EG10453

From BioE80 Boot
Jump to: navigation, search

Author Information

Luis Fernando Varela

Basic Information

  • ID: EG10453
  • Name: hisS
  • Organism: E. coli
  • Description:

Histidine-tRNA ligase (hisS) is a member of the family of aminoacyl-tRNA synthetases, which interpret the genetic code by covalently linking amino acids to their specific tRNA molecules. The reaction is driven by ATP hydrolysis. HisS belongs to the Class II aminoacyl tRNA synthetases, which share three regions of homology [Eriani90, Cusack91].

HisRS is a dimer in solution [Kalousek74]. The C-terminal domain of the protein is required for dimerization, while the N-terminal domain contains most of the catalytic activity. The two domains do not complement each other in trans [Augustine97]. Minimal active site fragments likely representing the ancestral "urzyme" have been studied [Li11h].

Specificity determinants within tRNAHis that are important for recognition by HisRS have been identified; the unique G-1:C73 base pair was found to play a crucial role [Himeno89, Yan94, Fromant00, Rosen04a, Guth07]. Specificity determinants and residues within HisRS that are important for catalytic activity have been investigated [Yan95, Ruhlmann97, Bovee99, Hawko01, Connolly04, Guth07], and a model for the catalytic cycle was proposed [Guth07]. The C-terminal domain of HisRS was found to be largely responsible for recognition of the tRNAHis anticodon [Yan96].

Crystal structures of hisRS have been determined, and a reaction mechanism was proposed [Arnez95, Arnez97]. Various types of experiments support a substrate-assisted concerted reaction mechanism [Guth05]. Catalysis may occur at sites alternating between the two monomers; conformational changes may be rate-limiting for product formation [Guth07, Guth09]. The mechanisms of substrate discrimination [Banik09, Banik10] and the aminoacylation reaction [Banik11] have been modeled.

  • DNA Length: 1272 base pairs.
  • DNA sequence:

ATG GCA AAA AAT ATC CAG GCA ATC CGC GGT ATG AAC GAC TAC CTG CCT GGG GAA ACC GCC ATT TGG CAG CGC ATT GAG GGG ACT CTT AAA AAT GTA TTG GGG TCC TAT GGT TAT AGC GAA ATT CGC TTA CCT ATT GTG GAG CAA ACC CCC TTA TTC AAG CGT GCC ATC GGA GAG GTC ACC GAC GTG GTC GAA AAG GAA ATG TAT ACT TTT GAA GAT CGC AAC GGG GAC AGT CTT ACA CTG CGT CCC GAA GGC ACC GCA GGC TGT GTC CGC GCA GGG ATC GAG CAC GGT TTG TTA TAC AAC CAG GAG CAG CGT TTG TGG TAC ATC GGA CCT ATG TTC CGT CAC GAA CGT CCA CAA AAG GGC CGT TAC CGC CAG TTC CAT CAG TTA GGT TGT GAA GTG TTT GGT CTT CAA GGA CCA GAT ATT GAC GCG GAA TTA ATT ATG CTT ACG GCC CGT TGG TGG CGT GCC TTA GGT ATT TCC GAA CAT GTT ACC TTA GAG CTT AAT AGT ATT GGA AGT CTG GAA GCG CGT GCG AAT TAC CGC GAT GCT TTA GTA GCG TTC CTT GAA CAA CAC AAA GAA AAA TTG GAT GAG GAC TGT AAG CGC CGT ATG TAT ACT AAT CCC CTT CGT GTA CTT GAT AGT AAG AAC CCC GAG GTG CAG GCG CTT CTG AAC GAT GCA CCT GCG TTA GGT GAC TAT TTG GAT GAG GAA AGT CGT GAG CAC TTT GCG GGA CTG TGT AAG TTG TTA GAG AGC GCT GGA ATC GCA TAC ACC GTG AAC CAA CGC TTA GTC CGT GGT CTT GAC TAT TAC AAT CGC ACT GTA TTC GAA TGG GTA ACC AAC TCT TTG GGC TCT CAG GGC ACT GTT TGC GCG GGG GGG CGC TAT GAC GGC TTA GTC GAG CAG CTG GGA GGA CGT GCT ACG CCT GCT GTC GGA TTT GCA ATG GGC TTA GAA CGC TTG GTG TTA TTG GTT CAA GCG GTA AAT CCT GAA TTC AAG GCT GAT CCT GTC GTA GAT ATT TAT CTG GTG GCT TCC GGA GCA GAC ACG CAA AGC GCT GCA ATG GCT TTG GCC GAG CGT CTG CGC GAC GAG CTT CCG GGG GTG AAG CTT ATG ACG AAC CAT GGC GGT GGA AAT TTC AAG AAG CAA TTT GCA CGC GCC GAT AAA TGG GGG GCT CGT GTA GCG GTG GTC CTG GGC GAA TCT GAG GTT GCG AAT GGC ACG GCT GTC GTC AAA GAT TTG CGT TCC GGG GAA CAG ACG GCT GTC GCA CAA GAT AGT GTG GCC GCA CAC TTA CGC ACC TTA TTG GGG

  • Amino Acid length: 424 amino acids.
  • Amino Acid sequence:

MAKNIQAIRG MNDYLPGETA IWQRIEGTLK NVLGSYGYSE IRLPIVEQTP LFKRAIGEVT DVVEKEMYTF EDRNGDSLTL RPEGTAGCVR AGIEHGLLYN QEQRLWYIGP MFRHERPQKG RYRQFHQLGC EVFGLQGPDI DAELIMLTAR WWRALGISEH VTLELNSIGS LEARANYRDA LVAFLEQHKE KLDEDCKRRM YTNPLRVLDS KNPEVQALLN DAPALGDYLD EESREHFAGL CKLLESAGIA YTVNQRLVRG LDYYNRTVFE WVTNSLGSQG TVCAGGRYDG LVEQLGGRAT PAVGFAMGLE RLVLLVQAVN PEFKADPVVD IYLVASGADT QSAAMALAER LRDELPGVKL MTNHGGGNFK KQFARADKWG ARVAVVLGES EVANGTAVVK DLRSGEQTAV AQDSVAAHLR TLLG

Function and Homologs

  • Product: Histidine--tRNA ligase
  • Closest homologous proteins: The top (max three) homologous proteins to this protein, as identified by BLAST searches.
    • histidyl-tRNA synthetase [Escherichia coli N40513], Max score: 871 / Query Cover: 100% / E-Value: 0.0 / Ident: 99%, OMI59605.1
    • histidine--tRNA ligase [Shigella boydii], Max score: 869 /Query Cover: 100% / E-Value: 0.0 / Ident: 99%, WP_078167048.1
    • Chain A, Histidyl-Trna Synthetase, Max score: 869 / Query Cover: 100% / E-Value: 0.0 / Ident: 99%, 1HTT_A

Expression

  • Expression Level: low
  • Expression Level Hypothesis: This interprets the genetic code by covalently linking amino acids to their specific tRNA molecules, which seems to be something that we don't need too frequently.
  • Expression Level References and Description: M. genitalium model data
  • Expression Time: Right at the beginning
  • Expression Level Hypothesis: Since this is a member of the family of aminoacyl-tRNA synthetases, I think that ATP binding is crucial for all the work that needs to be done, since it is the energy source of the cell, so it is needed right at the beginning.
  • Expression Time References and Description: I made my own hypothesis by taking conclusions from the protein's functionality.

Gene Context

  • Other Components: thrS
  • Possible Dependencies: L-histidine
  • Process: histidyl-tRNA aminoacylation
    • Inputs: ATP, L-histidine, tRNA(His)
    • Outputs: AMP, diphosphate, L-histidyl-tRNA(His)
  • Reference: F4MPL9

Construct

We will handle this - not part of your assignment

  • Synthesis Score: The synthesis score: 1, 2,3
  • Predicted Translation Rate: Prediction of construct translation rate from the RBS calculator
  • Design Notes and Details:
  • GenBank File: A link to the GenBank file. file