The ARE sequences, which were found in signalling gene clusters and neighbouring cryptic biosynthetic gene clusters, can be further refined to generate a consensus ARE motif by resubmitting promoter sequences into the MEME program (Figure 3-8 and Appendix B). The refined consensus ARE sequence motifs are highly conserved within and across the Streptomyces systems (Figure 3-8). The conserved pattern is more noticeable in the motifs for S. coelicolor, S. venezuelae and S. avermitilis, where they share the semi-palindromic sequence, AANATACCTTCNNGAAGGTATNTT (palindromic sequence is highlighted in red) (Figure 3-8).
Few differences in the refined consensus ARE sequence motif were observed in
S. hygroscopicus; for example, the S. hygroscopicus consensus motif contains a longer variable spacer of 2 bp and extremities of the motif are represented by a conserved AAA/TTT sequence instead of the ATA/TAT sequence as in S. coelicolor, S. venezuelae and S. avermitilis consensus motifs(Figure 3-8).
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Figure 3-8 – Refined consensus ARE motifs for S. coelicolor, S. venezuelae,
S. avermitilis and S. hygroscopicus.
To investigate possible relationships between the consensus ARE motifs and HTH DNA-binding domains, TetR repressors were aligned using Clustal Omega. 144 From alignment results, amino acids proposed to be involved in interactions with DNA bases were different in S. hygroscopicus TetR repressors (Figure 3-9).
In the SHJG_7322 repressor, an alanine residue was found in position 48 of α-helix 3, instead of glycine, which is conserved in the other analogous MmyR repressors. Based on the E. coli TetR repressor, this specific amino acid residue was shown to be involved in interactions with DNA bases (Figure 3-9). Although alanine and
S. coelicolor A3(2)
S. venezuelaeATCC 10712
S. avermitilisMA-4680
S. hygroscopicussubsp. Jinggangensis 5008
E-value: 2.0e-013
E- value: 5.3e-018
E-value: 1.3e-011
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glycine only differ by a single methyl group, these differences may significantly alter affinity to the ARE sequences. Furthermore, amino acid residues in α-helix 3, also known as the recognition helix, were shown to be primarily involved in the binding to extremities of their respective DNA sequence. 70
As for the analogous MmfR repressors, amino acid residues in α-helix 3 are all conserved; however, the residue proposed to be involved with DNA contact in α-helix 2 all differ (Figure 3-9). SAV_2268, MmfR and SgnR repressors contain amino acids with non-polar side chains (isoleucine, valine and leucine amino acids respectively), whilst in ShbR1, a methionine amino acid was found, which consists of a straight-chain with a sulphur atom (Figure 3-9). Based on the E. coli TetR repressor, this specific amino acid residue was shown to be involved in interactions with beginnings of the variable spacer. The methionine at position 49 of α-helix 2 in ShbR1 may explain differences in the variable spacer region. Although amino acid residues required for DNA binding are based on the E. coli TetR repressor, preliminary bioinformatic analysis has provided insights into observed variation in the refined consensus ARE motifs. It should be noted that the amino acid residue differences in SHJG_7322 and ShbR1 are not conserved in the two repressors and implies they may bind to different DNA sequences (Figure 3-9).
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Figure 3-9 –Clustal Omega alignment of analogous (a) MmyR and (b) MmfR
repressors reveals DNA-binding domains are highly conserved; however, small variations were identified and could contribute to the differences in the consensus ARE motifs. Amino acid residues involved in DNA contact and phosphate backbone are highlighted in red and green respectively (based on E. coli TetR repressor).
Early bioinformatic analyses of the S. coelicolor methylenomycin biosynthetic gene cluster published by O’Rourke and co-workers, identified highly conserved 18 bp sequences in the promoters of key regulatory genes (Figure 1-19). 57 MEME enrichment analysis successfully identify the same conserved sequences as well as further extend the consensus semi-palindromic sequence to 24 bp (Figure 3-8). MEME enrichment analysis was also successful in discovering ARE sequences in
S. venezuelae, S. avermitilis and S. hygroscopicus, which were previously unknown. 62
The prediction of TetR repressor binding sites upstream of putative transcriptional activators is of particular interest, as it provides specific targets to genetically manipulate in order to overproduce specific compounds from cryptic biosynthetic gene clusters. The S. hygroscopicus gene cluster contains two putative transcriptional activator genes (shjg_7324 and shjg_7325, which encode for
TetR RLNRESVIDAALELLNETGIDGLTTRKLAQKLGIEQPTLYWHVKNKRALL ShbR1 IRSRGNILLAAARAFAQRGYPAVTMLDVAELSGMTKGAVYFHFTNKEALA SAV_2268 IRTRGTILNAAAAAFATDGFPQVTIKDIADGAEMTKGAVYFHFPNKEALA MmfR IKTRAQILEAASEIFASRGYRGASVKDVAERVGMTKGAVYFHFPSKESLA SgnR RRTKVHILQSAAELFAERGYATVTLQDVAERAEMTKGAVYFHYTNKEALA .: :: :* : * : .:*: : : ::*:* .*.:* α1 α2 α3 TetR RLNRESVIDAALELLNETGIDGLTTRKLAQKLGIEQPTLYWHVKNKRALL MmyR MRTRDQVLDAAAEEFALHGYAGTNLATVAVRTGMTKGALYGHFPSKKALA SHJG_7322 RRTHELLLDAAAAEFVRHGYVGANLQRVAAEANLTKGALYAHFTSKRLLA GbnR RMTYDLVLGAAAAEFALHGFAGTNLADITARTGLTKGALYGHFSSKADLL SAV_2270 LRTYDRVLDAAAYEFARYGYTNANLQNIADRIRLTKGALYGHFANKEELA . : :: ** : * . :: . : : :** *. .* * a) b) α4 DNA-binding domain HTH
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TylR-like transcriptional regulator and streptomycin biosynthesis operon regulator respectively) and since the ARE sequence was only discovered in the shjg_7324
promoter, overexpression of shjg_7324 is expected to unlock the production of specific natural compounds.
Binding of analogous MmfR repressors to ARE sequences in S. coelicolor,
S. venezuelae and S. avermitilis have been demonstrated through a combination of
in vivo luciferase assays (Styles, K. Personal communication), in vitro EMSA (Malet, N., Harrison, P., Zhou, S., and Fullwood, A. Personal communication) and have ultimately led to the crystallisation of MmfR with MMF2 (Dean, R. et al. Manuscript in preparation). The MEME enrichment analysis provides an incredibly powerful approach for gaining insights into TetR repressor binding sites and to develop strategies to unlock the production of novel natural compounds.