Obviously. When you’re not changing the function, no mutations are required at all. So yes obviously to get a protein to perform a function it could not previously will require at least one mutations. But mutations do happen regardless of whether they are required to produce functional shifts or not. Proteins drift around in sequence space through neutral mutations to varying degrees. This is pretty much unavoidable.
We agree that mutation is required to get a protein to do something it could not before(*). We do not agree that it is generally the case that 3 or more specific ones with no other alternatives being possible, are required.
Fine with me, that is now your definition of a “new binding site”. A protein that bind a molecule it could not bind before. Do you happen to know of any such examples? And even better still, do you happen to have references that substantiate your still unsupported belief that such functional shifts generally require 3 or more specific mutations with no other such mutational combinations being possible?
No. It’s still binding the same molecules, only weaker.
A promoter sequence is a molecule of DNA. A new promoter is a different molecule. Changing preference of a DNA binding transcription factor for one promoter sequence to another is a change in binding site for that protein transcription factor.
No, you are ignoring the sentence that immediately follows. Please understand that you are confusing the number of steps separating the extant descendant SEQUENCE and the very distant reconstructed ancestor SEQUENCE with the number of steps it generally takes to evolve the descendant FUNCTION from the function of the ancestor.
As they say, there are multiple such functional shifts that require only one or two mutations. Look at Extended Data Figure 5:
The ancestral sequence is the one in the middle of fig 5(a). The blue circles are the ones with the ancestral function, and the green circles are the sequences with the new functions. From the ancestral sequence, one out of seven sequences reached with a single mutation has the new function. 1 in 7 result in a functional shift, the other are viable pathways for the protein to drift among. Which it then probably did.
Notice another thing. In that paper they are only testing all possible L=4 variants in the DNA binding domain of the protein. But many other residues in the protein are also likely to affect it’s binding preferences(as is also stated in the introduction and discussion).
The ancestral RH (EGKA) and derived RH (GSKV) can access many SRE-specific outcomes by short paths in AncSR1+11P.
a , Concentric rings contain RH genotypes of minimum path length 1, 2, or 3 steps from AncSR1+11P:EGKA (center). The historical outcome (GSKV, boxed, bottom) is accessible through a three-step path (EGKA – GGKA – GGKV – GSKV). Alternative SRE-specific outcomes accessible in three or fewer steps are in green. Lines connect genotypes separated by a single nonsynonymous nucleotide mutation; lines among genotypes in the outer ring are not shown for clarity. Orange arrows indicate paths of significantly increasing SRE mean fluorescence. b , For trajectories indicated by orange arrows in ( a ), SRE mean fluorescence is shown versus mutational distance from AncSR1+11P:EGKA (with x-axis jitter to avoid overplotting). Gray lines connect variants separated by single-nucleotide mutations. Error bars, 90% confidence intervals. Green dashed line, activity of AncSR1+11P:GSKV on SRE. c , For the SRE-specific outcomes accessed in orange paths in ( a ), the probability of each outcome under models where the probability of taking a step depends on the relative increase in SRE mean fluorescence (correlated fixation model), or where any SRE-enhancing step is equally likely (equal fixation model)8. d , The historical outcome (GSKV) has SRE-specific single-mutant neighbors. Concentric rings contain SRE-specific RH genotypes of path length 1 or 2 steps from AncSR1+11P:GSKV (center). Lines connect genotypes separated by a single nonsynonymous nucleotide mutation; lines among genotypes in the outer ring are not shown for clarity. e , The distribution of SRE mean fluorescence of SRE-specific neighbors of AncSR1+11P:GSKV illustrated in ( d ). Error bars, 90% confidence intervals.
Pick any particular blue or green circle on Fig 5 (a) and see that many of them have multiple 1-mutation functional shifts available to them. One of the neighbors to the ancestor has 5 out of 14, meaning that over a third of viable mutants from that sequence result in a shift in function with 1 mutation. Another has 2 out of 6, again implying roughly a third of 1 amino-acid substitution mutants result in a functional shift.
Read for comprehension. Understand that evolution wasn’t searching for a specific target sequence. Nor even a target function. Rather mutations just come and go over time as proteins drift around through fitness-neutral pathways, and among mutants that come and go are some with functional consequences that get to stick around if they positively affect fitness, until they themselves in turn are replaced with other neutral variants, or even better mutants.
(*) Just a small caveat. Some proteins can perform functions they normally cannot simply by changing the physical conditions around them. Well-known examples are so-called hot-start DNA polymerases(or some enzymes used by hyperthermophiles in general are some times inactive under mesophilic conditions) used in PCR which require increased temperature to activate. It is entirely possible many proteins with known functions have other functions we don’t know about that could be brought about by changing things like water activity, pH, temperature, and so on, without requiring any changes to the protein’s amino acid sequence.