Biological Dynamics Group

1. Biological Dynamics Group Central Dogma: DNA->RNA->Protein

2. Genes I • Genes consist of DNA that contains a promoter, a DNA sequence that enables a gene to be transcribed , and a coding sequence, which determines what the gene produces. • In our case, the riHC (nucleoside hydrolase) enzyme is produced during translation.

3. DNA • DNA is a long polymer of simple units called nucleotides.

4. DNA bonding • Phosphodiester bonds (covalent) • Between 3’OH and 5’phosphate on the adjacent nucleotide • Complementary (noncovalent) bonding between bases • A hydrogen bonds with T (or U in RNA). • G hydrogen bonds with C. • Forming a double helix with antiparallel strands • We use restriction enzymes to cut the riHC DNA.

5. Genetic (DNA) Elements • Genome • Chromosome • E. coli has one. • Extrachromosomal genetic elements • Plasmids • Usually small, circular dsDNA (10kbp) • We can cut a plasmid and insert the rihC gene by a ligation reaction.

6. On the way to protein! • The plasmid must have a promoter sequence recognized by RNA polymerase, which then initiates transcription. • DNA->RNA=transcription • The coding sequence is copied in a process called transcription, producing a RNA copy of the gene's information. • This RNA can then direct the synthesis of proteins or translation via the genetic code.

7. RNA->Protein=Translation • RNA that will be translated into protein is called messenger RNA (mRNA). • There are two other types of RNA that are important to translation. • tRNA transfers the next amino acid (aa) to the growing peptide chain. • rRNA is part of the ribosome complex.

8. Translation and Mutations • The ribosomes “read” triplets of the mRNA. • The genetic code is a triplet code. • If a change (mutation) occurs in the DNA code, then a different aa may be incorporated into the protein chain. • To change the active site of rihC we have engineered specific point mutations. • A point mutation is a single base change.

9. PCR • We engineer point mutations using the polymerase chain reaction (PCR) • PCR is DNA replication in vitro. • dsDNA is “melted” • ssDNA primers bind to C’ regions • DNA polymerase extends the template • Repeated through 30 cycles

10. Site-directed mutagenesis • The primers containing the desired mutation are utilized. • A mis-match during the first cycle in binding the template DNAstrand, after that first round occurs. • After successive cycles, the mutated strand would exponentially grow, and after 25 cycles, would outnumber the original 8 million : 1.

11. Ligation and Transformation • The mutated PCR product is then ligated into a linearized plasmid with C’ ends via the enzyme DNA ligase that forms phosphodiester bonds. • The ligated DNA is then transformed into a special strain of E. coli that will express the mutated rihC gene.

12. rihC Nucleic Acid sequence 27293..28207 • 27241 taagttatgc gaaaatgccg gtcttgttac cggcattttt tatggagaaa acatgcgttt • 27301 acctatcttc ctcgatactg accccggcat tgacgatgcc gtcgccattg ccgccgcgat • 27361 ttttgcaccc gaactcgacc tgcaactgat gaccaccgtc gcgggtaatg tctcggttga • 27421 gaaaactacc cgcaatgccc tgcaactgct gcatttctgg aatgcggaga ttccgctcgc • 27481 ccaaggggcc gctgtgccac tggtacgcgc accgcgtgat gcggcatctg tgcacggcga • 27541 atcgggaatg gctggctacg actttgttga gcacaaccga aagccgctcg ggataccggc • 27601 gtttctggcg attcgggatg ccctgatgcg tgcaccagag cctgttaccc tggtggccat • 27661 cggcccgtta accaatattg cgctgttact ttcacaatgc ccggaatgca agccgtatat • 27721 tcgccgtctg gtgatcatgg gtggttctgc cggacgcggc aactgtacgc caaacgccga • 27781 gtttaatatt gctgccgatc cagaagctgc tgcctgtgtc ttccgcagtg gtattgaaat • 27841 cgtcatgtgc ggtttggatg tcaccaatca ggcaatatta actcctgact atctctctac • 27901 actgccgcag ttaaaccgta ccgggaaaat gcttcacgcc ctgtttagcc actaccgtag • 27961 cggcagtatg caaagcggct tgcgaatgca cgatctctgc gccatcgcct ggctggtgcg • 28021 cccggacctg ttcactctca aaccctgttt tgtggcagtg gaaactcagg gcgaatttac • 28081 ctcaggcacg acggtggttg atatcgacgg ttgcctgggc aagccagcca atgtacaggt • 28141 ggcattggat ctggatgtga aaggcttcca gcagtgggtg gctgaggtgc tggctctggc • 28201 gtcgtaacct

13. Primers for site-directed mutagenesis • 14 -rihC F-ATA CTG ACC CCG GAA TTG CCG ATG • rihC R-CAT CGG CAA TTC CGG GGT CAG TAT • 15 -rihC F-ATA CTG ACC CCG GAA TTG CCG ATG • rihC R-GGC GGC GTC AAT TCC GGG GTC AGT AT • 164-rihC F-CGC CGA GGC TAA TAT TGC TGC • rihC R-GCA GCA ATA TTA GCC TCG GCG • 222-rihC F-CAC TAC GCT AGC GGC AGT ATG • rihC R-CAT ACT GCC GCT AGC GTA GTG • 233-rihC F-TGC GAA TGG CCG ATC TCT GCG • rihC R-CGC AGA GAT CGG CCA TTC GCA • 234-rihC F-TGC GAA TGC ACG CCC TCT GCG • -rihC R-CGC AGA GGG CGT GCA TTC GCA • 241-rihC F-ATC GCC TGG GCG GTG CGC CCG GA • -rihC R-TCC GGG CGC ACC GCC CAG GCG AT • 242-rihC F-ATC GCC TGG CTG GCG CGC CCG GA • -rihC R-TCC GGG CGC GCC AGC CAG GCG AT

14. Genetic code -> AA code

15. One letter code

16. rihC Amino Acid sequence • 0 MRLPIFLDTD PGIDDAVAIA AAIFAPELDL QLMTTVAGNV SVEKTTRNAL QLLHFWNAEI • 61 PLAQGAAVPL VRAPRDAASV HGESGMAGYD FVEHNRKPLG IPAFLAIRDA LMRAPEPVTL • 121 VAIGPLTNIA LLLSQCPECK PYIRRLVIMG GSAGRGNCTP NAEFNIAADP EAAACVFRSG • 181 IEIVMCGLDV TNQAILTPDY LSTLPQLNRT GKMLHALFSH YRSGSMQSGL RMHDLCAIAW • 241 LVRPDLFTLK PCFVAVETQG EFTSGTTVVD IDGCLGKPAN VQVALDLDVK GFQQWVAEVL • 301 ALAS