Harvard iGEM 2005 Molecular Genetics A Quick Primer Yin Li

Yin LiHarvard iGEM 2005 The Central Dogma DNA (Genes) mRNA Proteins Transcription Translation

Yin LiHarvard iGEM 2005 DNA nucleotide: Single-stranded DNA (ssDNA): 5'-P vs. 3'-OH Double-stranded DNA (dsDNA) has antiparallel strands in a double helix. Watson-Crick pairing (H-bonds): Purines to pyrimidines: Adenine = Thymine Guanine  Cytosine DNA

Yin LiHarvard iGEM 2005 RNA RNA nucleotide: 2'-OH (ribose sugar) instead of 2'-H (as in deoxyribose) Uracil instead of thymine Three kinds: Messenger RNA: carry genetic information from DNA Ribosomal RNA: components of ribosomes, where protein synthesis occurs Transfer RNA: mediates pairing of mRNA information to amino acids

Yin LiHarvard iGEM 2005 Proteins Amino acid: Polypeptide: polymer of amino acids N-terminus vs. C-terminus 4 Levels of Structure: Primary Structure: amino acid sequence Secondary Structure: alpha helices & beta sheets Tertiary Structure: overall shape (conformation) of single polypeptide Quaternary Structure: multimeric (>1) proteins Conformation defines function. Amino Group Carboxyl Group Functional Group: Polar, non-polar

Yin LiHarvard iGEM 2005 Genetic Code Codon: a triplet of DNA or RNA nucleotides, encoding one amino acid Redundancy of the code More than one codon for each amino acid Last letter of the codon is often interchangeable (wobble) Special codons: Start codon (also methionine): ATG (AUG) Stop codons: TAA, TAG, TGA (UAA, UAG, UGA)

Yin LiHarvard iGEM 2005 DNA Replication Semiconservative replication 1. Initiation: A short RNA primer is first synthesized by an RNA polymerase (primase) 2. Elongation: dNTPs added to the 3'-OH by DNA polymerase 5' to 3' direction of elongation Leading strand continuously replicated Lagging strand discontinuously replicated 3. Proofreading

Yin LiHarvard iGEM 2005 Mutations Silent/synonymous: same amino acid Missense: new amino acid Nonsense: becomes stop codon Frame shift: insertion or deletion of bases s.t. the codon frame, when read from ATG, shifts

Yin LiHarvard iGEM 2005 Transcription 1. Promoter recognition RNA polymerase and transcription factors (TFs) need to bind to promoter region for transcription to occur Consensus sequences, e.g. TATA box 2. Chain initiation & elongation RNA synthesis begins at transcription start site, next to the promoter Again: 5' to 3' elongation 3. Termination Chain-termination sequence (often forms hairpin loop)

Yin LiHarvard iGEM 2005 mRNA Transcription produces a primary transcript Serves as mRNA in prokaryotes Processed in eukaryotes: 1. 5' 7-methyl-guanosine cap 2. 3' poly(A) tail 3. Intronic splicing (exons vs. introns) 5' and 3' untranslated regions (UTRs) Open reading frame (ORF) start at AUG and end in the first stop codon mRNA stability varies

Yin LiHarvard iGEM 2005 Translation: 1. Initiation: Prokaryotes: ribosome-binding site (RBS) on mRNA is recognized by small 30S ribosome Eukaryotes: initiation complex (including small 40S ribosome) latches onto 5' cap 2. Scanning (eukaryotes only): initiation complex scans 5'-3' for the start codon AUG 3. Beginning of synthesis: Prokaryotes: large 50S ribosome is recruited and nearby AUG is recognized Eukaryotes: initiation complex dropped, large 60S ribosomal subunit recruited 4. Elongation: tRNAs are sequentially recruited 5. Termination: stop codon is recognized, special tRNA carrying release factor (RF) binds with ribosome

Yin LiHarvard iGEM 2005 The Central Dogma Revisited: Information Transfer & Sites of Regulation Transcription RNA Processing mRNA Degradation Translation Promoter TAC ATG Stop TSSCTS AUG Stop5' UTR3' UTRORF Stop 5' 3' NC DNA mRNA Protein Template DNA Post-Translational Modifications

Yin LiHarvard iGEM 2005 Transcriptional Regulation Promoter strength depends on Consensus sequences within promoter (e.g. TATA box) Eukaryotes: enhancer and silencer regions Negative regulation: basal promoter activity ON, needs to be turned OFF. Inducible: repressor is normally active; inducer required to remove repression. Repressible: (apo)repressor is normally inactive; co-repressor required to activate repressor Positive regulation: basal promoter activity OFF; needs to be turned ON. Autoregulation: Positive: protein product induces further transcription Negative: protein product inhibits further transcription

Yin LiHarvard iGEM 2005 The E. coli lac Operon of Jacob & Monod The components of lactose metabolism are adjacent genes governed by a single promoter (lacP): lacZ: beta-galactosidase (hydrolyzes lactose) lacY: lactose permease (permits lactose entry) lacA: thiogalactoside transacetylase Inducible negative regulation: lacI: product is a repressor protein that normally binds to the operator (lacO) region of operon to inhibit transcription The lactose isomer allolactose and synthetic analogues (e.g. IPTG) binds to repressor and permits transcription Positive regulation: Cyclic AMP concentrations indirectly regulated by glucose metabolism (s.t. high [glucose] results in low [cAMP]) crp: cAMP receptor protein (CRP), which, when complexed with cAMP, binds to lacP and permits transcription. lacOlacPlacZlacYlacA lacI