* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Biochemistry 6/e
RNA interference wikipedia , lookup
Agarose gel electrophoresis wikipedia , lookup
Promoter (genetics) wikipedia , lookup
Maurice Wilkins wikipedia , lookup
Community fingerprinting wikipedia , lookup
Expanded genetic code wikipedia , lookup
Biochemistry wikipedia , lookup
Molecular cloning wikipedia , lookup
RNA polymerase II holoenzyme wikipedia , lookup
Gel electrophoresis of nucleic acids wikipedia , lookup
Polyadenylation wikipedia , lookup
RNA silencing wikipedia , lookup
Silencer (genetics) wikipedia , lookup
Cre-Lox recombination wikipedia , lookup
Molecular evolution wikipedia , lookup
Eukaryotic transcription wikipedia , lookup
Transcriptional regulation wikipedia , lookup
Genetic code wikipedia , lookup
DNA supercoil wikipedia , lookup
Non-coding DNA wikipedia , lookup
Messenger RNA wikipedia , lookup
Gene expression wikipedia , lookup
Artificial gene synthesis wikipedia , lookup
Non-coding RNA wikipedia , lookup
Epitranscriptome wikipedia , lookup
Biochemistry I (CHE 418 β / 5418 Reading Assignment Berg et. al (2012) Chapter 4 Central Dogma of Molecular Biology Replication DNA Transcription RNA Translation Protein mRNA. tRNA, rRNA, snRNA Nucleus – Replication - DNA directed DNA synthesis – Transcription - DNA directed RNA synthesis · Processing of mRNA capping, polyadenylation, splicing • Cytoplasm – Translation - RNA directed Protein synthesis Functions of Nucleic Acids Building blocks of DNA and RNA – DNA = Genetic material – RNA = Adapter molecule between DNA and protein Transport chemical energy within the cell – ATP Signal molecule cyclic AMP Nucleic Acids • Nucleic Acid - linear, non-branched polymer of nucleotides Classes of Nucleic Acids – RNA = ribonucleic acid – DNA = 2' deoxyribonucleic acid Nucleotide • Nucleotide contains: – Pentose sugar – Nitrogenous base – Phosphate – One or more Adenosine Triphosphate (ATP) Nucleotide • Pentose sugar – Carbons are numbered with primes to differentiate between carbons / nitrogens of nitrogenous bases β-D-2-Deoxyribose Ribose DNA Only RNA Only Nucleotide • phosphate group Pyrimidine Bases Pyrimidine Thymine (T) 5-methyl-2,4-dioxypyrimidine Cytosine (C ) 4-amino-2-oxypyrimidine Uracil (U) 2,4-dioxypyrimidine DNA ONLY DNA and RNA RNA ONLY Purine Bases Purine Adenine (A) 6 – aminopurine Guanine (G) 2- amino-6-oxypurine DNA and RNA DNA and RNA Sugar Phosphate Backbone • Nucleotides connected by 3’ to 5’ phosphodiester bond – Imparts uniform negative charge to DNA / RNA • Negative charge repels nucleophilic species (e.g. hydroxyl) thus the phosphodiester bond resists hydrolytic attack. • Separation by agarose gel electrophoresis – Creates 3’ and 5’ end (directionality) • Convention: Nucleotide sequences are written 5’ to 3,’ L to R Bases are attached to sugar by Beta Glycosidic linkage • N-9 of purine and N-1 of pyrimidine Nucleotide Nucleoside = sugar + nitrogenous base, Nucleotide = sugar + nitrogenous base + phosphate. Adenosine (A nucleoside) Adenosine monophosphate (A nucleotide) What data did Watson and Crick use to determine the structure of DNA • • • • X ray diffraction photograph of DNA crystals Chargaff’s rules Bond angles from reference books Built models Erwin Chargaff’s “Rules” • Edwin Chargaff determined the composition of DNA from many organisms – [A] = [T] – [G] = [C] DNA is a Helix • X ray diffraction photograph – Maurice Wilkins and Rosalind Franklin – Two chains that wind in a regular helical structure. Watson and Crick (Complementary) Base Pairing G C A T Nucleotide content determines melting point of DNA. Double Helix • B form – Diameter of helix = 20.0 Å (2.00 nm) – 10.4 base pairs / turn; 34 Å (3.4 nm) – 1 base pair 3.4 Å (0.34 nm) • Note – – – – – Complementary base pairing Major grove Minor grove Antiparallel Hydrogen bonding between complementary base pairs. DNA held together by hydrogen bonding and Hydrophobic interactions • Hydrogen bonding between base pairs – 4 – 21 kJ / mol (1 – 5 kcal/mol) • Hydrophobic interactions (van der Walls) due to base stacking. – 2 – 4 kJ / mol (0.5 – 1.0 kcal / mol) Forms of DNA • B form – “Normal” form – Watson and Crick form • A form – “dehydrated” B form – nucleotide tilted 20o relative to helical axis • Z form (“zig zag”) – stretches of alternating purine / pyrimidines – base pairs flip 180o – Left handed helix DNA is Organized into Genes • Gene – discrete, functional unit of DNA – when expressed, (transcribed) yields a functional product • rRNA, tRNA, snRNA • mRNA - translated into a polypeptide sequence. – Open reading frame - long stretch of nucleotides that can encode polypeptide due to absence of stop codons. Chromatosomes Pack to Form Chromatin Fibers Histones H1, H2A, H2B, H3, H4 Histones contain (>20%) arg and lys ---basic amino acids Karyotype • Photograph of chromosomes from a single organism • Arranged by size (largest to smallest) • Homo sapiens – 46 chromosomes – 23 pairs • 3 billion base pairs (hapliod) • 25,000 genes Chromosome Contains • Centromere site that connects sister chromatids • Kinetochore attachment site of spindle to chromosome • Telomere - nucleotide repeat at end of linear chromosome – TTAGGG x 1000 – synthesized by telomerase Properties of DNA • Melt / Anneal / Reanneal • Hypochromic effects • Supercoiled / Relaxed dsDNA can Reversibly Melt • Heating DNA breaks hydrogen bonding between base pairs. – Acid or base also works • Tm = melting temperature – Half the helical structure is lost • Single stranded DNA absorbs light more efficiently than double stranded DNA Hypochromic effect (Hypochromism) • DNA can melt and then re-anneal. • If sequences are similar, they will reanneal or hybridize. DNA exist as Linear or Circular Molecules • Prokaryotic, Mitochondrial and Chloroplast genomes are circular – Circular molecules may exist in topological isomers • Relaxed • Supercoiled • Eukaryotic genomes are linear molecules Single Stranded Nucleic Acids can form complex structures • Stem Loops are produced by H-bonding between complementary regions in DNA and RNA. • Mismatches are observed Single Stranded Nucleic Acids can form complex structures • Hydrogen bonding stabilizes more complex structures. • Often observed in ribosomal RNA molecules Replication "It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material.” Watson, J.D. and Crick, F.H.C., Molecular Structure of Nucleic Acids: A Structure for Deoxyribose Nucleic Acid, Nature, 171, pp. 737-738, (1953). DNA may be labeled with • Grow E. coli in media containing 15NH4Cl and 14NH Cl. 4 • Purify DNA • Separate DNA using density gradient equilibrium sedimentation – CsCl gradient from 1.66 – 1.76 g / cm 15N Testing the Semiconservative Replication Hypothesis • Matthew Meselson and Franklin Stahl (1958) – Grew E. coli in 15NH4Cl until DNA was completely labeled. – Transferred E. coli to 14NH4Cl containing media. – Followed labeling pattern of DNA through several generations using density gradient equilibrium sedimentation DNA Replication • DNA directed DNA synthesis • DNA Polymerase – adds deoxyribonucleotide units to an existing DNA molecule in a template directed fashion in the 5’ to 3’ direction. • E. coli DNA Pol I isolated by Author Kornberg (1958) – DNA Polymerase requires • • • • Four dNTPs (dA,T,dG,dC) Divalent cation (Mg2+) Template DNA Primer provides 3’ OH DNA Polymerase Reaction Mechanism • Nucleophilic attack by the 3’ OH on the alpha phosphate group of dNTP • PPi (pyrophophosphate) is hydrolyzed to Pi + Pi (orthophosphate) Types of RNA • Types of RNA – – – – – Ribosomal RNA (rRNA)– part of the ribosome Transfer RNA (tRNA) Messenger RNA (mRNA)– sequence translated into protein sequence. Small nuclear RNA (snRNA) – involved in splicing (spliceosome) Micro RNA (mi RNA) – small RNA complementary to mRNA that inhibits translation of the mRNA – Small interfering RNA (siRNA) – small RNA that binds to mRNA causing destruction of mRNA Transcription • DNA directed RNA synthesis • RNA Polymerase – adds ribonucleoside triphosphate units to an existing DNA molecule in a template directed fashion in the 5’ to 3’ direction. – RNA Polymerase requires: • • • • • Four NTPs (A,U,G,C) Divalent cation (Mg2+) Template DNA NO primer required Lacks endo and exo nuclease activity One prokaryotic RNA Pol Three eukaryotic RNA Pol RNA Pol I RNA Pol II • RNA molecules are complementary mRNA to the DNA template. RNA Pol III Structure of RNA • Genes may or may not be transcribed depending on the needs of particular cell type. • gene is a functional region of DNA – expressed genes are “TURNED ON” – unexpressed genes are “TURNED OFF” RNA Polymerase Reaction Mechanism • Nucleophilic attack by the 3’ OH on the alpha phosphate group of NTP (ribonucleoside triphosphates) • PPi (pyrophophosphate) is hydrolyzed to Pi + Pi (orthophosphate) RNA molecules are complementary to the DNA template. • mRNA is complementary to template strand • mRNA is identical (except for U to T changes) to the coding strand. Prokaryotic Promotor – Pribnow box (also called TATA box) • 5’TATAAT 3’ centered at -9/-10 – designated by the 5’ to 3’ sequence on the NONtemplate strand » 8 to 10 nucleotides left (5’ or upstream) of transcriptional start site (designated +1 --- there is no 0 nucleotde) – -35 sequence • 5’TTGACA3’ centered -35 from Eukaryotic Promotor • Class II genes – those synthesized by RNA Pol II. • Pre mRNA and snRNA • Parts – TATA or Hogness box – GC box (GGGCGG) – CAAT box Transcriptional Termination • Rho dependent – Involves protein called Rho • Rho independent – Involves stem loop structure in mRNA – Stem loop is followed by UUUs mRNA • Prokaryotic mRNA are polycistronic – May encode two or more proteins • Eukaryotic mRNA are monocistronic – Encode only one protein • Eukaryotic mRNA are Posttranscriptionally Modified Capping – attachment of 7-methylguansine using 5’ to 5’triphosphate linkage • Polyadenylation – attachment of 40 to several hundred adenine nucleotides to 3’ end of mRNA • Splicing – removal of introns Amino acids are attached to 3’ end of tRNA • Aminoacyl-tRNA synthestase – attaches amino acid to tRNA Translation • Stages of Translation – Initiation • assemble and align ribosome, mRNA, and tRNAfMet – Elongation • template directed synthesis of proteins – Termination • termination factors halt protein synthesis • ribosome, mRNA and new protein dissociate • Orientation of Translation – Ribosomes move 5’ to 3’ along mRNA – Protein is synthesized N to C Genetic Code • Marshall Nirenberg, Har Gobind Khorana, Frances Crick • Specific- Unambiguous – specific codon always codes for SAME amino acid – Three nucleotides (codon) = one amino acid • 61 codons encode amino acids – Codons encoding one amino acid usually differ in the last base. • 3 codons encode stop codons (UAA, UAG, UGA) • Universal – conserved from species to species • main exception = mitochondria • Redundant (also called degenerate) – amino acid may have more than one codon • Nonoverlapping and comma less (no puctuation) – read from fixed starting point (AUG) – lacks punctuation between codons Codon Usage Table • mRNA are “read” three nucleotides (codon) at a time starting from a fixed point. Translational Start Site • AUG encodes Met (n-terminal amino acid). – Prokaryotes use a Shine-Dalgarno sequence to align a ribosome on the mRNA upstream ( 5’) of AUG – Eukaryotes use the 5’Cap to align the ribosome on the mRNA Mitochondrial Genetic Code differs from the Universal Code Eukaryotic mRNA contain Exons and Introns Philip Sharp and Richard Roberts (1977) • Exons – coding regions • Introns –noncoding regions – intervening sequences Introns were discovered by hydridizing mRNA to genomic DNA Splicing • Removal of introns • Spliceosome – specific proteins and small nuclear RNA. • Most introns start with GU and end with AG WHY UNDERSTAND TRANSLATION? • Many Antibiotics kill bacteria by inhibiting prokaryotic translation!!!