CopG Repressor Protein

John DePowell, '02 and
Timur Senguen, '03

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Contents:

I. Introduction

Bacterial plasmid replication is highly regulated, and stable maintenance in the host depends on constant plasmid copy number.  Without regulation of plasmid copy number, too many plasmids are produced thus reducing needed resources for other processes in the bacteria.  Replication is controlled by the availability of the rep gene-encoded initiator of replication protein (Rep).  In the streptococcal 5536 bp pMV158 plasmid, the synthesis of the RepB initiator protein is under the control of the products of two genes, rnaII and copG.

The copG gene codes for the transcriptional repressor CopG.  This protein is the smallest naturally occurring transcriptional repressor described as of its discovery date.  It has been purified as a homodimer of identical 45 amino acid subunits.  The protein represses its own synthesis as well as RepB protein by binding to the copG-repB promoter region.  As a result of binding, the DNA is bent 60º over the course of four successive DNA turns.  Binding of the DNA can hinder host RNA polymerase binding. 

II. General Structure

CopG is a dimer composed of two chemically identical polypeptide chains, A and B,  each 45 amino acids in length
<>.  Each monomer contains a helix-turn-helix motif <> which is stabilized by an intramolecular hydrophobic cluster formed by the side chains of 7 residues: Leu17, Met20, and Met24 (from helix A) and Leu26, Met 31, Ile32, and Val34 (from the posterior turn and helix B) <>.  The dimer is formed by 20 hydrogen bonds, 56 van der Waals contacts and one salt bridge.  This association of the two molecules leads to the formation of a two-stranded twisted antiparallel ribbon, based on 10 inter main-chain hydrogen bonds from Met1 to Glu11 of each monomer <>.  This ribbon along with the HTH results in a ribbon-helix-helix motif for each monomer symmetric homodimer. 

III. CopG in Complex with DNA

The structure of CopG in complex with double stranded 19-bp DNA reveals a tetramer <> formed from the association of two dimers <>.   CopG interacts with DNA-bases via the N-terminal b-ribbon <>, and the DNA-backbone phosphate groups via residues at the N-terminus of helix B of each monomer <>.  This process causes the DNA to bend by 60º in the nucleic acid moiety.  This bend is produced by compressions of both minor and major grooves facing the protein <>.  The minor groove at the center of the operator becomes extremely narrow (1.9 A); the major groove is compressed to about 3/4 (8.6 A) of it's usual size (11.7 A).  The DNA backbone follows a smooth path, excecpt for the zone close to the center of the operator, where the minor groove is compressed and the base pairs are rather inclined <>.  Here is a cartoon of the tetramer in complex with DNA4, Fig. 2D from Gomis-Ruth, F.X. et al.  Open the link in a new window.  This cartoon clearly shows the tetramer in contact with and bending the DNA. Back to the CopG-DNA complex PDB.

IV. Base Recognition Contacts

The CopG molecule comes into contact with the bases of DNA via the N-terminal b-ribbon.  Both the DNA and the CopG have twofold symmetry, but each of the two b-ribbons of CopG contacts different bases, not matching the twofold symmetry of the DNA sequence.   The asymmetrical recognition contacts of the two b-ribbons is exemplified by the different interactions of Thr6 of each strand.   For example,Thr6A  acts as a H-bond donor to Thy-6 whereas Thr6B contacts Cyt-5 as a H-bond acceptor <>.  Other H-bonding base interactions include Arg4B to Thy-7 <> and Arg4A to Gua-4 and Gua-5 <>. There is also a stacking interaction where a methyl group of Thy-3 is sandwiched between methyl groups of Thr6B and Thr8B <>.

V. Backbone Interactions

Further contacts exist between CopG and the DNA backbone.  These backbone interactions are not directly dependent on DNA sequence.  They consist of H-bonds between the side chains of Thr8, Ser29 and Lys28 of each monomer with the DNA phosphate groups <>.  In addition, some phosphates of the DNA backbone establish contacts with the protein main-chain amide nitrogens of Lys28A and Ser29A.  These two amino acids are on first turn of helix B right after the turn of the HTH motif <>.  Thus the phosphate group N-caps the helix and is located on the axis of the helix dipole .  In monomer B, helix B also points its N-terminus towards a DNA phosphate group, but in this case, there are no direct H-bonds.
 


VI. Implications for the Members of the Cop Family of Plamid Repressors

CopG is the prototype for a whole family of Cop repressors of plasmid origin.  Consensus analysis shows that 14 members of this family are compatible with the structure described above; differences in length are accounted for by longer N- and C-terminals.  All members of the family show certain similarities: they display the glycine mediated turn connecting the two helices as well as similar residues at key positions, such as the hydrophobic pocket.  It is assumed that due to these similarities all members of the Cop-family share the same RHH-motif.  Furthermore, some unrelated hypothetical gene products of bacteria and viruses display significant similarities, sharing the same domain architechture witht he Cop family.
 

VII. References

1.  del Solar,G.H.,  Giraldo,R.,  Ruiz-Echevarria,M.J.,  Espinosa,M.,  Diaz-Orejas,R.  (1998) Replication and control of circular bacterial plasmids.  Microbiol. and Mol. Biol. Rev., 62, 434-464.

2.  del Solar,G.H.,   perez-Martin,J. and Espinosa,M. (1995) Replication control of plasmid pLS1: efficient regulation of plasmid copy number is exerted by the combined action of two plasmid components, CopG and RNA II.  Mol. Microbiol., 18, 913-924.

3.  Gomis-Ruth,F.X.,   Sola,M.,   Perez-Luque,R.,   Acebo,P.,  Alda,M.T.,  Gonzalez,A.,  Espinosa,M.,  del Solar,G., and Coll,M.  (1998) Overexpression, purification, crystallization, and preliminary X-ray diffraction analysis of the pMV158-encoded plasmid transcriptional repressor protein CopG. FEBS Lett., 425, 161-165.

4.  Gomis-Ruth,F.X.,   Sola,M.,  Acebo,P.,  Parraga,A.,  Guasch,A.,  Eritja,R.,  Gonzalez,A.,  Espinosa,M.,  del Solar,G.,  Coll,M.  (1998)  The structure of plasmid-encoded transcriptional repressor CopG unliganded and bound to its operator.  The EMBO Journal, 17, 7404-7415.