the question of why nature chose phosphate 
 for life on earth has 
 been around for many years . phosphate 
 diesters are especially adapted to link two nucleotides and still 
 ionize , helping to stabilize the dna and rna for carrying and processing 
 genetic information . arsenic acid , which is the most closely related 
 to phosphoric acid , was thought to be unsuitable because of the fast 
 hydrolysis of arsenic esters . diphoshate 
 arsenate ( adp  arsenate ) can be synthesized by beef heart submitochondrial 
 particles . furthermore , a recent finding 
 of a bacterial strain ( gfaj-1 ) that can rely on arsenic instead of 
 phosphorus raised the questions of if and how arsenate can replace 
 phosphate in biomolecules that are essential to sustain cell life . the possibility of arsenate - based cellular life 
 has been questioned , mostly in the form of comments and a literature review . while the gfaj-1 strain observations await further experimental 
 validation , computational approaches can be effective tools to address 
 scientific questions related to the arsenate assuming that 
 arsenate can replace phosphate in vital nucleotides , 
 we computationally probe if arsenate nucleotides can retain the structural 
 and functional features of phosphate nucleotides . in order to have 
 a broad representation of the molecular systems in the cell and to 
 ensure biological relevance rather than particular protein / dna / rna 
 sequences of the gfaj-1 cell , we combine quantum mechanical , 
 molecular mechanical and molecular dynamics simulations to investigate 
 arsenate  phosphate replacement in three selected types of molecules 
 ( atp / adp as energy source and replication regulation ; dna  protein 
 complexes for dna replication and transcription initiation ; a trna the hydrolysis 
 of atp / adp as the energy source is coupled to most 
 enzymatic reactions in life , and is involved in processes such as 
 regulation of dna replication . we investigated 
 three bacterial dna  protein complexes which are essential for 
 dna replication and transcription . fis selects 
 targets mainly through indirect recognition involving dna bending , 
 using the minor groove shape and the sequence - dependent conformational 
 change over adjacent major groove surfaces . dna complex provides a test whether the combination 
 of geometry and sequence specificity still allow an arsenate backbone . the second factor is the integration host factor ( ihf ) , which functions 
 in nucleoid structuring , chromosome replication and dna rearrangements , 
 and transcription regulation in many 
 prokaryotic processes . ihf contacts the 
 dna exclusively via the phosphodiester backbone in the minor groove , 
 introducing a u - turn into the dna . thus , the ihf  dna complex 
 provides a comparison of phosphodiester versus arsenatediester backbones 
 in dna recognition . the third complex is the f plasmid rep protein 
 ( repe ) dimer in complex with the repe operator dna . repe is essential 
 for stringent regulation of the plasmid copy number in esherichia coli . in 
 the repe dna complex , the two dna binding sites are separated 
 by 100  ) . thus , repe  dna provides a test for cumulative 
 structural changes due to as p replacement in dna . to investigate 
 a possible phosphate - arsenate substitution in trna , we selected the 
 bacterial trna lysidine synthetase complex ( tils ) , which ensures translational 
 fidelity . we used the crystal structure 
 of the thermus thermophilius 70s ribosome 
 bound to rf2 as the starting conformation . 
 in this translation termination state , a step just before ribosome 
 recycling , the interactions between 30s and 50s could be sensitive 
 to energy perturbation . finally , we compared the experimentally observed 
 exafs spectra with the theoretical exafs spectra for the arsenate 
 dna and rna . pure dna and rna with phosphate and arsenate backbones 
 could have 
 similar conformations and the ability to retain the watson  crick 
 base pairs . ( b ) trajectory 
 of the number of hydrogen bonds between watson  crick base pairs 
 in the dna . ( d ) trajectory 
 of the number of hydrogen bonds between watson  crick base pairs 
 in the trna ( pdb 3a2k , chain c ) . dnaa is a large component of the initiation complex 
 at the oric region in bacterial replication initiation . md simulations 
 indicated that adenosine diarsenate ( adas ) may still regulate dnaa 
 conformation change . dnaa 
 complex is represented by red ribbons ( pdb code : 1l8q ) ; the snapshot 
 from simulations of adp  dnaa is depicted by blue ribbons , and 
 the snapshot from simulations of the adas ( b ) superimposition of the snapshot from the simulations of 
 adp  dnaa ( blue ribbon ) and the apo form dnaa ( green ribbon ) . ( c ) rmsds along the trajectories with respect to the crystal structure 
 of adp ( d ) adas interacts 
 with dnaa less strongly than adp with dnaa . in this study , the density functional theory was 
 used to calculate 
 the free energy change of the hydrolysis of adenosine phosphates and 
 adenosine arsenates . molecular mechanical force fields for the arsenates 
 systems were parametrized using intensive density functional calculations , 
 and then molecular dynamics simulations were employed to investigate 
 the structural and energy changes due to phosphate - arsenate replacement 
 in the selected molecular complexes . earlier quantum mechanical calculations 
 indicated that arsenate dna and phosphate dna may have similar conformational 
 properties . consistently , our work confirms 
 that partial replacement of phosphate by arsenate in dna can retain 
 certain structural and functional properties ; however , this is unlikely 
 to hold for the ribosome . our study also indicates a perfect match 
 between proteins and phosphate in terms of structure and interaction 
 energy , which are superior to those between proteins and arsenate . the 
 theoretical free energy changes of hydrolysis of adenosine triarsenate 
 ( atas ) and adenosine diarsenate ( adas ) were based on qm calculations 
 with large basis set ( 6 - 311++g * * ) and include solvation effects . the 
 full geometry optimizations and harmonic vibrational frequency calculations 
 were performed at the b3lyp/6 - 311++g * * level of theory . vibrational 
 energy corrections and entropies were used to calculate the free energy 
 change of the hydrolysis reactions . implicit solvent effects with 
 water as the solvent were also considered by the polarizable continuum 
 model ( pcm ) using the continuous surface charge formalism . gaussian09 , density functional theory at the b3lyp/6 - 311++g * * level was used 
 to calculate the energy change of atas hydrolysis . b3lyp/6 - 31+g * level 
 of theory was used to add arsenate nucleotides into charmm27 force 
 field , due to the arsenate  phosphate replacement . the parametrization 
 of all - atom empirical force field for arsenate nucleic acids were 
 based on modification of the current charmm27 force field for phosphate 
 nucleic acids . phosphate replacement were recalibrated to quantum 
 mechanical calculations of the model compounds listed in supporting information , sup - figure 1 . the corresponding 
 phosphate compounds in the supporting information , sup - figure 1 , were used in the original charmm27 force field parametrization . the full geometry optimizations , harmonic vibrational frequency calculations , 
 and potential energy surface for torsion angles were performed at 
 the b3lyp/6 - 31+g * level of theory using the gaussian09 program . bond lengths and angles related to as atom were 
 fitted to average values obtained from the model compounds . the force 
 constants were fitted to the harmonic vibrational frequency of dimethyl 
 arsenate ( supporting information , sup - figure 
 1a ) . atomic charges were based on nbo calculations at the b3lyp/6 - 31+g * 
 level , adjusted by fitting the potential energy curve of h2o---(ch3)2aso4 interactions ( supporting information , 
 sup - figure 2 ) . it can be seen that the potential energy curves for 
 the arsenate and phosphate are almost identical , with arsenate shifted 
 to a slightly longer r0 distance , due to the large vdw 
 radius of the arsenic atom . the partial charges of as , o = as , 
 and o  as were 1.58 , 0.59 , and 0.8 , very closed 
 to that of the related phosphate charges ( 1.50 , 0.57 , 0.78 ) . similarly , the force fields for adenosine triarsenate ( atas ) and adenosine 
 diarsenate ( adas ) were also parametrized . sup - figure 3 , supporting information , compares the vibrational 
 frequencies obtained by density functional theory calculations and 
 our optimized arsenate force field calculations for the atas . supporting information table 1 compares selected 
 force field parameters for arsenate and phosphate compounds . besides 
 the longer as  o bond , the optimized arsenate parameters are 
 very similar to phosphate nucleotides used in the charmm force field . md simulations 
 were performed using the namd package and the charmm 27 force field , with constant 
 pressure ensembles ( npt ) at 1 atm and the temperature at 300 k. the time step was 2 fs with a shake constraint 
 on all bonds with hydrogen atoms . productive 
 md runs were performed after 5000 steps of minimizations and three 
 150 ps heating and equilibration runs . the starting conformations of the pure dna / rna and 
 protein  nucleic acid complexes were based on x - ray crystal 
 structures of the phosphate system . the pdb codes for fis  dna , ihf  dna , and repe  dna complexes are 3iv5 , 1ihf , and 2z9o , respectively . the 
 trna  protein complex is the bacillus subtilis trna in complex with geobacillus kaustophilus lysidine synthetase ( pdb 3a2k ) . ribosome crystal structures are taken from the thermus thermophilus 70s ribosome bound to release 
 factor 2 ( pdb codes , 2x9r and 2x9s ) . the simulated molecules were solvated 
 in a tip3p water box with a margin of at least 
 15  from any edge of the water 
 box . magnesium , 
 potassium and chloride ions were also added to the simulated system 
 to obtain different ionic conditions . pure dna system ( 7bna ) was neutralized 
 with 22 sodium ions . in the simulation of the trna tils complex , 
 three ionic concentrations were tested . for low ionic conditions , 
 33 sodium , 20 magnesium , and 5 chloride ions for the high ionic concentration , the 
 effective ion concentration is around 0.2 m ( with 167 sodium , 20 magnesium , 
 20 potassium , and 160 chloride ions ) . the system with protonated histidines 
 is similar to the low ionic concentration setup , with 17 sodium , 20 
 magnesium , and 6 chloride ions . the high ionic concentration ( 0.25 
 m ) was used for pure trna simulations . for the ribosomal systems , 
 in addition to the existing magnesium 
 ions in the crystal structure , we also added 400 magnesium ions , 2397 
 sodium ions , and 400 chloride ions to neutralize the overall charges 
 in ribosome . 497327 water molecules are added to solvate the ribosome 
 complex . before the production run , we have pre - equilibrated the systems 
 with mg and other ions in the solution . the ribosomes were fixed and 
 only water and ions were allowed to move in the md simulations for 
 the first 4 ns , which were not included in the productive simulations . , we 
 used the program ionize ( theoretical and computational biophysics 
 group , university of illinois at urbana  champaign , http://www.ks.uiuc.edu/development/mdtools/ionize/license.html ) to place magnesium and sodium ions around the negatively charge 
 regions in the rna . following the 
 md simulations , first , we calculate the buried surface 
 areas for two interacting partners , with a surface tension coefficient 
 of 0.015 kcal/(mola ) , as recommended by haberthuer 
 and caflish . second , we include the ions 
 within the first solvation shell ( 5  ) of solute in the interaction 
 energy calculation , to consider the ionic shielding effect in the 
 electrostatic interactions . the inclusion of ions in the interaction 
 energy may provide better interaction energies for highly charged 
 nucleotides , especially for the trna and ribosome systems . the 
 normalized correlation matrix was obtained by computing the covariances 
 of the spatial atom displacements of 60 ns md trajectories for selected 
 pairs of atoms:12we use p or as atom in the dna ( or rna ) backbone 
 to represent the motions in dna ( or rna ) . the c carbons were 
 used to represent protein structure . after we obtain the normalized 
 correlation matrix , we compare phosphate and arsenate complexes to 
 obtain the linear correlation between each correlation matrix element . the rmsd analysis of dynamic trajectories was obtained using starting 
 crystal structures as reference . theoretical as k - edge ( 11867 ev ) exafs spectra for the arsenate dna 
 and rna were simulated with ifeffit algorithms in feff8 , using the 
 graphical utility artemis . all non - hydrogen 
 atoms within an 8  distance from as atoms were extracted from 
 the dna or rna complexes . the average spectra were obtained by averaging 
 all simulations with as atoms in the dna system ( linear 7bna dodecamer , 
 fis  dna , ihf the spectra 
 for the ribosome were averaged from 60 arsenic positions in the 30s 
 and 60 arsenic positions in the 50s subunits . separate averages using 
 only 60 arsenic positions in 30s or only 60 arsenic positions in 50s 
 subunits converged , indicating that the sampling is representative 
 of the ribosome structures . our quantum mechanical calculations 
 reveal the as = o bond length is 1.653  and as o 
 is 1.823  ; thus , the average bond lengths of as = o and 
 as  o is 1.74  , which is very close to 1.73  , obtained 
 from fitting of exahs spectra . as can 
 be seen in sup - figure 4 , supporting information , the vibrational frequencies obtained by density functional theory 
 calculations and our optimized arsenate force field calculations agree 
 very well , indicating a good fit of the force constants . in molecular 
 mechanics calculations , molecular vibrational frequencies are mostly 
 dominated by force constants of bond stretch and bending , and the 
 low frequency vibrational modes are usually mixed with bending and 
 torsional motions of chemical bonds . the fitting of the potential 
 energy surfaces for the torsion angles of compounds a , b , c , and d 
 ( see sup - figure 1,supporting information ) are reported in sup - figure 5 , supporting information . in the model compound torsion scans , only the specific torsion 
 angle is fixed , and all other coordinates have no constraints . in 
 the force field parametrization , it is essential to fit the intrinsic 
 molecular mechanics surface to the quantum mechanical surface , and 
 keep the location of the molecular mechanics global minimum for a 
 particular dihedral at the same point as the molecular mechanics global 
 minimum . as can be seen in sup - figure 5 , supporting 
 information , the majority of the molecular mechanics curves 
 overlap the quantum mechanical surface , including the  and 
  which are important for rna structures . the deviations of 
 two torsional angles , o c3 , 
 are also by less than 0.5 kcal / mol , which is within the range of the 
 accuracy of quantum mechanical calculations . often , different levels 
 of quantum mechanical calculations and optimized torsion profiles 
 for nucleic acids force field could deviate by more than 0.5 kcal / mol . recently , the charmm 27 force field has been 
 improved leading to the charmm36 force field which has a better treatment of nucleic acids . in this study , we 
 only modified the force field parameters related to phosphate - arsenate 
 substitution ; all other parameters are still charmm 27 . first , 
 we compare pure dna and rna with phosphate and arsenate backbones . 
 for dna , we selected a linear b  dna dodecamer ( the 7bna dodecamer ) , 
 which wolfe - simon et al . used as the representative dna structure 
 to compare with the observed exafs spectra . for rna , we simulated a pure trna , which forms the trna lysidine 
 synthetase complex ( tils ) . as can be 
 seen in figure 1 , both phosphate and arsenate 
 dna are very stable during the 60 ns simulations , with the arsenate 
 dna being slightly more flexible than the phosphate dna ( figure 1a ) . both phosphate and arsenate dna are able to 
 keep all watson  crick base pairs ( figure 1b ) . in the trna simulations , both phosphate and arsenate trnas have 
 larger rmsd fluctuations than dna ( figure 1c ) , which is a typical behavior for trna molecule in solution . crick base pairs are also very stable for both phosphate 
 and arsenate trna , and only a few opened during simulation ( figure 1d ) . overall , our simulations of two dna and rna 
 systems indicated that the arsenate dna and rna could have similar 
 conformations and the ability to keep the watson crick base 
 pairs , consistent with recent quantum mechanical studies . can atas or adenosine diphoshate 
 arsenate ( adp  as ) in the gfaj1 
 cell provide energy comparable to that of atp ? to compare the hydrolysis 
 energies of atp and its arsenate analogues , we calculated the theoretical 
 free energy changes for five reactions ( table 1 ) . hydrolysis of atas releases less energy than atp . the free energy 
 change obtained for hydrolysis of adp  as to adp and as ( reaction 
 2 ) was calculated to be 3.0 kcal / mol , while hydrolysis of 
 atas into adenosine diarsenate ( adas ) gives 2.2 kcal / mol . hydrolysis of atas into adenosine monoarsenate ( amas ) can yield a 
 larger free energy change ( 8.1 kcal / mol ) . the calculated free 
 energy changes for the hydrolysis of atp are 4.9 kcal / mol 
 ( reaction 1 ) and 11.5 kcal / mol ( reaction 4 ) . the product of atp hydrolysis can be used to regulate 
 the initiation 
 of dna replication , which is a key event in the cell cycle of all 
 organisms . in bacteria , replication initiation occurs at the oric region which is recognized by the atp  dnaa and 
 adp  dnaa complexes . following replication 
 initiation , atp  dnaa reforms to reinitiate a dna replication 
 cycle . dnaa interaction should 
 be sufficiently strong for dnaa oligomerization ; yet , it should also 
 be sufficiently weak to allow adp replacement by atp . we used molecular 
 dynamics ( md ) simulations to test if adas in complex with dnaa can 
 regulate dna replication similar to adp . adp binds dnaa in a pocket 
 between domains iiia and iiib ( figure 2a ) . 
 without adp , the domains separate , resulting in misorientation of 
 domains iiib and iv with respect to iiia ( figure 2b ) . when we replaced adp by adas , the interaction between 
 domains iiia and iiib in the adas dnaa complex still appeared 
 relatively stable ; however , domain iv became more flexible than in 
 the adp dnaa ( figure 2d ) , indicating 
 that it is easier to replace adas than adp during the reinitiation 
 of dna replication . dna complexes in both phosphate / arsenate forms 
 are stable during the simulations ( figure 3 , parts a and e ) , deviating little from the initial structure . the 
 rmsds of the md relaxed snapshots at 50 ns of the arsenate and phosphate 
 dna - fis complexes are 2.9  from the crystal structure 
 ( figures 3a and 3e , 
 table 2 ) . if we only focus on dna dynamics , 
 the covariance matrices for phosphate and arsenate dna are correlated 
 with r = 0.74 ( sup - figure 6a , supporting 
 information ) . dna complex presents a large difference 
 between phosphate and arsenate dna ( figure 3b ) . while both ihf  and  subunits maintain their interaction 
 with the u - shaped phosphate dna , the  subunit interacts more 
 weakly with arsenate dna ( green structures , figure 3b ) . however , the overall rmsds for both phosphate and arsenate 
 ihf  dna complexes are similar ( sup - figure 7a , supporting information , table 2 ) . the 
 structural deviations of repe  dna ( figure 3c ) are also larger than those of fis  dna , with rmsd 
 of 3  for phosphate dna and 4  for arsenate dna 
 ( sup - figure 7b , supporting information , 
 table 2 ) . arsenic dna ( dna(as))-protein complexes 
 are more stable than arsenic 
 rna ( rna(as))protein complexes . in the figure , the crystal 
 structures of phosphate dna protein complexes are represented 
 by red lines ( dna ) and ribbons ( proteins ) ; snapshots from simulations 
 of phosphate dna ( dna(p))protein complexes are correspondingly 
 in blue ; snapshots from simulations of the arsenate dna / rna  protein 
 complexes are in green . ( a ) the dna(as)fis protein ( green ) 
 complex does not deviate significantly from the dna(p)protein 
 complexes . ( b ) weaker arsenate dna(as)ihf protein ( green ) 
 interaction leads to larger dna structural fluctuations . ( c ) the repe 
 protein is able to accommodate the cumulative dna structural changes 
 introduced by arsenate phosphate replacement in a 37 bp dna 
 fragment , with the two ends separated by 100  . ( d ) the trna(as)-tils 
 complex deviates extensively from the phosphate crystal structure . tils crystallized with trna(p ) is represented by red ribbon ; green 
 ribbon represents tils complexed with trna(as ) . trna(p ) is represented 
 by sticks , while trna(as ) is shown as yellow surface to highlight 
 the large differences between phosphate and arsenate trna ( e ) two independent simulations for fis  dna complex 
 indicate that arsenate dna protein complex is slightly more flexible 
 than phosphate dna protein complex . ( f ) arsenate dna interacts less 
 strongly with protein than phosphate dna protein complex in two simulations 
 of fis  dna complex . three ionic conditions are listed 
 for trna  tils complex : hsp , protonated histidines in tils ; 
 high , high ionic concentration ; low , low ionic concentration . average rmsd from crystal structure 
 for whole complex , and rmsd for nucleotides are in parentheses . interaction energies are averaged 
 over the last 30 ns in the md simulation trajectories ; values in parentheses 
 are the interaction energies averaged over the last 15 ns . the agreement 
 between the interaction energies averaged over different time periods 
 indicates convergence in the simulations . the interaction energies between the protein and arsenate 
 dna are 
 slightly smaller than those with phosphate dna ( table 2 ) . the differences are about 2 , 0.5 , and 5 kcal / mol , for the 
 fis  dna , ihf  dna , repe  dna complexes , respectively . the average interactions from the two simulations are within 0.6 kcal / mol 
 for fis  dna ( phosphate ) and 1.0 kcal / mol for fis  dna 
 ( arsenate ) . for 
 the ihf  dna complex , dna wraps around the ihf proteins ( figure 3b ) . therefore , the phosphate - arsenate substitution 
 may not induce geometrical strain in the dna , leading to almost identical 
 protein  dna interaction energies for the phosphate and arsenate 
 dna ( sup - figure 7b , supporting information ) . the binding distance between two repe monomers in the repe  dna 
 complexes separates to almost 100  ( figure 3c ) ; thus , here phosphate - arsenate substitution could have 
 larger effects leading to much weaker protein  dna interactions 
 in the case of the arsenate dna ( sup - figure 7d , supporting information ) . the smaller protein dna 
 interaction energy for arsenate 
 dna is correlated with its higher exposure to water and cations ( table 2 ) . overall , even though the arsenate  dna 
 protein interaction energy is lower than that of the phosphate  dna 
 counterpart , their dynamics are similar , with correlation coefficients 
 ranging from r = 0.56 to 0.42 ( sup - figures 
 6 and 8 , supporting information ) . in summary , 
 we found that the structural and dynamical properties of arsenate 
 dna and phosphate dna do not present marked differences . however , 
 the interaction of the arsenate dna with the protein is weaker than 
 that of phosphate dna . we simulated three ionic conditions ( low and high salt concentrations , 
 and protonated histidines in the tils protein ) . trna interaction 
 energies are similar at high ionic concentration and with protonated 
 tils histidines ( table 2 and sup - figure 7 , supporting information ) , and both are stronger 
 than the tils arsenate substitution weakens the tils  trna interaction , making 
 the gap slightly larger than that observed for protein the arsenate substitution 
 in the trna system leads to larger structural changes and the dynamics 
 of the trna and the tils protein also change ( sup - figures 7 and 8 , supporting information ) . tils 
 interaction is only r = 0.3 ( sup - figure 
 6 , supporting information ) . similar to 
 the dna systems , the arsenate trna has higher solvent exposure and 
 contact with cations ( table 2 ) . the difference 
 in the dynamics of phosphate and arsenate trnas raises a pivotal question : 
 can an arsenate ribosome exist and be functional ? the interaction 
 energy difference between the small - sized trna  protein complexes 
 could be magnified in the largest cellular rna to address the question of the differences in the energy landscape 
 and dynamics of the assembly between ribosomes made of phosphates 
 and arsenates , we conducted massive md simulations of phosphate and 
 arsenate ribosomes . both phosphate and arsenate ribosomes underwent 
 large conformational 
 changes ( figure 4 ) after 40 ns simulation at 
 300 k. during the first 10 ns , the rmsds of both phosphate and arsenate 
 ribosomes are small , with the structures similar to the crystal structure 
 ( figure 4c ) . because arsenate is heavier , one 
 would expect that the arsenate ribosome would have slower conformational 
 dynamics . however , after 10 ns , the structural change of the arsenate 
 ribosome becomes larger than that of the phosphate , mainly because 
 of the change in the arsenate 30s . both phosphate and arsenate 50s 
 present large rmsds , mostly because of the portion extruding from 
 the main 50s body , like the l1 stalk where the l1 ribosomal protein 
 binds ( figure 4 , parts a and b ) , which is known 
 to be very flexible . apart from this 
 region , the main body of 50s has smaller rmsd than the 30s subunit 
 ( figure 4c ) . the rna backbone follows the 
 same trend as the entire unit , but with a slightly lower magnitude . these conformational changes of both the phosphate and arsenate ribosomes 
 may be mixed with structural relaxation and intrinsic motion since 
 the ribosome is a large molecular machine with large global motions . as can be seen in figure 4d , converged motions can be observed at the p - site trna already after 
 20 ns , with arsenate trna having smaller rmsd than phosphate trna . the e - site trna is more flexible , consistent with the anisotropic 
 network model analysis that the e - site trna has different dynamics 
 than the p - site trna . subunits of arsenate 
 ribosome ( ribosome(as ) ) are more repulsive 
 than those of phosphate ribosome ( ribosome(p ) ) , making it more difficult 
 to assemble arsenate rrna into the functional 70s ribosome than its 
 phosphate counterpart . ( a and b ) snapshots from simulations of the 
 ribosome(p ) and ribosome(as ) , respectively . the two trnas are shown in 
 surface presentation , with the p - site trna sitting between the e - site 
 trna and rf2 . ( c ) rmsds ( root 
 mean squared deviations ) of ribosome(p ) and ribosome(as ) from the 
 starting crystal structures during the simulations . ( d ) e - site trna 
 is more flexible than p - site trna . the trnas , rf2 , and mrna are not included in 
 the calculation . including these four molecules makes 30s and 50s 
 more repulsive for the arsenate system . the rmsd differences between the phosphate and 
 arsenate ribosomes 
 coincide with the interaction energy change of the s16 ribosomal protein 
 with its surrounding rrna , with the s16-arsenate rna interaction weakening 
 after 10 ns ( sup - figure 9a , supporting information ) . several other ribosomal proteins , 
 s4 , s17 , and s20 are also important for global stabilization of rrna 
 structure , and the interaction of arsenate rrna with all these 
 proteins is weaker than for phosphate rrna ( sup - figure 9 , supporting information ) . the consistent weaker 
 interaction of arsenate rrna with ribosomal proteins may hamper the 
 30s assembly . if however arsenate rrna could successfully fold 
 into 30s and 50s 
 subunits , could arsenate 30s and 50s associate as phosphate 30s and 
 50s do ? as can be seen in figure 4d , while the phosphate 30s and 50s spend most of their time in an attractive 
 energy landscape , the arsenate 30s and 50s could be repulsive to each 
 other . assuming that arsenate 30s and 50s can associate 
 into 70s , we examine 
 the energy and dynamics of the p - site and e - site trnas , mrna , and 
 the rf2 protein ( sup - figure 9 , supporting information ) . in the p - site , arsenate trna has similar dynamic and interaction 
 energies with rf2 and other molecules ; however , in the e - site , the 
 arsenate trna is more repulsive to other molecules than its phosphate 
 counterpart . overall , it appears that it is more difficult 
 to assemble arsenate rrna into the 30s/50s subunits and into the full 
 70s ribosome than its phosphate counterpart . this raises the question 
 of whether an arsenate ribosome has been observed in the arsenate 
 cell . to answer 
 this question , we compare the experimentally observed exafs spectra 
 with the theoretical exafs spectra for the arsenate dna and rna . wolfe - simon 
 et al . recorded and fitted as k - edge ( 11867 ev ) exafs spectra . there 
 are three characteristic peaks with decreasing magnitude , with the 
 first peak reflecting the as  o bond geometry , and second and 
 third peaks reflecting nonbonded atoms around as ( figure 5a ) . our theoretical spectra of the 70s ribosome(as ) do 
 not match the experimental exafs , implying that the arsenate 70s does 
 not contribute to the main arsenate source of experimental exafs . ( a ) theoretical curves of two as atoms in the 7bna dodecamer dna(as ) 
 ( blue and green lines ) compared to the observed exafs curve ( black 
 line ) and the wolfe - simon s fitting ( red line ) . ( b ) the theoretical 
 curves averaged from the 7bna dodecamer dna ( green line ) and the 70s 
 ( blue line ) versus the observed exafs curve ( black line ) and the wolfe - simon s 
 fitting ( red line ) . ( c ) two typical theoretical curves from linear 
 7bna dodecamer dna ( green line ) and 70s ribosome ( red line ) . ( d ) theoretical 
 curve from bent dna ( red line ) is between that of linear dna ( green 
 line ) and 70s ribosome ( blue line ) . the experimental exafs 
 and wolfe - simon s fitting are taken from ref ( 3 ) . on the basis of the structures of arsenate dna 
 and rna simulated 
 in this study , we calculated the phase and amplitude of arsenate with 
 the ifeffit algorithms in feff8 , using the program artemis . dna 
 dodecamer ( the 7bna dodecamer ) as the representative dna structure 
 to compare with the observed exafs spectra . we also used the same 
 refined 7bna dodecamer structure as a benchmark ( with the appropriate 
 as = o and as  o distances ) . we found that two as atoms 
 may generate theoretical curves that coincide with the observed exafs 
 curve and the wolfe - simon s fitting ( figure 5a ) . the fitting curves averaged from all as atoms in the 7bna 
 dodecamer also have three distinct peaks ( figure 5b ) . bacterial dna is mostly bent due to packing , binding 
 with proteins , 
 and high salt concentration . therefore , 
 the linear 7bna dodecamer may not be a good candidate to represent 
 cellular dna structures . more importantly , for normal bacterial 
 cells , the ribosome constitutes about 25% of the total mass . thus , 
 ribosome structures should be included in a comparison between cellular 
 polynucleotide structures and experimental observations . we 
 then tested the nonlinear dna structures described above and the 70s 
 ribosome . we found that the spectra of bent dna and rna around the 
 regions of peaks 2 and 3 are totally different from those of linear 
 dna . for example , most as atoms in the ribosome have a typical peak 
 around 2.3  , whereas there is a valley between peaks 2 and 3 
 for linear dna ( figure 5c ) . the simulated exafs 
 spectra for nonlinear dna progressively change from a linear dna pattern 
 to that of rna . for a bent dna ( repe , figure 3c ) , the position and shape of peak 1 overlap with those of rrna , 
 and the spectra around the 23 region lie between those of 
 linear dna and rrna ( figure 5d ) . overall , 
 the theoretical exafs spectra of the as 70s ribosome ( blue line , figure 5b ) do not match the experimental 
 exafs curve . thus , our results demonstrate that arsenate 
 70s ribosome does not contribute to the main arsenate source in wolfe - simon s 
 bacterial cell . another indication of a negligible concentration of 
 arsenate 70s ribosome comes from comparison of nonbonding as c nonbonding 
 distances have been characterized to be 2.35 and 2.92  . however , 
 in our simulated arsenate ribosome , the radial distribution for as c 
 distances is 2.8  and there is no density around 2.35  
 ( sup - figure 10 , supporting information ) . the possible existence of 
 arsenate dna and rna in the gfaj-1 cell has attracted considerable scientific debate 
 as to whether and how arsenate analogues of phosphate can exist . because of problems associated with chemical stability , current 
 experimental approaches encounter difficulties in fully assessing 
 the characteristics of arsenate dna and rna . instead , we test computationally 
 possible arsenate  phosphate replacement by examining key molecular 
 systems ; however , because of the large size of the molecules our theoretical 
 protocols are also limited . for the atp and adenosine triarsenate 
 ( atas ) hydrolysis , we used the highest level of quantum mechanical 
 calculations feasible given current computational power ( density functional 
 theory at b3lyp/6 - 311++g * * level ) . the solvation treatment with state 
 of - the - art current quantum mechanical methods also involves approximations . 
 as can be seen in table 1 , while the calculated 
 free energy change for reaction 4 is lower than the experimental value 
 by only 0.6 kcal / mol , the free energy change for reaction 
 1 deviates significantly from the experimental measurement . however , we successfully reproduced the trend 
 of the hydrolysis reactions , i.e. , hydrolysis to amp ( reaction 4 ) 
 provides more energy than hydrolysis to adp ( reaction 1 ) . therefore , 
 we expect that the relative energies of hydrolysis of atp and atas 
 would reflect a trend : hydrolysis of adenosine triarsenate ( atas ) 
 provides 23 kcal / mol less energy than atp . it is still 
 computationally challenging to calculate the absolute 
 protein interaction energy using molecular mechanics simulations . nevertheless , our current study revealed that arsenate nucleotides 
 would interact with proteins less strongly than phosphate nucleotides . as can be seen in table 2 , our two independent 
 simulations of the fis dna complex agree quite well : the interaction 
 energies which are estimated from different time periods deviated 
 from each other by less than 1.0 kcal / mol . overall , the interaction 
 energy differences between phosphate and arsenate dna are about one 
 to two times the standard deviations , indicating that the differences 
 between the phosphate and arsenate cases are significant . the smaller 
 protein  nucleotide interaction energy for arsenate dna / rna 
 could be explained by the larger volume of the arsenate nucleotide , 
 which weakens electrostatic interactions . our observation of the higher 
 exposure of arsenate dna / rna to water and cations also reflects the 
 effects of the larger volume of arsenate . in principle , because the 
 arsenate nucleotides possess larger volumes , the solvation energy 
 changes due to the buried surface area can compensate for the weak 
 arsenate nucleotide - protein interaction ; however , our study indicates 
 that the solvation effect can not offset the weaker electrostatic 
 interaction . large scale molecular dynamics simulations can 
 provide insights 
 into biological systems ; including dna 
 flexibility , dna  protein interactions , trna and trna  protein 
 complexes , and the mechanism of ribosomal 
 function . the computational methods used are sufficiently accurate to reproduce 
 experimental structures and conformational 
 dynamics of a variety of molecules . our 
 evaluation of the structural consequences of phosphate - arsenate substitutions 
 in selected crucial processes in the cell may help in understanding 
 the biological consequences . at the level of small nucleotide hydrolysis 
 as energy source , we found that hydrolysis of adenosine triarsenate 
 ( atas ) provides 23 kcal / mol less energy than atp . thus , while 
 atas hydrolysis may provide the gfaj-1 cell sufficient energy for 
 some reactions , because ribosome assembly requires many energy - consuming 
 atp  dependent enzymatic reactions , the lower free energy obtained in atas hydrolysis may lead to an 
 energetic strain that hampers ribosome assembly . on the other hand , 
 at the level of gene replication and transcription , we found that 
 the small adenosine diarsenate molecule may be able to similarly regulate 
 the dnaa protein conformational dynamics . while arsenate dna interacts 
 with proteins less strongly than phosphate dna , complexes of proteins 
 with arsenate or phosphate dna may share similar dynamics . at 
 the level of translation we found major problems . arsenate - substituted 
 rna not only interacts with proteins less strongly than phosphate 
 rna , but also presents changes in conformational dynamics . the most 
 detrimental arsenate substitution outcome that we observe is that 
 the 30s and 50s may become too repulsive to assemble into a functional 
 70s ribosome . in principle , mutations in the proteins and in the rna 
 could make up for some of the structural and energetic changes caused 
 by arsenate replacement . however , while such mutations may be possible 
 in arsenate dna  protein interactions , this is not the case 
 for the ribosome . to further confirm our doubts regarding the existence 
 of an arsenate ribosome our comparison of the experimental exafs spectra of the arsenic bacteria 
 with theoretical exafs spectra for arsenate dna and rrna only finds 
 evidence for a possible existence of linear arsenate dna fragments 
 in the dried gfaj-1 cell , and the characteristic ribosomal structure 
 has not been observed . we hypothesize that the main reason for the 
 slow growth of the gfaj-1 cell could be the small number of phosphate 
 ribosomes that survived during cellular divisions . nature 
 has chosen phosphate not only for its energetic 
 function and its stability ; our study points to a perfect match between 
 proteins and phosphate in terms of structure and interaction energy , 
 which are superior to those between proteins and arsenate . apparently , 
 evolution has optimized the inter - relationship between proteins and 
 dna / rna , which requires overall changes at the molecular and systems 
 biology levels when replacing phosphate by arsenate .
a recent finding of a bacterial strain ( gfaj-1 ) that 
 can rely on 
 arsenic instead of phosphorus raised the questions of if and how arsenate 
 can replace phosphate in biomolecules that are essential to sustain 
 cell life . 
 apart from questions related to chemical stability , there 
 are those of the structural and functional consequences of phosphate - arsenate 
 substitutions in vital nucleotides in gfaj1-like cells . in this study 
 we selected three types of molecules ( atp / adp as energy source and 
 replication regulation ; dna  protein complexes for dna replication 
 and transcription initiation ; and a trna  protein complex and 
 ribosome for protein synthesis ) to computationally probe if arsenate 
 nucleotides can retain the structural and functional features of phosphate 
 nucleotides . 
 hydrolysis of adenosine triarsenate provides 23 
 kcal / mol less energy than atp hydrolysis . 
 arsenate dna / rna interacts 
 with proteins slightly less strongly than phosphate dna / rna , mainly 
 due to the weaker electrostatic interactions of arsenate . 
 we observed 
 that the weaker arsenate rna 
 protein interactions may hamper 
 rrna assembly into a functional ribosome . 
 we further compared the 
 experimental exafs spectra of the arsenic bacteria with theoretical 
 exafs spectra for arsenate dna and rrna . 
 our results demonstrate that 
 while it is possible that dried gfaj-1 cells contain linear arsenate 
 dna , the arsenate 70s ribosome does not contribute to the main arsenate 
 depository in the gfaj-1 cell . 
 our study indicates that evolution 
 has optimized the inter - relationship between proteins and dna / rna , 
 which requires overall changes at the molecular and systems biology 
 levels when replacing phosphate by arsenate .