terminator, whereas in k 29 the extended Kozak sequence is locked into a stem with the CDS. This causes an increase in MFE and consequently a higher fluorescence. However, unlike in these three sequences, the extended Kozak sequence of k 27 did not contain any adenine.
The fluorescence level of k 27 was slightly higher than that of k 29 , i. The five sequences considered so far k 26 , k 27 , k 29 — k 31 have in common an extended Kozak region rich in guanine that was sequestered into a stem in the MFE mRNA secondary structure. The MFE of k 26 was the lowest, as its upstream region was also sequestered into a stem. The other four sequences showed very similar MFE values but rather different fluorescence levels. The other group of sequences affected by multiple mutations with respect to k 1 had only adenines in the extended Kozak sequence and a variable number of guanines in the upstream region.
Although the MFE of k 35 was clearly higher than that of k 28 and k 34 Table 2 , the three sequences gave rise to similar mRNA structures where at least five guanines of the upstream region plus the first adenine downstream were locked into a stem due to base-pairing interactions with the CYC1 terminator see Fig.
Interestingly, both the MFE and fluorescence level of k 28 were comparable to those of k 27 and k Hence, even if the Kozak sequence was free of pairing interactions, the sequestering of the upstream region into a stem was enough to guarantee a clear drop in protein expression. This is further confirmation of the role played by the nucleotides upstream of the Kozak sequence in tuning protein expression.
A different MFE mRNA secondary structure was obtained for k 33 four guanines, intermixed with adenines , in which half of the extended Kozak sequence and almost the whole upstream region were involved in base-pairing interactions with the CDS, giving rise to a long stem. However, compared to k 35 , where only five nucleotides of the upstream region were locked into a stem with the CYC1 terminator, k 33 showed a higher MFE as well as a higher fluorescence level Fig.
Finally, for k 32 , k 36 , and k 37 with four, three, and two guanines in the upstream region, respectively RNAfold returned the same MFE as for k 1. The corresponding mRNA secondary structures were all characterized by the presence of the the giant hairpin see Additional file 1.
Compared to our experimental data, this result was plausible only for k 37 but in apparent disagreement with the measurements for k 32 and k 36 , whose fluorescence levels were significantly lower than that of k 1 Fig. Therefore, it can be argued that in vivo k 32 and k 1 share the same MFE and mRNA secondary structure, as suggested by the in silico simulations.
In contrast to the multiple point mutations, of the single point mutations on k 1 , only k 4 caused a modification in the structure of the giant hairpin and a consequent decrease in the MFE. According to our data, this minimal change has no effect on fluorescence expression. All the other point mutations that induced a fluorescence level significantly higher than that of k 1 namely, k 16 , k 47 — k 51 , and k 53 — k 55 were characterized by the same MFE and corresponding mRNA secondary structure as k 1 , according to the RNAfold simulations.
As for S. In contrast, two other works [ 18 , 19 ] showed that several adjacent guanines placed just upstream of the START codon induced a significant drop in protein synthesis. We fused pCYC1min to a reporter protein and quantified the strength of 58 synthetic leader sequences using fluorescence measurements.
In a previous report, Dvir et al. We called this sequence k 1. In contrast, a statistically significant enhancement in fluorescence was achieved by single point mutations in k 1. In particular, a guanine the least frequent nucleotide in S. Hence, single point mutations on an adenine background seem to constitute a novel technique for improving translational strength. We also studied the effects that multiple mutations to guanine can have on gene expression. Here, the starting point was a leader sequence containing the least frequent nucleotides in highly expressed S.
This sequence, here termed k 26 , switched off fluorescence expression. This was the first hint that mutations in the upstream region or, more generally, outside the Kozak sequence can markedly affect gene expression. Furthermore, we showed that gene expression can be tuned just by varying the number of adjacent guanines in the upstream region while keeping the extended Kozak sequence made of adenines only.
This represents another possible approach to engineering synthetic promoters that differ in their translational strength. We also noticed that four guanines intermixed with adenines reduce translation initiation less than four guanines in a row. This confirms that the effect of point mutations multiple or single on gene expression is highly context-dependent. Therefore, in this configuration, both the extended Kozak sequence and the upstream region are free from base-pairing interactions.
Moreover, this mRNA secondary structure returns the highest MFE among the 59 sequences analyzed in this work and seems to foster protein synthesis. Multiple mutations to guanine and cytosine cause either the extended Kozak sequence or the upstream region to be locked into a stem due to base-pairing interactions with the CDS or the CYC1 terminator.
As a result, the giant hairpin is destroyed, the MFE is lowered, and fluorescence expression is decreased to different extents. Therefore, the leader configuration upstream of the Kozak sequence has a marked influence on translation initiation. As in the reference work by Dvir et al. Hence, the results shown here might be not completely valid for other S.
As in the CYC1 gene, these modifications might lead to very high increases or decreases in translation initiation.
This notion should be taken into account to improve the modelling of basic modules for eukaryotic gene circuits as we previously described in [ 21 ]. Our hope is that this work will emphasize the fact that part characterization—a fundamental concept in Synthetic Biology—is still far from being achieved and more basic experiments on standard biological parts and subparts are required for a comprehensive description of the basic components of synthetic gene circuits.
Only with such accurate knowledge could Synthetic Biology be regarded as a proper engineering discipline. Backbones for all the plasmids used in this work were either the yeast integrative shuttle-vector pRSII Addgene, a gift from Steven Haase [ 22 ] or the modified version pMM, where the BsaI site in the ampicillin resistance gene and the BpiI site in the URA3 marker were removed via silent mutations.
A slightly different version termed yEGFPgg where the BsaI site was removed through a silent mutation was used in the plasmids assembled using the Golden Gate method [ 25 ]. Future development. As a toolbox to recoginize functional sites in DNA sequences, DNAFSMiner is expected to provide functions to identify other functional sites, such as splice site and etc.
We are working on this. On the other hand, we are planning to incrementally expand the system with newer sequences, in particular, to make it work on additional organisms not covered by the current datasets.
TIS Miner—overall accuracy, sensitivity, specificity and precision under different thresholds of the score based on the validation results on Human Chromosome data Liu et al.
Kozak, M. Nucleic Acids Res. Legendre, M. BMC Genomics 4 7. Liu, H. In-Silico Biol. Salamov, A. Bioinformatics 14 — Tabaska, J. Gene 77 — Oxford University Press is a department of the University of Oxford. It furthers the University's objective of excellence in research, scholarship, and education by publishing worldwide. Sign In or Create an Account. Sign In. Advanced Search. Search Menu.
Article Navigation. Close mobile search navigation Article Navigation. Volume Article Contents Abstract. Huiqing Liu , Huiqing Liu. Oxford Academic. Google Scholar. Hao Han. Jinyan Li.
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