*4.2. Site-Specific Substitutions*

The combinatorial approach utilizes the fact that mutual substitutions of the predicted regulatory motifs are introduced into the pairs of 5'UTRs with the same lengths but with experimentally confirmed significant difference in translation efficiency (Figure 7a). Matsuura et al. [8] successfully used this approach to identify new cis-regulatory elements in 5'UTRs that influence the differential translation of *A. thaliana* transcripts in response to heat shock (HS). The genome-wide analysis of the changes in polysome loading of the transcripts in Arabidopsis cell culture allowed for selection of a set of genes with different translational responses to HS. The 5'UTR nucleotide sequences of the transcripts that change the level of reporter protein in the protoplasts affected by HS were used to predict the regulatory elements in 5'UTRs with the help of partial least square (PLS) method. These computational predictions suggested that two short regions residing in the vicinity of 5'-proximal region of the 5'UTR can play an important role in the relative activity of reporter protein and, thus, may be regarded as cis-regulatory region candidates. In order to experimentally confirm the predictions on the importance of these 5'UTR regions in differential translation control, a series of mutual substitutions of these regions in the pairs of 5'UTRs with equal length but different translation efficiencies were analyzed. Analysis of the reporter gives convincing evidence that the 5'-proximal region of the 5'UTR plays a key role and that certain specific determinants in 5'UTR mediate the differential translation in response to HS [8].

Site-specific mutagenesis makes it possible to introduce substitutions of one or a group of nucleotides within strictly defined regions of nucleic acid sequences. Comparison of the levels of a reporter protein translated when controlled by a native regulatory region (for example, 5'UTR) and the same regulatory regions but with the introduced mutations (Figure 7b) demonstrates how the modification of the primary sequences of nucleotide contexts and/or their secondary structures modulates the translation of specific mRNAs. This approach, along with other molecular methods (Western blotting, qPCR, polysome fractionation, and so on), has emerged to be most efficient when studying both the mechanism underlying formation of RNA G-quadruplex in the 5'UTR of the SUPPRESSOR OF MAX2 1-LIKE4/5 (SMXL4/5) mRNAs and the clarification of the role of a specialized structure, the regulator of phloem formation. In particular, a novel zinc finger protein, JUL, was identified; it specifically bound to consecutive guanine repeats in the 5'UTR of SMXL4/5 and induced RNA G-quadruplex. Moreover, convincing experimental data that both JUL1 and G-quadruplex are necessary for strong translation suppression rather than a single-stranded G-rich element have been obtained using the strategy of reporter systems. This suggests that the suppression of translation is caused by either JUL1-mediated formation of G-quadruplex or the G-quadruplex/JUL1 complex recruits an unknown translational suppressor [53].

Site-specific mutagenesis has emerged to be efficient for clarifying the role of the TAGGGTTT motif, overrepresented in the 5'UTRs of the transcripts regulated at the level of translation. A comparative study of two constructs, one with a native 5'UTR carrying this motif and the other with the 5'UTRs carrying mutations in this motif, has shown that the transcripts with the native 5'UTR are more efficiently translated provided that the number of transcript are equal. Thus, it is experimentally proved using reporter genes that the TAGGGTTT cis-element regulates expression of the gene particularly at the level of translation [33].
