Dependence of the E. coli promoter strength and physical parameters upon the nucleotide sequence

Dependence of the E. coli promoter strength and physical parameters upon the nucleotide sequence 1 first-author 2 †E-mail:Yury.G.Shckorbatov@univer.kharkov.ua †E-mail:Yury.G.Shckorbatov@univer.kharkov.ua Yury.G.Shckorbatov@univer.kharkov.ua National Scientific Center, Kharkov Physical-Technical Institute, Kharkov 61108, Ukraine National Scientific Center, Kharkov Physical-Technical Institute, Kharkov 61108, Ukraine Institute of Biology, Kharkov National University, Kharkov 61077, Ukraine Institute of Biology, Kharkov National University, Kharkov 61077, Ukraine The energy of interaction between complementary nucleotides in promoter sequences of E. coli was calculated and visualized. The graphic method for presentation of energy properties of promoter sequences was elaborated on. Data obtained indicated that energy distribution through the length of promoter sequence results in picture with minima at −35, −8 and +7 regions corresponding to areas with elevated AT (adenine-thymine) content. The most important difference from the random sequences area is related to −8. Four promoter groups and their energy properties were revealed. The promoters with minimal and maximal energy of interaction between complementary nucleotides have low strengths, the strongest promoters correspond to promoter clusters characterized by intermediate energy values. DNA sequence Promoter strength Nucleotide pair energy −35 sequence −10 sequence +7 sequence INTRODUCTION 1 Bacterial protein-coding genes must be differentially expressed during the cell cycle, in response to a wide variety of extracellular signals. Initiation of transcription by RNA polymerase (RNAP) requires cis-acting DNA elements including core promoters. Core prokaryotic promoters, especially from Escherichia coli for RNA polymerase complexes with the factor σ 70 usually are situated between −60 to +20 base pair from the transcription start site (+1), have two most important transcription initiation sites: at the −35 position and at −10 region (the Pribnow box). The sequences of −10 and −35 sites may affect the binding of RNA polymerase and the formation of open complexes (Babb et al., 2004 2004 ). The Escherichia coli RNAP core enzyme can initiate the elongation stage of transcription, but only the holoenzyme containing a σ factor trigers the specific transcription initiation. Promoter recognition by the holoenzyme containing the major σ factor ( σ 70 ) occurs through interactions of σ with up to three promoter modules. The notion of promoter strength was introduced in order to evaluate the promoter ability to initiate transcription. The problem of the connection between promoter strength and its structure was intensively investigated in the 1980s. The −10 hexamer (consensus sequence 5′-TATAAT-3′) is recognized by σ region 2.3~2.4 (Burr et al., 2000 2000 ); the extended −10 region (consensus 5′-TGTGn-3′) is recognized by σ region 3.0 (Murakami et al., 2002 2002 ); and the −35 hexamer (consensus 5′-TTGACA-3′) is recognized by σ region 4.2 (Campbell et al., 2002 2002 ). The C-terminal domains of the two α subunits ( α CTDs) at some promoters interact with specific sequences referred to as upstream elements located upstream of the −35 hexamer (Gourse et al., 2000 2000 ). The rate-limiting step in transcriptional initiation typically is opening the promoter DNA to expose the template strand. Promoter mutations are known to reduce opening rates. Junction binding activity is contained within the sigma factor component of the holoenzyme (Guo and Gralla, 1998 1998 ). The site −11 is known to be critical for open complex formation. It is highly conserved in promoters and substitutions there have by far the strongest effect in diminishing rates of open complex formation (Roberts and Roberts, 1996 1996 ). The promoter strength may be determined by different ways. Using the in vitro mixed transcription system Kajitani and Ishihama ( 1983 1983 ) determined the two parameters of the promoter strength, i.e., the rate of open complex formation between RNA polymerase and promoter, and the saturation level of the open complex formation at equilibrium. Vogel et al.( 2002 2002 ) defined the overall promoter strength as the rate at which the open complex RP o of RNAP· σ 54 (R) at a given promoter P is formed in a multi-step reaction R+P⇔RP c ⇔…⇔RP o . MATERIALS AND METHODS 1 DNA sequences 2 We obtained 106 Escherichia coli promoter sequences using σ 70 subunit from the Regulon database (©2004, CIFN/UNAM all Rights Reserved. RegulonDB DataBase V. 4.0, 02-FEB-05) thanks to the courtesy of the Regulon database administration. All promoter sequences were transcribed with the aid of σ 70 . Promoter strength (the promoter ability to initiate transcription) was measured with the help of fluorescent labelling method in microarray experiments on the total transcripts of E. coli . Promoter strength was determined in arbitrary units reflecting the fluorescence intensity (Kanehisa et al., 2004 2004 ; Mori et al., 2000 2000 ). Promoter strength data obtained from KEGG EXPRESSION database ( http://www.genome.jp/kegg/expression/ http://www.genome.jp/kegg/expression/ ) which contains microarray data obtained by the Japanese research community. Orientation of promoter sequence in genome was determined as forward or reverse depending the gene position in the genome. As far as we know forward and reverse orientation is not connected with gene functioning. The number of random sequences analyzed as the control variant was equal to 30. The number of forward promoter sequences was equal to 28 and the number of reverse sequences was equal to 34. Computer analysis 2 We suggest the notion of promoter energy that is determined as a sum of energy of interaction of each nucleotide pair in promoter divided by nucleotide number. For analysis of AT-contents and energy of pair interaction in promoter sequences we applied the “sliding-window” method. The AT-contents and energy of pair interaction at the site of ten nucleotide pairs (window length) were summarized and the mean value of these parameters were estimated. On every next step the analyzed site at one base pair was shifted. The data for every stage are presented in the figures. Computer programs for obtaining random DNA sequences, programs for promoter sequences energy estimation, program for slide-window data investigation, program for estimation of standard errors and t -criterion were elaborated by Berezhnoy. Cluster analysis was realized by the computer program STADIA 3.0 (Borland Corporation, USA). RESULTS 1 The names of promoters of E. coli and their corresponding numbers in our investigation are presented in Table 1 1 . Number of promoter sequence in the figures Number of forward promoter sequence in Table 2 2 Name of forward promoter sequence Number of reverse promoter sequence in Table 2 2 Name of reverse promoter sequence 1 1 AccA 29 AccD 2 2 AccB 30 Alas 3 3 Adk 31 AspC 4 4 Cfap1 32 AstCp1 5 5 ClpAp1 33 AtpI 6 6 Cmk 34 CedAp 7 7 CorA 35 CysE 8 8 Efpp 36 DapA 9 9 Frrp 37 DapD 10 10 FxsAp 38 DppA 11 11 GalRp 39 DrpA 12 12 GlnS 40 FtsJp1 13 13 ManA 41 Gnd 14 14 MraZp 42 HepAp 15 15 NohAp 43 Hiss 16 16 Pgi 44 HscB 17 17 Phe 45 Lep 18 18 PurA 46 LysP 19 19 Rep 47 MenAp 20 20 RplJ 48 NanAp 21 21 RplK 49 OtsB 22 22 RpoB 50 Pdx 23 23 RpoN 51 PheS 24 24 SbcB 52 PntA 25 25 ThrA 53 Pthp 26 26 TufB 54 PutA 27 27 Ung 55 RplT 28 28 YhcA 56 RpsJ 29 57 Smp 30 58 Spc 31 59 Str 32 60 SufAp 33 61 Upp 34 62 XseBp In the Fig. 1 1 are data for specific mean energy of complementary base pair interaction in different promoters. These data vary in chaotic manner. The distribution of the energy of interaction between base pairs through the length of promoter sequence was measured by the method of sliding window. The mean data for forward and reverse promoter sequences (number of forward sequences equals 28 and number of reverse ones equals 34) are presented in Fig. 2 2 . The mean energy of nucleotide pair is −29.33 kJ/mol for AT-pair and −70.35 kJ/mol for GC-pair (Kudritskaya and Danilov, 1976 1976 ). As Fig. 2 2 data show that the distribution of pair free energy of interaction in promoter sequences have three minimums. The one in the area between −40 and −30 window position relative to the beginning of transcription point (+1), a second one between −15 and −10 position, the last one between −4 and +10 position. These windows are situated in the most important areas of promoter sequence, and correspond to consensus sequences at −10, −35 and +10. The mean contents of AT-pairs is elevated in three areas: −35, −8 and +7 window position (Fig. 3 3 ). The t -criterion data on differences in nucleotide contents between forward and random sequences and between reverse and random sequences are presented in Fig. 4 4 . As one can see t -criterion for difference in nucleotide contents has two maximum in the area near −35 and −10 window position. In this area the energy differences between random and forward or reverse sequences are the most pronounced (Fig. 2 2 ) because of the elevated concentration of AT-pairs (Fig. 3 3 ). The mean data on promoter strength are presented in Fig. 5 5 . With the help of cluster analysis using the method of Euclidian distances determination we analyzed all promoter sequences by the character of mean energy of base pair interaction per nucleotide pair. The obtained data are presented in Table 2 2 . We suggested that promoter sequences are divided into 3 and 4 clusters. We proposed such subdivision because existence of less than two clusters is impossible and the promoter sequences number was not large enough to divide our set of promoter sequences reliably in the more than 4 clusters. As one can see all clusters differ significantly in the mean energy of the complementary nucleotide interaction parameter. Our data indicate that the energy differences between clusters do not directly correspond to differences in their strengths. The promoters in clusters with minimal and maximal energy (for instance 1 and 3 if we suggested 3 clusters or 1 and 4 in the case of 4 clusters) have low strengths. The strongest promoters have intermediate energy values (cluster 2 and 2, 3 correspondingly). 2 Clusters quantity 2 Cluster number 2 Forward sequences 2 Reverse sequences Mean energy of base pair interaction (kJ/mol) Mean strength Mean energy of base pair interaction (kJ/mol) Mean strength 3 3 1 44.1±0.45 0.75±0.09 46.4±0.48 0.38±0.05 2 53.5±0.56 0.95±0.08 47.2±0.68 2.16±0.11 3 49.3±0.37 2.90±0.10 51.2±0.55 1.03±0.10 4 4 1 44.1±0.45 0.75±0.09 45.4±0.76 0.37±0.04 2 52.6±0.44 1.01±0.08 47.2±0.68 2.16±0.11 3 49.3±0.37 2.90±0.10 49.4±0.32 1.10±0.06 4 58.0±0.00 0.71±0.01 55.4±0.27 0.99±0.15 It has long been known that DNA must be locally melted in order to be transcribed (Spassky et al., 1985 1985 ). Most transcription regulators act at the steps leading up to DNA melting (Gralla, 1996 1996 ). The base-specific interaction between defined segments of DNA and the σ 70 subunit of the RNAP leads to separation of base pairs (primarily nontemplate strand bases in the −10 promoter region) and exposure of the template strand for RNA synthesis (Roberts and Roberts, 1996 1996 ). A short segment is melted to make the template strand accessible to the catalytic core (Gourse et al., 2000 2000 ). In this process holoenzyme first binds to the promoter to form a closed complex and then opens a segment roughly from position −11 to +3 (Kainz and Roberts, 1992 1992 ). The sequences on the nontemplate strand of the −10 consensus element, which extends from −12 to −7, are known to have important influence (Roberts and Roberts, 1996 1996 ). Both the sigma and core components of RNAP may take part in the melting reaction. Mutations of RNAP subunits can affect promoter melting (Jones et al., 1992 1992 ; Juang and Helmann, 1994 1994 ). This α subunit of RNAP binds to upstream element DNA using minor groove as well as backbone contacts. The functional groups in the −10 and −35 hexamers are involved in the interaction with the σ subunit (Ross et al., 2001 2001 ). Our own results indicated that energy of base pairs interaction in promoter sequences is significantly decreased in the region between −45 and +7 (Fig. 2 2 ). This phenomenon is connected with elevated AT-contents in this area (Fig. 3 3 ). The decreased energy of pairs interaction leads to easier melting of these regions. Visualization of these data makes clearer the physical bases of different functional roles of different promoter regions. The validity of these differences is proved by data of Fig. 3 3 , where the t -criterion of differences of random sequences is presented. The mean base pair energy per promoter sequence differs between promoters. This indicates that conformational changes in the DNA that accompany initiation of transcription such as promoter melting are determined by the polymerase rather than the DNA sequence (Meier et al., 1995 1995 ). CONCLUSION 1 The process of transcription is regulated in a very complex manner. But in spite of this we suppose that on the general level of promoter structure it may be revealed that some simple laws that involved in gene regulation. This work attempts to find simple general laws to explain differences in promoter strengths. We elaborated the graphic method for presentation of the energy properties of promoter sequences. Our data indicate that energy distribution throughout the promoter sequence is minimal at −35, −8 and +7 (Fig. 2 2 ). The obtained results do not depend on promoter orientation in the genome and are similar for forward and reverse sequences. 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Binding affinity of Escherichia coli RNA polymerase σ 54 holoenzyme for the glnAp2, nifH and nifL promoters . Nucleic Acids Research , 2002 . 30 ( 18 ): 4094 - 4101 . DOI: 10.1093/nar/gkf519 http://doi.org/10.1093/nar/gkf519 . Binding affinity of Escherichia coli RNA polymerase σ54 holoenzyme for the glnAp2, nifH and nifL promoters The authors are very indebted to Dr. H. Kiryu for data concerning promoter strengths and very useful information. 10.1631/jzus.2005.B1063 2005-07-07 2005-09-07 2005-10-28 2022-01-05 Copyright © 2005, Journal of Zhejiang University Science 2005 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 11 6B