Albany 2015:Book of Abstracts

Albany 2015
Conversation 19
June 9-13 2015
©Adenine Press (2012)

Compensatory Evolution in Response to a Novel RNA Polymerase: Electrostatic Properties of Promoters May Lead the Adaptation in T7-T3 bacteriophages

It is known that not only the consensus sequence text is essential for RNA polymerase-promoter recognition, but some additional information can be coded in physical properties of DNA. Especially electrostatic interactions between promoter DNA and RNA polymerase is of considerable importance in regulating promoter function.

Here we report the analysis of electrostatic properties of promoters, described in the paper of J.J. Bull, R. Springman and I.J. Molineux, where a bacteriophage genome of the obligate lytic phage T7 by replacing its RNA polymerase (RNAP) gene with that of a relative phage T3 was forced to evolve a new system of regulation.

T3 RNAP was supplied in trans by the bacterial host to a T7 genome lacking its own RNAP gene and the phage population was continually propagated on native bacteria throughout the adaptation. Evolution of the T3 RNAP gene was thereby prevented, and selection was for the evolution of regulatory signals throughout the phage genome. T3 RNAP transcribes from T7 promoters only at low levels, but a single mutation in the promoter confers high expression, providing a ready mechanism for reevolution of gene expression in this system.

More than 30 mutations were observed in the evolved genome, but changes were found in only 9 of the 16 promoters, and several coding changes occurred in genes with no known contacts with the RNAP.

Mutations in promoters:
    replication promoter (phiOL) -394 A -> C, del T 403 (to T3 consensus );
    class II promoter phi1.5 -7768 A -> C (to T3 consensus);
    class II promoter phi1.6 - 7884 G -> A (not to T3 consensus);
    class II promoter phi2.5 -9105 T -> A (to T3 consensus);
    class III promoter phi6.5 - 18534 G -> A (not to T3 consensus) & 18543 T -> A (to T3 consensus);
    class III promoter phi9 -21863 T -> A (to T3 consensus);
    class III promoter phi10 -22893 G -> A (not to T3 consensus) & 22902 T -> A (to T3 consensus);
    class III promoter phi13 -27265 C -> T (from T3 consensus);
    replication promoter (phiOR) - 39218 G -> A (not to T3 consensus) & 39227 T -> A (to T3 consensus).

We found that the main differences in the electrostatic profiles of promoters of T7 phage, from the one hand, and that of T3 and mutated T7, from the other, lie in the starting point region (shown to be electrostatically distinctive to different classes of T7 promoters). In -2 - -5 b.p. (5-15 Å) electrostatic potential of T7 promoters is considerably less than that of T3 and mutant (Fig. 1). Mutated promoters demonstrated the largest potential shift, though non-mutated also showed some potential gain due to mutations in flanking regions.

Though fitness is an integral indicator of promoter activity strength, we can still make an assumption that the differential recognition of promoters by T7 and T3 RNA polymerases can by driven by their electrostatic properties.


Fig. 1. Distribution of electrostatic potential around T7, T3 and T7 mutant promoters, averaged by groups:

  1. T3 promoters

  2. T7 promoters, mutated in the experiment

  3. T7 promoters, not mutated in the experiment

  4. T7 mutant promoters, mutated in the experiment

  5. T7 mutant promoters, not mutated in the experiment

Vertical axe: top electrostatic potential in ēÅmiddle- group standard deviation; bottom - GC content in % with 3 b.p. window. Horizontal axe: sequence length in Åaligned around start point (vertical line). DEPPDB (deppdb.psn.ru) and its tools were used to make the analysis.This research has been supported by RFBR grant 14-44-03683.

    S.G. Kamzolova, A.A. Sorokin, T.D. Dzhelyadin P.M., Beskaravainy, A.A. Osypov. (2005) Electrostatic potentials of E. coli genome DNA, J. Biomol. Struct. Dyn. 23(3), 341-346.

    A. A. Osypov, G.G. Krutinin, S. G. Kamzolova. (2010) DEPPDB-DNA Electrostatic Potential Properties Database. Electrostatic Properties of Genome DNA, J Bioinform Comput Biol, 8(3), 413-25

    A. A. Osypov, G.G. Krutinin, E.A. Krutinina, S. G. Kamzolova. (2012) DEPPDB -DNA Electrostatic Potential Properties Database. Electrostatic Properties of Genome DNA elements, J Bioinform Comput Biol, 10(2) 1241004

    J.J. Bull, R. Springman, I.J. Molineux. (2007) Compensatory evolution in response to a novel RNA polymerase: orthologous replacement of a central network gene, Molecular Biology and Evolution, 24(4), 900-908

    SG Kamzolova, PM Beskaravainy, AA Osypov, TR Dzhelyadin, EA Temlyakova, AA Sorokin, (2014), Electrostatic map of T7 DNA: comparative analysis of functional and electrostatic properties of T7 RNA polymerase-specific promoters, J Biomol Struct Dyn, 32(8), 1184-1192

    Y. Mandel-Gutfreund. (2013) Novel geometric approaches to uniquely characterize DNA-binding interfaces, J. Biomol. Struct. Dyn. 31 SI Supplement: 1, 47-48. DOI: 10.1080/07391102.2013.786508

    S. Pala, D. Dasgupta. (2013) Differential scanning calorimetric approach to study the effect of melting region upon transcription initiation by T7 RNA polymerase and role of high affinity GTP binding, J. Biomol. Struct. Dyn. 31(3), 288-298

Alexander A. Osypov
Svetlana G. Kamzolova

Institute of Cell Biophysics of RAS
Pushchino Moscow Region, Russia, 142290

Ph: +7(929) 606-9828