MMSYN1 0833 paomart

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Author Information

Paola Martinez

Basic Information

  • ID: MMSYN1_0833
  • Name: rplI (polypeptide: 50S ribosomal protein L9)
  • Organism: JCVI-Syn3.0
  • Description:

Overview & Function: This protein is part of the 50S subunit of the ribosome (EcoCyc). It has been found to be homologous to the amino acid code of L9 proteins found in E. Coli and the chloroplasts of Arabidopsis and pea (PubMed). There is only one copy of it in each chromosome. L9 is considered to be a large ribosomal protein that is found in all eubacteria even though it is not essential to life. This protein has been found to be responsible for keeping track of the reading frame when there is a pause during the translation process. It is especially efficient in preventing "ribosomal hopping" during translation. Strains that do not contain this protein have higher indices of stalled ribosome, frameshifting, and stop-hopping (EcoCyc).

Location: This protein is found on the surface of the ribosome and binds to the 23S rRNA. It is the first reconstitution intermediate of its subunit. Sequence similarities have been found between this protein and other proteins which has led to it's inclusion in a family of ribosomal proteins (InterPro).

Proposed Models: Multiple models of L9 have been proposed. Some have the show that L9 is in contact with other ribosome subunits such as the 30S. Other show that it consists of N-terminal and C-terminal that are joined by alpha helical domains. Depending on the state of the tRNA there can be crosslinking of tRNA in the A site of the ribosome to L9.

Studies: There have been in depth studies on L9 and they have found that when this protein is removed or lost there is a decrease in translation fidelity. To perform these studies they identified pathways that were affected by L9 and identified which mutants of E. coli required L9 to sustain life. Studies have found a second type of mutations that are dependant on L9 that significantly reduce or inactivate the elongation factor P which is a prokaryotic protein translation factor that is necessary for the synthesis of peptide bonds in ribosomes (Wikipedia). Even further studies suggest that there are mutant cases in which L9 replenishes monosomes and lowers the amount of immature rRNA in 30S particles. Scientists have also found that L9 makes the 16S maturation in wild=type cells work much more effectively (LOPS).

  • DNA Length: 444 base pairs.
  • DNA sequence:

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

  • Amino Acid length: 147 amino acids.
  • Amino Acid sequence:

MKVIFLKDVPNQGKKNEIKEVSDGYARNYLLPNQLVKIATNNSIQTLKDHLKADQEEKELAKAQTKQIKKTLEELTLHFKLQTNDDKVFGSISSQDIVNQLKDVHRIEIDKKKFIHFKNINKIGINYVKVKLDFGIEAIIKIDVKEV

Function and Homologs

  • Product: 50S subunit of the ribosome involved in transcription fidelity.
  • Module: Part of the Ribosome (largest ribosomal protein).
  • Closest homologous proteins: Note that since the protein itself belongs to Mycoplasma mycoides, Mycoplasma mycoides was excluded from the search.

0833BLAST.png

    • 50S ribosomal protein L9 [Mycoplasma feriruminatoris], Max score: 277/Query Cover: 94% /E-Value: 3e-94 /Ident: 100%, WP_008363087.1
    • 50S ribosomal protein L9 [Mycoplasma capricolum], Max score: 275 /Query Cover: 100% /E-Value: 2e-93 /Ident: 94, WP_011387003.1
    • 50S ribosomal protein L9 [Mycoplasma leachii], Max score: 275 /Query Cover: 100% /E-Value: 2e-93 /Ident: 95%, WP_013447466.1
  • Equivalent E. coli: EG10870 (functional equivalent)
    • It's important to note that I tried blasting the protein sequence of MMSYN1_0833 with the NCBI website and selected "Escherichia coli (taxid:562)" as the organism with which I wanted to limit the search set and only came up with one hit called "Chain F, Crystal Structure Of A Streptomycin Dependent Ribosome From Escherichia Coli, 50s Subunit Of 70s Ribosome". This hit was the E. coli analog because it belong to the same super family, however, it is interesting that it had a very low query and identity scores (36% and 43% respectively). Because the query and identity scores where so low, I wanted to double check that the blast was right and instead look for a functional equivalent through research. After extensive research, I have determined RL9_ECOLI to be the analog if MMSYN1_0833 because I figured out that RL9_ECOLI performs the same function in E. coli as MMSYN1_0833 in Mycoplasma mycoides.

Expression

  • Expression Level: High
  • Expression Level Hypothesis: I selected a 'high' level of expression even though its expression level information was not on the spreadsheet. I chose this expression level because this protein is a ribosome protein. Thus, it's probably really important because it's a necessary component of the ribosome and the ribosome is in charge of protein synthesis! If it's very important, its expression level must be high.
  • Expression Level References and Description: Unfortunately, expression level information was not available for this gene in UniProt or EcoCyc. However, I discussed what expression level was most adequate with a TA during Office Hours and then corroborated this information by researching and finding the information in this book.
  • Expression Time: Right at beginning
  • Expression Level Hypothesis: We expect the gene will be expressed at the beginning because in order for anything else to happen in the cell we need translation to already be occurring. Since this is a ribosomal protein, without it, translation cannot occur. It seems logical to have all the ribosomes created right at the beginning so that you can later on make everything else at a faster rate (the more ribosomes that you have, the more efficient your cell will be).
  • Expression Time References and Description: The ribosome is essential for translation which begins to occur during interphase. Interphase happens right at the beginning. After talking to TAs in OH and discussing possibilities, I concluded that the expression of this protein should be at the beginning so that the cell can properly develop.

Gene Context

  • Other Components: L6_bact: ribosomal protein L6 MMSYN1 0656
  • Possible Dependencies: The ribosome depends on RNA polymerase because if RNA polymerase doesn't translate the DNA into mRNA, the ribosome won't have any codons to translate to create proteins.
  • Process: Translation Elongation (specifically transcription fidelity)

Construct

  • Synthesis Score: The synthesis score of your construct: 1, 2,3
  • Predicted Translation Rate: Prediction of construct translation rate from the RBS calculator
  • Design Notes and Details: For example, had to use a rare codon to fix folding energy;
  • GenBank File: A link to the GenBank file. file