Foundations of Molecular Biology Nucleotide and Nucleic Acid Structure

Nucleotide and Nucleic Acid Structure Introduction to Cells & Microscopy Structural Components of Nucleotides Base Sugar Phosphate Glycosidic bond H NUCLEOTIDE H 1 RNA DNA Table 3-1 Nucleic acid – polymer of nucleotides – directionality 5’à3’ When you write a sequence: ATCG It is assumed that the 5’-end is on the left and the 3’-end is on the right, unless otherwise labeled. Phosphodiester bonds 5’-ATCG-3’ 3’-GCTA-5’ same molecule Composition of DNA? 2 Chargaff’s Rules DNA is Double Stranded Helix 3 Figure 3-8 Computer-simulated space-filling model of DNA. 4 • The crucial piece of evidence for DNA structure came from X-ray “crystallography.” Wilkins learned how to purify DNA and make regular fiber patterns. Rosalind Franklin performed the X-ray diffraction and deduced there was a helix. • Francis Crick saw the data at a seminar Wilkins gave and also deduced there was a helix and the size parameters. • James Watson discovered how the bases went together (complementarity) using Chargaff rules (A=T, G=C). • Watson & Crick published their structure in 1953. Beautiful example of how structure predicted function. 5 SUMMARY (34 Å) sugar–phosphate backbone (phosphodiester bonds) Right-handed, antiparallel, doublestranded helix. With the “base complementarity,” it explains genetic material: • Storage of genetic information • Replication • Information retrival Central Dogma of Molecular Biology Introduction to Cells & Microscopy 6 From DNA to Protein: Gene Expression • Central Dogma: from Genes to Proteins • Replication of the genes (DNAàDNA) • Transcribing the information (DNAàRNA) • Translating the nucleotide sequence into protein sequence (RNAàProtein) – The Genetic Code – Protein Biosynthesis Central Dogma The central dogma of molecular biology Replication Information Flow 7 Replication DNA replication is semiconservative (Meselson-Stahl Expt) 8 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 9 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’ g 3’) Pyrophosphatase 34 Transcription 10 Central Dogma The central dogma of molecular biology Replication Messenger RNA (mRNA) Transfer RNA (tRNA) Ribosomal RNA (rRNA) 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 11 Central Dogma The central dogma of molecular biology Replication Transcription Messenger RNA (mRNA) Transfer RNA (tRNA) Ribosomal RNA (rRNA) 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! 12 Transcription • Production of mRNA transcript by RNA polymerase Transcription: Where to start? 5' Flanking Coding Region 3'-flanking prokaryotes Promoter eukaryotes The consensus sequence for each element in human genes (N is any nucleotide) 13 Transcription Translation 14 Central Dogma The central dogma of molecular biology Replication Translation 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) 15 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. 16 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 17 Translation: tRNA Template for mRNA – read 3’à5’ tRNA anticodon N C 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. 18 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 19 Translation: Protein Biosynthesis; Elongation EF-Tu GTP Decoding (GTP hydrolysis) Peptidyltransferase Translation: Protein Biosynthesis; Elongation ELONGATION Translocation (GTP hydrolysis) 20 Central Dogma The central dogma of molecular biology Replication 21