Prokaryotic DNA-Binding Proteins and HTH Motif

Prokaryotic DNA-binding Proteins

• Prokaryotic DNA-binding proteins are key regulators of gene expression
• These proteins recognize specific DNA sequences to control gene activity
• Structural motifs like helix-turn-helix (HTH) enable DNA binding
• The lambda phage genetic switch serves as a model for DNA recognition

DNA Recognition in Procaryotes by Helix-Turn-Helix Motifs

• The helix-turn-helix (HTH) motif is a protein structure consisting of two α-helices connected by a short β-turn
• The recognition helix binds in the major groove of B-DNA
• The stability helix stabilizes the motif's interaction with DNA
• Recognition α-helices of subunits are separated by 34Å
• Recognition α-helices bind in the major groove of the DNA

DNA Binding Mechanism of the HTH Motif

• Palindromic operator regions allow effective protein binding
• Each subunit binds to almost identical halves of the palindrome
• Identical bases in the first three base pairs create a general recognition site for operators
• Residues of recognition α-helix interact with DNA base pairs
• Different base pairs (e.g., T14'-A1), have particular amino acid interactions (e.g., hydrogen bond of Gln 28 with adenine)
• Gln 29 is essential for sequence specificity

Differential DNA Binding by Repressor and Cro Proteins

• Protein-DNA interactions determine DNA conformation
• Binding of protein dimer induces structural changes in B-DNA
• DNA sugar-phosphate interactions stabilize these changes
• Protein subunits anchor to the major groove through hydrogen bonds with DNA phosphate groups
• Protein loops extend into the minor groove, creating additional contacts with phosphates
• Repressor prefers A-T base pairs; Cro is indifferent
• Sequence of central base pairs influences interactions
• A-T pairs fit better in the narrowed minor groove

HTH Motifs in Prokaryotes

• Lac repressor molecule binds into two successive major grooves of DNA
• The CAP-cyclic AMP-DNA complex illustrates HTH motif binding in prokaryotes

Take Home Messages (DNA Recognition in Prokaryotes by Helix-Turn-Helix Motifs)

• The helix-turn-helix motif includes a recognition helix and a stability helix connected by a short turn
• Subunit interactions ensure the appropriate distance and orientation for binding
• Sequence-specific interaction between the recognition helix and DNA facilitate identification of the palindromic operator region
• Hydrogen bonds between sugar-phosphate backbone and protein and DNA distortion facilitate close interaction between regions and DNA-binding proteins

Take Home Messages (Tetracycline-induced gene expression)

• Tetracycline induces expression of tetA by binding to and changing TetR conformation
• TetR is a homodimer with two domains (HTH-motif and regulatory domain)
• TetR-teto interactions are sequence-specific and have high structural complementarity
• TetR and teto interactions involve hydrogen bonds.
• No overall DNA bending, kink at base pair 2 is compensated

Induction by Tetracycline and Mg2+

• Conformational changes throughout the entire protein occur due to tetracycline (or Mg2+) binding
• Mg2+ displaces certain amino acid residues
• Whole a6 helix shifts due to conformational change

Comparison of TetR to Lacl

• Both TetR and Lacl are DNA-binding proteins that control gene expression
• Similarities include dimeric nature; HTH-motif; DNA-binding and inducer-binding domains
• Differences include TetR’s conformational changes upon induction, kink; Lacl’s distinct DNA bending upon induction

Structure of TetR

• TetR is a homodimer
• It has 10 α-helices per polypeptide chain
• TetR has two separate domains, DNA-binding and regulatory domain
• [MgTc]+ binding pocket in the regulatory domain

Sequence Specific Interactions

• All base pairs (except 0) contribute to the sequence-specific interactions between repressor and operator
• Each HTH-motif binds the major groove of the palindromic half-operator

Explore the critical role of prokaryotic DNA-binding proteins in regulating gene expression. This quiz focuses on the helix-turn-helix (HTH) motif and its mechanism of DNA recognition. Understand how these proteins interact with DNA sequences to control gene activity.