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Central dogma of molecular biology • Settled opinion • Principle or tenet • Doctrine laid down with authority Central dogma of molecular biology Central dogma of molecular biology Central dogma of molecular biology Nucleic acids Central dogma of molecular biology amino acids Stuff responsible for hereditary information • Must be stable – Information that makes life GO has stood the test of time (evolution) • Must replicate accurately – Most mutations are deleterious • Must be capable of change – Without change (something of a lottery) there would be no variability, and no evolution DNA (and RNA) are nucleic acids A, G, C, T/U • polymers consisting of monomers termed nucleotides. • nucleotides: a molecule composed of: – a pentose sugar – a phosphate group – and an organic molecule called a nitrogenous base. Polynucleotide Phosphodiester Backbones Mb long “Bases” Adenine Thymine Guanine Cytosine 3’! 5’! Phosphodiester Bonds Note 5’- 3’Orientation Bases: DNA 34 5 2 1 6 6 5 1 2 3 4 7 8 9 Pyrimidines (one ring, long name) Bases: DNA Bases: DNA Purines (two rings, short name) Chargaff’s Rules • Base composition varies among species • BUT: there are regularities. • The amount of guanine always equals the amount of cytosine. • The amount of thymine always equals the amount of adenine. • The amount of adenine + guanine = 50% of the total. (So 50% of the bases are purines). • The amount of cytosine + thymine = 50% of the total. (So 50% of the bases are pyrimidines). Using Chargaff’s rules • The % of A in DNA isolated from human liver is observed to be 30%. What is the expected percentage of • (a) T • (b) G • (c) C Using Chargaff’s rules • The % of A in DNA isolated from human liver is observed to be 30%. What is the expected percentage of • (a) T Because the amount of A=T, then amount of T should be ~30%. • (b) G • (c) C G+C together make up the remainder: 100% - 30% -30% = 40%, divided by 2 = 20% 20% Base pairing The DNA molecule is a helix • X-ray crystallography revealed a helical structure. • This beautiful image was taken and analyzed by Rosalind Franklin. Book alert! Aha! • Watson and Crick formed a great intuitive partnership. • They depended on A LOT of other work. • They could see the forest for the trees. DNA structure 5’ → 3’ 5’ → 3’ DNA structure 5’ → 3’ 5’ → 3’ DNA structure 5’ → 3’ 5’ → 3’ Bases: DNA Bases: RNA DNA nucleotides RNA nucleotides RNA nucleotides Compare DNA and RNA • Why 2’ OH in RNA? • Why is RNA single stranded? • Is RNA always single stranded? • Why Uracil instead of Thymine in RNA? RNA structure • Inherently unstable because the 2’-OH group is reactive, and so the backbone of RNA breaks down pretty easily • Except where RNA is a genome (some viruses) RNA molecules are shorter than DNA, and transient. RNA structure • RNA molecules are less stable BUT can make some pretty fancy secondary structures (by internal complementarity) that can be catalytic. RNA structure • Why the 2’-OH in RNA? – RNA is transient; the stored information is stable (DNA) but the expressed information is transient (RNA) – because you don’t need all the information all the time… • Is RNA always single stranded? – No! Sometimes it can form very elaborate secondary structures, which can even be catalytic! In other words, you don’t always need protein to catalyze things…maybe once upon a time, information storage, information propagation and information expression were all carried out by the same molecule (“RNA world”) – The 2’-OH increases the stability of of an intra-stand duplex by locking it into a more compact A-from helix and may also be important in RNA-protein interactions • Why Uracil instead of Thymine in RNA? – …later…. RNA function DNA RNA mRNA tRNA protein rRNA Central dogma of molecular biology Nucleic acids Central dogma of molecular biology Permanent chemical carrier of genetic instructions GENOTYPE Nucleic acids Short-term copy of genetic instructions Genetic information is copied over (transcribed) Everything is protein, or is made by protein: PHENOTYPE Translation from one chemical language to another DNA replication (synthesis) • Double stranded anti-parallel molecule. • Know the structure of the bases in the context of a nucleotide (deoxyribose nucleic acid). • Understand how DNA structure differs from RNA structure, and why. • Learn how DNA is replicated. • Learn how DNA is proof-read. • What kinds of errors can occur? • What are the consequences of these errors? How does DNA replicate? Semi-conservative model The DNA double helix is anti-parallel It can be opened up to create templates for copying • Single stranded DNA can serve as a template for highfidelity replication. • Also as a template for making mRNA (transcription). Cell division would go nowhere without DNA replication Cell division would go nowhere without DNA replication Cell division would go nowhere without DNA replication One replicated chromosome (consisting of two sister chromatids) One (unreplicated) chromosome 5’-AAAGGCTGATCA-3’! ’ ’! 5 -AAAGGCTGATCA-3 3’-TTTCCGACTAGT-5’! 3’-TTTCCGACTAGT-5’! S phase 5’-AAAGGCTGATCA-3’! 3’-TTTCCGACTAGT-5’! Mitosis, meiosis I Chromosomes are LONG pieces of DNA Origins of replication: multiple points along a chromosome where replication begins Origins of replication: multiple points along a chromosome where replication begins Replication has to plough through chromatin Level 1: nucleosome formation Level 2: 30 nm fiber Level 3: Nuclear scaffolding Level 4: Mitotic (metaphase) chromosome What do you need for DNA replication? • • • • Template Enzymes Primer dNTP’s (d=dexyribose, N= base A,T,G,C) • Energy Proteins for DNA replication: Single-stranded binding proteins • Proteins that stabilize the unwound single stranded DNA Enzymes for DNA replication: Helicases • DNA helicases. • Enzymes that unwind the DNA double helix for DNA replication • proteins bind to specific DNA sequences. • Dna/A, Dna/B, Dna/C. Enzymes for DNA replication: Primase • Primase: provides a short, complementary strand of RNA that is required for DNA synthesis from a naked DNA template. • There is no known DNA polymerase that can initiate synthesis of a DNA strand – they can only add nucleotides to a pre-existing strand. • Don’t confuse a template with a primer. DNA polymerase needs the 3’OH Enzymes for DNA replication: DNA polymerase III • DNA polymerase III • The enzyme that adds complementary nucleotides to the backbone, based on the sequence of the single stranded template. • Can only work 5’ to 3’. Enzymes for DNA replication: DNA polymerase III • Directionality of synthesis due to polarity of bond: incoming dNTP needs 3’OH to react with high energy phosphate bond. Enzymes for DNA replication: DNA polymerase III • Directionality of synthesis due to polarity of bond: incoming dNTP needs 3’OH to react with high energy phosphate bond. Energy for reaction carried on 5’ triphosphate of incoming free nucleotide Enzymes for DNA replication: DNA polymerase III Energy for reaction carried on 5’ triphosphate of incoming free nucleotide • Directionality of synthesis due to polarity of bond: incoming dNTP needs 3’OH to react with high energy phosphate bond. Central dogma of molecular biology nucleic acids Transcription • RNA molecules shorter than genomes! (But there are such things as RNA genomes!) • transient • NO primer • Proofreading? Different types of RNA Other kinds of RNA: dsRNA, siRNA, microRNA, pRNA etcRNA (I made this one up…) HUGE burgeoning field… Central dogma of molecular biology amino acids Protein structure – very diverse Protein structure – why so diverse? • A protein is a chain of amino acids, and there are twenty amino acids (that we know of…). • Amino acids have unique electrostatic and chemical properties. • There are energetically favorable ways amino acid chains can fold… A chain of amino acids makes a polypeptide A chain of amino acids makes a polypeptide Polypeptide chains fold into higher order structures called proteins • DNA is nucleic acid. • RNA is nucleic acid. • Proteins are made up of amino acids. • How do you turn nucleic acids into amino acids???? The Genetic Code: nucleic acids spell amino acids with three letters The Genetic Code U’s instead of T’s The Genetic Code We’re in triplets! The Genetic Code Special spellings for START and STOP The Genetic Code More than one way of spelling an amino acid The code is degenerate The Genetic Code • Triplet, unpunctuated and non-overlapping. – Reading frame depends where coding starts. Triplets in RNA are codons. • Degenerate: most amino acids are specified by more than one codon. – In fact only Met and Trp are specified by a single codon. • Degeneracy is usually found only in the third nucleotide of the codon. • The code is unambiguous: no codon specifies more than one amino acid. – Exceptions: UAA, UGA and UAG. • The code is universal. – Some organelle variants, some nuclear variants. Where does the code fit in? DNA RNA mRNA tRNA protein rRNA tRNA’s match the right amino acid with the right codon Read about “wobble” Protein synthesis takes place in the ribosome http://www.youtube.com/watch?v=D3fOXt4MrOM&feature=related The Ribosome is a ribozyme!!! The Ribosome is a ribozyme!!! The Ribosome is a ribozyme!!! The Ribosome is a ribozyme!!! Reading frames Reading frames Reading frames The first methionine in bacterial translation is N-formylmethionine Where to start?