Question

DNA Polymerase can distinguish between dNTPs and rNTPs because of discriminator amino acids in the enzyme's nucleotide-binding pocket. These amino acids occupy the space where the 2'OH group of an incoming rNTP would need to reside in order to properly position the substrates for catalysis. These discriminator amino acids usually have large R groups, which sterically exclude the ribose 2'OH. If you experimentally mutate/change the discriminator amino acids to glycines, predict the effect that this change might have on DNA polymerase.

Answer 1

DNA synthesis is catalysed by enzyme DNA polymerase. The structure of DNA polymerse resembles a 'palm' whose catalytic site is made up of - sheet that binds to divalent metal ions ( Mg ²⁺ or zn ²⁺). DNA polymerases use the same active site to add all the four dNTPs. At the same time DNA polymerase is also able to distinguish between rNTPs which is usually present at much higher concentrations than dNTPs. This recognition is in fact mediated by the steric exclusion of ribonucleotides from the enzymes active site.

The prime and the only difference between deoxy ribonucleotides (dNTPS) and ribonucleotides (rNTPs) is that, dNTPs has a sugar that is deoxy ribose which lacks oxygen at the second position of the sugar while in case of rNTPs the sugar is ribose,ie; the second position of sugar is occupied with an OH group (2' OH). In DNA polymerase, the active site ( ie; the nucleotide binding pocket) cannot occupy a 2'OH of the incomming nucleotide. This space is provided by two amino acids which are usually called as 'Discriminator amino acids' that makes Van der Waals forces with the sugar ring. The speciality of this discriminator amino acids are that they have a large R group. When this discriminator amino acids are replaced with Glycines, the problem is that they are the simplest amino acids with a small R group ( In glycine the R group is hydrogen atom itself which is extremely small) that makes the steric interaction weak or not possible. So, this reduces the enzymes capability to distinguish between rNTP and a dNTP leading to the incorporation of rNTPs which finally ends up in the termination of replication.