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Changing the transcriptional activity of genome regulatory loci by PCR-mutagenesis Bykov A. A., Shavkunov K.S., Ozoline O. N.
Department of Functional Genomics and Cellular Stress, Institute of Cell Biophysics, Russian Academy of Sciences, Pushchino, Moscow Region 142290, Russia forrozinho_cap@mail.ru

Transcription requires the presence of a single promoter upstream of a gene, but in many cases there are multiple transcription signals. Promoter clustering is common for rRNAs genes[1], plant mitochondrial genes[2], being quite a widely distributed phenomenon for vertebrate genomes, including the human[3] . It is also inherent to bacteria and, in particular, to E. coli. This clustering provides an additional possibility for adaptive regulation in different growth conditions and in different physiological processes
[1-5]

. Although the number of independently functioning promoters

in the cluster vary in the range 2-8 (exemplified in Fig. 1, left panel), in most cases 2 or 3 transcription signals are sufficient for adequate gene expression. Hence it was highly unexpected to find 434 loci in the genome of E. coli with extremely high density of promoter-like sites (8 signals in 100bp frame along 300 bp), predicted by promoter finder PlatProm (Fig. 1, right panel). We called them "promoter islands" (PIs) -specificity[7]. Promoter clusters usually show higher transcriptional activity than single promoters[8]. However, "islands", on the contrary, demonstrated very low propensity to initiate synthesis of fullsized transcripts. It was found that 85% of PIs are associated with foreign genes originated in the E.coli chromosome due to horizontal transfer from other bacteria function that is not typical for the promoter regions. The aim of this study was to estimate the changes occurring in the promoter distribution profile in comparison to a normal promoter, in the course of random mutagenesis with subsequent picking out up- and down-regulated mutated promoter derivatives. If RNA polymerase binding to multiple promoters prevents effective transcription, up-regulation in this model system should be accompanied by a decrease, rather than increase in the number of potential promoters. Two chromosomal loci were chosen for the experiment a PI associated with the appY gene and a regulatory region of the dps gene, containing several functional promoters
[10] [7,9] [6,7]

. Seventy-eight of them are composed of promoters,

recognized by 70-RNA-polymerase, while the remaining "islands" include promoters with different

. Low transcriptional activity of

PIs may therefore be evolutionary adapted to arrest expression of foreign genes, i.e. to perform a

(Fig. 1).

Fig. 1. Disposition of potential promoters in the regulatory region of dps (left) and "promoter island" of appY (right). Vertical bars above and below the X-axis show the predicted TSPs for the upper and lower strands, respectively. The height of the peaks reflects promoter score, calculated by PlatProm. Blue arrows denote the E.coli genes. Two central panels show PlatProm scans obtained for natural genomic sequences. Two upper and two lower panels demonstrate changes obtained upon selection of derivatives with higher and lower promoter activity, respectively. Mutations affected contexts of consensus promoter elements -35 (dps) and -10 (dps and appY) are indicated.

The experimental scheme is shown in Fig. 2. The first step includes PCR mutagenesis using GeneMorph II Random Mutagenesis Kit (Agilent Technologies), where approximately 1-5 ng of plasmid containing the fragment to be mutated is used as a template in amplification. The PCR program included 2 minutes preheating at 95°, then 30 cycles: 95° 30 sec (DNA melting), 55° 30 sec (primers annealing), 72° 1 min (DNA synthesis). PCR-fragments were gel-purified by QIAquick® PCR Purification Kit (Qiagen, Germany) subjected to restriction with XbaI and BglII (1.5 h at 37oC) (Thermo Scientific, USA) and ligated with T4 ligase (overnight at +4oC) (Thermo Scientific, USA) into the pET28b plasmid in front of a promoterless gfp. E. coli Top10 cells were transformed with the derived vector using 0.2 M CaCl2 and plated on LB agar plates supplemented with 80 mkg/ml kanamycin. Preliminary selection of the outgrown colonies was accomplished judging by the fluorescence level using a fluorescent microscope. The selected clones were then grown overnight at 37oC in liquid LB medium. The cells were harvested by centrifugation, resuspended in 10mM Tris-HCl (pH8.0), 1mM EDTA) and sonicated. Fluorescence intensity was measured for each supernatant sample individually and normalized on total protein concentration. Plasmids were extracted from cells with the highest and lowest fluorescence/protein ratio, sequenced and used in the next cycle of mutagenesis and sequencing.

Four rounds of PCR and negative selection accumulated 6 point mutations in the dps regulatory region, which decreased PlatProm scores for the main (Pdps) and three minor (P1, P1' and P2) promoters. As a result transcriptional activity in this region was completely suppressed (left bottom panel in Fig. 1).

Fig. 2. Schematic layout of the SELEX procedure

.

The dps promoter area selected for increased transcription contained 9 mutations (3 rounds), resulting in a 4-fold efficiency gain, with 12 possible RNA starts instead of 10 in the initial sequence (20% addition) and closer to consensus (TTGACA) substitution in the -35 element of P
dps

(left top

panel). Transcriptional activity of appY-associated PI was also suppressed to the background level upon four rounds of negative selection, which accumulated 11 point substitutions (right bottom panel). The amount of possible transcription start points (PSPs) decreased by 27%, though the fragment carrying the appY "promoter island" still contained 62 potential transcription signals on the top strand. Multiple promoters therefore can not guarantee active transcription. The positive selection for the "promoter island" of appY led to a 6.1-fold increase of the expression level (6 rounds). It was caused by 15 point substitutions and 3 single base pair deletions. These mutations also promote a decrease in the amount of possible TSPs from 85 to 63 and noticeable rise in the score of the promoter, located 85 base pairs upstream of the gene start (right top panel in Fig. 1). Thus, in case of dps promoter region the situation is very predictable in positive selection the amount and scores of individual starts were increased, while in negative selection their quantity and quality dropped down. But in case of the appY PI both positive and negative selection decresed the

amount of possible TSPs. Confirming a hypothesis assuming obstacles for effective transcription initiation connected with a high density of potential promoters, these data also suppose instability of this high promoter density. It is possible that a special genomic system maintains the excessive amount of transcription starts in "promoter islands" so as to keep expression of neighboring genes quenched, but ready for rapid activation if necessary. This research is supported by a grant of RFBR (13-04-00997). 1. Gonzalez-y-Merchand JA, Colston MJ, Cox RA. (1998) Roles of multiple promoters in transcription of ribosomal DNA: effects of growth conditions on precursor rRNA synthesis in mycobacteria. J Bacteriol. 180: 57565761 2. Kuhn K, Weihe A, Borner T. (2005) Multiple promoters are a common feature of mitochondrial genes in Arabidopsis. Nucleic Acids Res. 33: 337346 3. Xin D, Hu L, Kong X. (2008) Alternative promoters influence alternative splicing at the genomic level. PLoS One. 3:e2377 4. Choi J, Newman AP. (2006) A two-promoter system of gene expression in C. elegans. Dev Biol. 296(2) :537544 5. Mendoza-Vargas A, Olvera L, Olvera M, Grande R, Vega-Alvarado L, Taboada B, et al. (2009) Genome-wide identification of transcription start sites, promoters and transcription factor binding sites in E. coli. Plos One. 4(10):e7526 6. Shavkunov KS, Masulis IS, Tutukina MN, Deev AA, Ozoline ON. (2009) Gains and unexpected lessons from genome-scale promoter mapping. Nucleic Acids Res. 37: 4919 4931 7. Panyukov VV, Kiselev SS, Shavkunov KS, Masulis IS, Ozoline ON. (2013) Mixed promoter islands as genomic regions with specific structural and functional properties. Mathematical Biology and Bioinformatics. 8(2): t12t26 8. Glazunova OA, Kiselev SS, Shavkunov KS, Bykov AA, Panyukov VV, Ozoline ON. Promoter Islands in the Genome of E. coli: Comparative Analysis against AT-Rich Sequences. (2014) Mat. Biolog. Bioinform. 9(2): 563574 9. Panyukov VV, Ozoline ON. (2013) Promoters of Escherichia coli versus promoter islands: function and structure comparison. PLoS One. 8(5):e62601 10. Shvyreva US, Tutukina MN, Ozoline ON. (2013) Bacterioferritin: Properties, structural and functional organization of the dps gene regulatory region. Biophysics. 56(5): 795-802