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

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.DNA promoterDNA genetranscribed In our case, the riHC (nucleoside hydrolase) enzyme is produced during translation.

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

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.

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.

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

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.

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.

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

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

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.

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

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

Genetic code -> AA code

One letter code

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