EG10523

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Author Information

Angela Li (asli)

Basic Information

  • ID: EG10523
  • Name: rsmA
  • Organism: E. coli
  • UniProt ID: [[1]}
  • Description: Ribosomal RNA small subunit methyltransferase A (also known as ksgA) is a dimethyladenosine transferase. It is an enzyme that methylates two adjacent adenines in 16S rRNA (which is part of the small subunit of the ribosome), a process which typically occurs rather late in ribosome maturation. [1] The two A's are part of a hairpin loop that's near the 3' end of the 16S rRNA. rsmA is only able to methylate 30S ribosomal subunits in their translationally inactive confirmation. It interacts with the decoding site of the 30S subunit (the part that monitors the complementarity of tRNA and mRNA in protein translation. [2]. It has been suggested that when rsmA binds to the rRNA on the 30S subunit, it prevents the 30S subunit from entering the translation cycle. [3]

In addition, in E. Coli, the protein has a DNA glycosylase activity (enzymes that do base excision repair) and removes cytosines that are mispaired with oxidized thymines in the DNA. Thus there are some early research suggesting it may play a role in protection of DNA against oxidative stress. [4]

Inactivation of rsmA leads to resistance to kasugamycin (a particular antibiotic) without any substantial growth defects. In particular, it appears that the lack of methylation at the second adenosine (at position 1519) is responsible for the resistance phenotype. Cells that do not have this gene have altered ribosome profiles, greater sensitivity to cold, and deficiencies (as expected) in small subunit rRNA-processing. The only known mutation that leads to inactivation is a modification of the amino acid at position 66. Meanwhile, extreme overexpression of rsmA is toxic to cells.[5]

  • DNA Length: 822 base pairs.
  • DNA sequence:

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

  • Amino Acid length: 273 amino acids.
  • Amino Acid sequence:

MNNRVHQGHLARKRFGQNFLNDQFVIDSIVSAINPQKGQAMVEIGPGLAALTEPVGERLDQLTVIELDRDLAARLQTHPFLGPKLTIYQQDAMTFNFGELAEKMGQPLRVFGNLPYNISTPLMFHLFSYTDAIADMHFMLQKEVVNRLVAGPNSKAYGRLSVMAQYYCNVIPVLEVPPSAFTPPPKVDSAVVRLVPHATMPHPVKDVRVLSRITTEAFNQRRKTIRNSLGNLFSVEVLTGMGIDPAMRAENISVAQYCQMANYLAENAPLQES

Function and Homologs

  • Product: Ribosomal RNA small subunit methyltransferase A
  • Module: rRNA base methylation (GO term)
  • Closest homologous proteins: The top (max three) homologous proteins to this protein, as identified by BLAST searches.
    • ribosomal RNA small subunit methyltransferase A [Shigella], 564/100/0/99%, WP_001065372.1
    • ribosomal RNA small subunit methyltransferase A [Escherichia fergusonii], 564/100/0/99%, WP_001065375.1
    • dimethyladenosine transferase [Shigella flexneri K-315], 564/100/0/99%, EIQ27412.1
  • Equivalent E. coli / JCVI functional protein: MMSYN1_0004.

Expression

  • Expression Level: Medium
  • Expression Level Hypothesis: This gene is likely to be moderately expressed because on the one hand, there's been some evidence to suggest that it's involved in ribosome biogenesis. However, it likely plays more of a regulatory role -- for instance, it appears to bind immature 30S ribosomal subunits to prevent them from entering the translation cycle. Proteins that play a regulatory role typically don't have to be expressed as much.
    • Expression Level References and Description: Medium expression levels from the E Coli proteomic dataset (451 copies / cell). I also used [EcoCyc] and the references that it links, like [this one].
  • Expression Time: Early
  • Expression Time Hypothesis: Because it is potentially involved in both ribosome biogenesis and 30S subunit regulation, it plays a crucial role for the ribosome to function properly. Since ribosomes are the means by which proteins are made, it must be expressed early. Also it is considered an "ancient gene" and almost universally conserved, indicating its prime importance and thus its need to be expressed early on. Because of it's regulatory role, I thought the overall expression time was "early" rather than "right at the beginning" (i.e. there is not concrete evidence saying that it is required for ribosome biogenesis, so it doesn't have to be right away).
    • Expression Time References and Description: The [BioCyc page] and basic biology.

Gene Context

  • Other Components: This module only has one component in the sense that the rsmA protein product is an functional enzyme itself (i.e. it doesn't need any other subunits). Enzymes that perform related functions of rRNA methylation are rsmH and rmsG.
  • Possible Dependencies: Era modifies rRNA precursors into 16S rRNA. Therefore, it helps produce the rRNA which ksgA methylates.
  • Reactions: What reaction(s) does the functional module catalyze? Specify one reaction the module performs.
  • Process: rRNA methylation
    • Inputs: 4 S-adenosyl-L-methionine, adenine at position 1518, adenine at position 1519
    • Outputs: 4 S-adenosyl-L-homocysteine, N6-dimethyladenine at position 1518, N6-dimethyladenine at position 1519
    • Resource: [EcoCyc]

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