CAP-DNA complex bends 90 degree

Delandy H. Young

Mar 1999, UCLA Chem Dept

Table of Contents

-Introduction

-Structure of CAP-cAMP complex binding to B-DNA

-Transcriptional activation by CAP

-Sequence specific of binding

-Reference

-Introduction

Before the transcription of a strand of DNA to RNA, initiation is required to start the transcription. In prokaryotic, the lac operon has a weak (low-efficient) promoter. Such weak promoters evidently require some sort of help for efficient transcriptional initiation. This is where CAP-cAMP complex (push the button to see cAMP closer) comes in and binds to the lac operon. The binding stimulate transcriptional initiation. The CAP-cAMP complex binds to B-DNA (lac operon) and bend it 90 degrees which results in the direct interaction between B-DNA and RNA polymerase. CAP-cAMP complex acts as a positive regulator that turns on transcription.

-Structure of CAP-cAMP complex binding to B-DNA

Size of the molecules: CAP (catabolite gene activator protein) is a dimeric protein of indentical 210-residue subunits. Its large N-terminal domain binds cAMP (adenosine 3',5'-monophosphate) and the small C-terminal domains binds B-DNA. The section of B-DNA interact with CAP has 30 base pairs (bps).

The bending: Schultz, Shields and Steitz proposed that B-DNA is bent by the protein at ~90 degrees. In 1987, J Warwicker constructed CAP-DNA models that bent the DNA by 100-160 degrees. However, the DNA failed to bind with the protein complex. Thus, 90 degrees is the only possible angle for CAP-DNA complex . The bending, however, is not symmetric. One side of the DNA bending starts at bp G23 and T24 . The DNA on this side is bent at ~43 degrees, and it's essentially straight. On the other side, DNA is bent ~8 degrees toward the protein near bp A28 and C29 . That makes the angle ~51 degrees. Thus, a total of 94 degrees bending is formed. In addition, the bending is out of plane ~35 degrees. The protein-DNA interaction: As mentioned before, the N-terminal of CAP binds to B-DNA. The CAP dimer's two helix - turn - helix (HTH) motifs binds to major grooves of the DNA. HTH motifs are ~20-residues that form two alpha helices which cross at ~120 degrees. One helix is parallel to the DNA helix. The other helix is parallel to the major groove.

Source of the bending: Such large degree of bending requires energy. The main source of energy is from the hydrogen-bond and ionic interaction. The three bp at G23, T24 and G25 position hydrogen-bonding with three protein side chains. Such interaction provide evergy for the DNA bending. The ionic interaction of 13 protein and 11 DNA phosphate backbone is another source providing energy. In addition, electrostatic interaction of DNA phosphate and CAP protein compensate energy required to bend DNA for ~-17 to -20 kcal/mole.

-Sequence specific of binding

(I) HTH act as a "recognition helix". Three side chains from HTH hydrogen-bond directly to the three bps in the major groove of the DNA. The affinity of the interaction is very high. In 1991, Schultz, Shields and Steitz mutated Glu181 that binds to bp G23 to Val or Leu. However, the mutation removes the interaction. The result demonstrates the protein-DNA sequence discrimination against other side chains.

(II) The bending of DNA specifies the binding of protein. The small bend ~8 degrees occurs about 10 bps from the bending axis contributes to specific DNA binding through sequence-dependent distortion of DNA. Gartenberg and Crothers found that CAP binding sites contain AT bp at A28 and C29 position bend more than GC bps. GC bps are usually found at position 34. Thus, DNA bending specifies the protein-DNA binding.

-Transcriptional activation by CAP

The DNA bending by CAP-cAMP complex results in interaction with RNA polymerase. The protein bends the DNA and form a loop at position -61 DNA. DNA is adjacent to the -35 region of the RNA polymerase binding site. Such interaction suggest that CAP-cAMP complex catalyzes transcriptional initiation. Upon interaction, DNA is protected from deoxyribonuclease I digestion by RNA polymerase. This might be essential to transcription activation. Schultz, Schields, and Steitz proposed that DNA alone can also activate transcription. CAP binding sites can be replaced by A-tract sequences that bends DNA the same way as CAP, and activation of transcription still occurs.

-Reference

Steve C. Schultz, George C. Shields, Thomas A. Steitz; Crystal Structure of a CAP-DNA Complex: The DNA Is Bent by 90 degrees; in Science 253, 1001 (1991)