EG11254

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Author Information

Joey McKenna 05930701 (mckenj)

Basic Information

  • ID: EG11254
  • Name: rlmH
  • Organism: E. coli
  • Description: RlmH is a methyltransferase that catalyzes the addition of a methyl group in the N3 position of the pseudouridine (Ψ) residue at nucleotide 1915 in 23S rRNA [Purta08, Ero08]. The enzyme requires the intact ribosome and the presence of pseudouridine at position 1915 in 23S rRNA for activity [Ero08, Ero10]. m3Ψ1915 is the only currently known methylated pseudouridine residue in bacterial RNAs.

Computational docking between RlmH and the ribosome shows contacts with both ribosomal subunits [Purta08], consistent with the requirement for intact ribosomes for in vitro activity [Ero08].

RlmH is a member of the SPOUT superfamily of methyltransferases [Anantharaman02, Tkaczuk07] and belongs to the α/β knot superfamily of proteins [Mallam07, Tkaczuk07]. Thermodynamic and kinetic analysis of folding of RlmH shows that the protein folds via a simple three-state sequential mechanism [Mallam07]. Folding may involve a slipknot intermediate [Sulkowska09]. The RlmH polypeptide chain has a trefoil knot conformation even in the denatured state [Mallam10]. Backbone NMR assignments for urea-denatured RlmH have been reported [Hsieh13]. RlmH is a dimer in solution [Purta06, Mallam07, Mallam10].

RlmH activity is influenced by the presence of specific nucleotides in stem-loop 69 (H69) surrounding Ψ1915 in 23S RNA [Leppik12].

An rlmH deletion mutant lacks the m3Ψ1915 modification [Purta08, Ero08] and has a slightly lower growth rate than wild type [Purta08].

RlmH: "rRNA large subunit methyltransferase H" [Purta08, Ero08] Reference

  • DNA Length: 468 bp
  • DNA sequence:

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

  • Amino Acid length: 155 amino acids
  • Amino Acid sequence:

MKLQLVAVGTKMPDWVQTGFTEYLRRFPKDMPFELIEIPAGKRGKNADIKRILDKEGEQMLAAAGKNRIVTLDIPGKPWDTPQLAAELERWKLDGRDVSLLIGGPEGLSPACKAAAEQSWSLSALTLPHPLVRVLVAESLYRAWSITTNHPYHRE

Function and Homologs

  • Product: 23S rRNA m3Ψ1915 methyltransferase
  • Closest homologous proteins: The top (max three) homologous proteins to this protein, as identified by BLAST searches.
    • Name: 23S rRNA (pseudouridine(1915)-N(3))-methyltransferase RlmH [Shigella flexneri 2a str. 301], Max Score: 318/Query Cover: 100%/E-Value: 4e-110/Ident: 100% NP_706574.1
    • Name: MULTISPECIES: 23S rRNA (pseudouridine(1915)-N(3))-methyltransferase RlmH [Shigella], Max Score: 317/Query Cover: 100%/E-Value: 7e-110/Ident: 99% WP_000776102.1
  • Equivalent E. coli / JCVI functional protein: MMSYN1_0361

Expression

  • Expression Level: [medium].
  • Expression Level Hypothesis: In an ecoli gene, the RNA methyltransferase has a higher expression level because it occurs in a eukaryote. Based on the excel sheet of Ecoli genes, this gene was shown to operate at a medium expression level.
  • Expression Level References and Description: File:EcoliProteomicExpressionData.xlsx
  • Expression Time: [early].
  • Expression Level Hypothesis: After analyzing the central dogma and learning a bit about this gene, it seems necessary in the early stages of the lifecycle because it deals with RNA processing before it is translated into a protein.
  • Expression Time References and Description: Intuition

Gene Context

  • Other Components in the functional module. EG13427
  • Possible Dependencies: metal ion binding
  • Process: rRNA base methylation
    • Inputs: S-adenosyl-L-methionine, uracil(747) [[1]]
    • Outputs: S-adenosyl-L-homocysteine, 5-methyluracil(747)

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