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.

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

DNA

DNA is a long polymer of simple units called 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.

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.

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.

A mis-match during the first cycle in binding the template DNA strand, 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.

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 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

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