Lecture 2: Direct and Indirect Readout

Question 1

How does direct readout work?

Answer 1

Direct readout is when the amino acids of a protein directly correspond to a complementary sequence of nucleic acids in the major groove of the DNA helices.

• In B DNA the edges of bases are all different. They are decorated by hydrogen bonding groups and methyl groups of thymine.
• There is a distinct pattern of hydrogen bond acceptors and donors.
• For the TA base pair, the methyl group is often recognised by a hydrophobic pocket formed by amino acids like leucine.
• The pattern from T to A is MADA in the major groove.
• In the minor groove the recognition pattern is AA.
• In the major groove, the pattern is unsymmetrical. AT and TA can be distinguished.
• For the GC base pair, the patter in the groove is DAA (from C to G).
• CGs pattern in the minor groove is fairly symmetrical (ADA) but we can distinguish GC from CG.

Question 2

How does local shape readout work? Give examples.

Answer 2

Local shape readout refers to the binding of DNA to local structural elements.

• Arginines can bind to areas where the minor groove has narrowed.
• Lysines can also do this but it observed much less frequently.
• Kinks are often stabilised by protein side chains.
• For example, the Lac repressor forms a kink at the central CpG step. The major groove widens and the two leucines interact with the kinked base pair step.
• TBP shows Phe intercalation in the first and last base pair.

Question 3

How does global shape readout work?

Answer 3

Global shape readout works based on the overall shape of the DNA.

• Bending optimises hydrogen bond contacts.
• GC rich sequences favour the transition to A-DNA.
• In A-DNA the C3’ endo sugars are more accessible. C2’ endo sugars are buried in B-DNA.
• This contributes to the specificity of ZFs for GC-rich sequences.
• A-like conformation also causes the minor groove to widen and allows hydrophobic residues to be inserted.
• A-like conformation is generally recognised by ZFs.
• B to A transition is often observed for endonucleases.

Question 4

How do DNA kinks work?

Answer 4

DNA kinks are local disruption in an otherwise linear helix.

• It involves a partial or complete loss of stacking at a single base step.
• TA has the weakest stacking interaction. It’s often referred to as a hinge step.
• Kinks are often stabilised by proteins. They compensate for lack of stacking by the intercalation of hydrophobic side chains. This enhances the kink.
• TATA binding proteins inserts a Phe residue between the TpG step, thereby resulting in a roll value of 40 degrees (very high).
• IHF stabilises a kink between TpT by interaction of a proline residue.

Question 5

How do water-mediated hydrogen bonds work?

Answer 5

In some structures, the amino acids do not directly bind to the bases, a water is used to intervene.

• The bridging can be seen in enzymes as well as transcription factors.
• Water-mediated hydrogen bonds can be used for specific regions. They often reflect the position of hydrogen bonds and acceptors.

Question 6

How can hydrophobic contacts be used?

Answer 6

Hydrophobic contacts can be used in a number of ways.

• We can distinguish thymine from cytosine.
• The TBP/TATA box complex has a completely dehydrated minor groove. The contacts are mainly made via non-polar side chains. Very few hydrogen bonds can be observed.
• The majority of minor groove contacts are associated with dramatic widening and extensive hydrophobic contacts.

Question 7

How does minor groove narrowing occur?

Answer 7

Minor groove narrowing is one of the key methods in DNA-protein recognition.

• Minor groove width is affected by hydrogen bonding patterns and differential stacking.
• Minor groove narrowing is often caused by A-tracts, which are AT-rich sequences that exclude the TpA step (as it normally widens the groove).
• A tracts narrow the groove by leading the major groove to have more inter base pair hydrogen bonds.
• GC rich sequences lead to wider minor grooves.
• The SCR Hox protein uses the minor groove width and electrostatic potential to distinguish small differences in nucleotide sequence.
• Narrowing of the groove increases negative electrostatic potential, this can be detected by arginines.

Question 8

How do amino acids recognise structural features of DNA?

Answer 8

There is not a simple recognition code.

• Gln can directly interact with adenine (MADA). Arg can recognise guanine (DAA). They are both bidentate ligands. They affinity is entropically driven. They’re very specific.
• Serine and threonine can contact two bases at once. This is called bifurcated hydrogen bonding. They’re quite specific.
• Leucine can form a VDW binding pocket for the methyl group of the thymine.
• Arg and Lys can be used for indirect readout by detecting the phosphate.
• Gln, Asn (-NH2), Ser, Thr and peptide bonds can make hydrogen bonds to non-esterified phosphate oxygens. This can anchor the protein to DNA.