Ribosomal+RNA+Large+Subunit+Methyltransferase+Cfr+(Clostridium+botulinum)


 * *Target (protein/gene name): Ribosomal RNA Large Subunit Methyltransferase Cfr/ 23S rRNA (adenine2503-C8-methyltransferase) **
 * *NCBI Gene # or RefSeq#: 441771 **
 * *Protein ID (NP or XP #) or Wolbachia#: 2.1.1.224 **
 * *Organism: //Clostridium botulinum// (strain Hall / ATCC 3502 / NCTC 13319 / Type A) **


 * Etiologic Risk Group: 2 **


 * Disease Information: This bacteria releases a toxin that causes the disease, commonly known as botulism. There are five kinds of botulism infections, all of which can be fatal if not treated immediately: foodborne, wound, infant, adult intestinal, and iatrogenic. It is a rare disease in the United States, with an average of 145 cases per year. The most common forms are foodborne, infant, and wound botulism. Signs and symptoms include vision problems, muscle weakness, slurred speech and mouth dryness and in infants, poor muscle tone is common in those afflicted with the infant form of the infection. Botulism is prevented through proper food production and cooking methods. It can be treated with an emergency anti-toxin if an infection occurs. **

http://www.ncbi.nlm.nih.gov/protein/205829640/ http://www.uniprot.org/uniprot/A5I5U3 Is it a monomer or multimer as biological unit ** ?: ** **Monomer**
 * Link to TDR Targets page (if present): Not present **
 * Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.) **
 * Essentiality of this protein: Yes **
 * Complex of proteins?: No **
 * Druggable Target: This has been targeted in //Staphlococcus aureus// with gene erm, which is coexpressed with this protein (see BRENDA). **

http://www.brenda-enzymes.org/enzyme.php?ecno=2.1.1.224
 * *EC#:2.1.1.224 **
 * Link to BRENDA EC# page: **
 * -- ** Show screenshot of BRENDA enzyme mechanism schematic

2 × S-adenosyl-L-methionine ||~ + ||~ adenine(2503) in 23S rRNA ||~ + ||~ 2 × reduced [2Fe- 2S] ferredoxin ||~ = ||~ S-adenosyl-L-homocysteine ||~ + ||~ L-methionine ||~ + ||~ 5'-deoxyadenosine ||~ + ||~ 8-methyladenine(2503) in 23S rRNA ||~ + ||~ 2 × oxidized [2Fe- 2S] ferredoxin || ||
 * [[image:https://www.ebi.ac.uk/thornton-srv/databases/enzymes/templates/gif/1x1.gif width="5" height="1"]] || ||~ [[image:https://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/GetImage.pl?kegg_id=C00019]]

http://jb.asm.org/content/169/8/3857.full.pdf
 * Enzyme Assay information: **
 * New England Medical Corp. **
 * Reagents- MgCl2, KCl, Tris hydrochloride, ****10 mM dithiothreitol, 0.01 mM S-adenosyl-L-[methyl-3H]- methionine ([methyl-3H]AdoMet; 3.30 Ci/mmol), 10 pmol of B. subtilis 23S rRNA, and 5 p,l of methylase containing less than 10 ng of protein.**
 * Cost- $320.54 **

code format="sequence"  10        20         30         40         50 MKQTKTKYGK MKQIASNLKL PDYRYEQLTK AIFHQRIDNF HDMHILPKAL 60        70         80         90        100 RIALVNEFGK NVSSVTPIFS QDSKQAQKLL FELTDGERIE AVGLKYKQGW 110       120        130        140        150 ESFCISSQCG CSFGCRFCAT GSAGFKRNLT ADEITDQLLY FYFNDHRLNS 160       170        180        190        200 ISFMGMGEAF ANPELFDAVK ILTDQNLFGL SQRRITISTI GIIPGIQRLT 210       220        230        240        250 KEFPQVNLAF SLHSPFESQR SDLMPINKRF PLNEVMKTLD EHIIHTGRRV 260       270        280        290        300 FIAYIMLEGI NDSKEHAEAI IGLLRNRGSW EHLYHIDLIP YNSTDKTTFK 310       320        330        340 FQSSSAIKQF CSTLKKASIS ATVRTQFGSE ISAACGQLCY ENEL code TATTCATGATGTTGTGATTATCGGCTCAGGACCTGCTGCACATACTGCGGCTATTTATTTAG GAAGATCTTCATTGAAACCAGTTATGTATGAAGGGTTTATGGCAGGAGGAGTAGCTGCAGGAGGACAACT AACTACCACTACTATCATTGAGAATTTCCCAGGATTTCCAAACGGTATTGATGGAAATGAATTAATGATG GTATTAATGGTAGTTGGTGGAGGAGATGCAGCGATCGTATTGCGTATGCGATTTGGCCAAGTAGGCAACTGAGATCGCTATCGATGGTTGCATAGCA CTATGGAAGAAGCACTTCATTTAACAAAATATGGAAGTAAAGTTATTATTCTTCATCGAAGAGATGCTTT TAGGGCAAGTAAAACTATGCAAGAACGAGTATTAAATCATCCAAAGATTGAAGTAATATGGAATTCTGAA TTAGTAGAATTAGAAGGAGAT ATAGTCCAAATAGTAAATTCTTAGGTGGGATAGAGACTTGTGTGCTGGCAAGCTA ACAAGTTAAAACTGCTGATGATGGATATATTCTTACAGAAGGACCAAAAACTAGTGTTGATGGTGTATTT GCATGTGGGGATGTATGTGATAGAGTATATAGACAAGCTATTGTAGCTGCAGGAAGTGGTTGTATGGCAG CCTTAAGCTGTGAAAAATGGCTTCAAACTCATTAATTCAGTTTCTTTTTTAACA AACAAAATATGGAAGTAAAGTTATTATTCTTCATCGAAGAGATGCTTT TAGGGCAAGTAAAACTATGCAAGAACGAGTATTAAATCATCCAAAGATTGAAGTAATATGGAATTCTGAA TTAGTAGAATTAGAAGGAGAT ATAGTCCAAATAGTAAATTCTTAGGTGGGATAGAGACTTGTGTGCTGGCAAGCTA ACAAGTTAAAACTGCTGATGATGGATATATTCTTACAGAAGGACCAAAAACTAGTGTT
 * Structure (PDB or Homology model): 5HR6 **
 * Current Inhibitors: Erythromycin **
 * Expression Information (has it been expressed in bacterial cells): It has been expressed in the BL21Star //E.coli// strain. **
 * Purification Method : Recombinant His6-tagged Cfr from //E.coli// strain BL21Star by nickel affinity chromatography and gel filtration/C-terminally His6-tagged wild type enzyme and mutant enzymes C112A, C116A, and C119A **
 * Image of protein (PyMol with features delineated and shown separately): **
 * *Amino Acid Sequence (paste as text only - not as screenshot or as 'code'): **
 * Length of the protein in Amino Acids: 344 **
 * Molecular Weight of the protein in kiloDaltons: 39160.9 **
 * Molar Extinction coefficient of the protein at 280 nm wavelength: 26400 (assuming all pairs of Cys residues form cysteines) or 25900 (assuming all Cys residues are reduced) **
 * TMpred graph Image: **
 * *CDS Gene Sequence (paste as text only): **
 * *GC% Content for gene: 45% **
 * *CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only): **
 * *GC% Content for gene (codon optimized): 67% **

Do Not Need this info for Spring (but still copy these lines to your Target page for now) -- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
 * Primer design results for pNIC-Bsa4 cloning (list sequences of all of your ~40 nt long primers): **
 * ( link to DNA Works output text file - ** that should be saved in your Google Docs folder after you did the primer design protocol)

**
 * Primer design results for 'tail' primers (this is just 2 sequences): **