IntroductionNucleotide to Cells & Microscopy and Nucleic Acid Structure Adapted from Prof. Dean Tolan • Outline of today’s supplemental lecture • Nucleotide and nucleic acid structure • Central Dogma • Replication • Transcription • Translation • Quiz at the end of the lecture Structural Components of Nucleotides Glycosidic bond Table 3-1 Nucleic acid – polymer of nucleotides – directionality 5’3’ When you write a sequence: ATCG It is assumed that the 5’-end is on the left and the 3’-end is on Phosphodiester bond the right, unless otherwise labeled. 5’-ATCG-3’ Chargaff’s Rules B-Form DNA http://higheredbcs.wiley.com/legacy/college/voet/0470129301/kinemages/exercise_2.html Figure 3-8 Computer-simulated space-filling model of DNA. Video: Computer-simulated space-filling model of DNA. SUMMARY (34 Å) Right-handed, antiparallel, double- sugar–phosphate stranded helix. With the “base backbone complementarity,” it explains genetic material: (phosphodiester • Storage of genetic information bonds) • Replication • Information retrival IntroductionCentral to Cells Dogma & Microscopy of Molecular Biology From DNA to Protein: Gene Expression • Central Dogma: from Genes to Proteins • Replication of the genes (DNADNA) • Transcribing the information (DNARNA) • Translating the nucleotide sequence into protein sequence (RNAProtein) – The Genetic Code – Protein Biosynthesis Central Dogma The central dogma of molecular biology Replication Information Flow Replication DNA replication is semiconservative (Meselson-Stahl Expt) DNA Replication Arthur Kornberg showed that DNA contains information for its own replication. He combined in a test tube: DNA, the four deoxyribonucleoside triphosphates (dNTPs–monomers), DNA polymerase, salts (Mg+2), and buffer. The DNA served as a template for synthesis of new DNA. Each New DNA Strand Grows from Its 5´ End to Its 3´ End Each New DNA Strand Grows from Its 5´ End to Its 3´ End ALL polymerases add nucleotides to the 3’ end (Direction is termed 5’ 3’) Pyrophosphatase 34 Transcription Central Dogma The central dogma of molecular biology Replication Messenger RNA (mRNA) Transfer RNA (tRNA) Ribosomal RNA (rRNA) • What is the relationship between a DNA sequence and an amino acid sequence? Central Dogma RNA is key to this process: • Messenger RNA (mRNA)—carries copy of a DNA sequence to site of protein synthesis at the ribosome • Transfer RNA (tRNA)—carries amino acids for polypeptide assembly • Ribosomal RNA (rRNA)—catalyzes peptide bond formation and provides structure for the ribosome Transcription Transcription components: • A DNA template for base pairings—one of the two strands of DNA • Nucleoside triphosphates (ATP,GTP,CTP,UTP) as substrates • An RNA polymerase enzyme Transcription process: • RNA polymerase unwinds DNA about ten base pairs at a time; reads template in 3’ to 5’ direction, synthesizes RNA in the 5’ to 3’ direction. • The RNA transcript is antiparallel to the DNA template strand, and adds nucleotides to its 3’ end. • NTPs incorporate NMP and PPi is a product! Transcription • Production of mRNA transcript by RNA polymerase Transcription: Where to start? 5' FlankingCoding Region 3'-flanking prokaryotes Promoter eukaryotes The consensus sequence for each element in human genes (N is any nucleotide) Transcription Translation Central Dogma The central dogma of molecular biology Replication Messenger RNA (mRNA) Transfer RNA (tRNA) Ribosomal RNA (rRNA) • What is the relationship between a DNA sequence and an amino acid sequence? • The Code • The Adaptors (tRNA) • The Ribosome (rRNA + rProteins) Translation: The Genetic Code The genetic code: Specifies which amino acids will be used to build a protein Codon: A sequence of three bases—each codon specifies a particular amino acid. Start codon: AUG—initiation signal for translation. Stop codons: UAA, UAG, UGA—stop translation and polypeptide is released. Translation: The Genetic Code The genetic code is redundant. The genetic code is universal. Translation: tRNA tRNAs must deliver amino acids corresponding to each codon The conformation (three-dimensional shape) of tRNA results from base pairing (hydrogen bonding) within the molecule. 3‘-end is the amino-acid attachment site—binds covalently. At the other end (middle of the tRNA sequence) is the Anticodon—site of base pairing with mRNA. Unique for each species of tRNA. Translation: tRNA Translation: tRNA Template for mRNA – read 3’5’ tRNA anticodon NC Translation: Ribosome Ribosome: the workbench—holds mRNA and charged tRNAs in the correct positions to allow assembly of polypeptide chain. Ribosomes are not specific, they can make any type of protein. Translation: Protein Biosynthesis: Ribosome Structure Ribosomes have two subunits, large and small. When not active in translation, the subunits exist separately. • The small subunit (40S) has one ribosomal RNA (rRNA) (18S) and 33 proteins. • The large subunit (60S) has three molecules of rRNA (28S, 5.8S, 5S) and 49 different proteins. • Ribosomal subunits are held together by ionic and hydrophobic forces (not covalent bonds) (80S). Translation: Ribosome Translation: Protein Biosynthesis; Elongation EF-Tu GTP Decoding (GTP hydrolysis) Peptidyltransferase Translation: Protein Biosynthesis; Elongation ELONGATION Translocation (GTP hydrolysis) Central Dogma The central dogma of molecular biology Replication Animated videos of DNA structure and Central Dogma.