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DNA Molecular Biology of the Gene Genes • biological blueprints • give us attributes & traits • every nucleus, in every cell carries genetic blueprint • every cell has all information needed to make a complete you • genes are located on chromosomes • humans have 46 • each containing thousands of genes Genes • share genes with all living organisms • 98% match chimpanzees • 99.9% match all other humans • differences exist at particular sites • causes each of us to be unique • differences maybe as small as one base substitution in one gene Genes & DNA • genes are made of DNA – deoxyribonucleic acid • macromolecule • made of 4 different nucleotides • paired in a precise manner • order of nucleotides is genetic code • each 3 combinations of nucleotides = one amino acid • DNA gives instructions to make proteins • smallest chromosome-Y has 50 million nucleotides • largest has 250 million DNA • nucleic acid • macromolecule composed of smaller subunits –nucleotides • contains • carbon sugar-deoxyribose • nitrogenous base • 1-3 PO4 groups • contains 4 different nucleotides • each with different nitrogenous base • bases are found in 2 major groups • Purines – double ring structures – adenine (A) – guanine (G) • Pyrimidines – Single ring structures – thymine (T) – cytosine (C) DNA NUCLEOTIDES Sugar-Phosphate Backbone • bases are linked via dehydration synthesis into phosphodiester bonds • phosphate of one nucleotide covalently bonds to sugar of next • forms sugar-PO4 backbone • nitrogenous bases are arranged as appendages along backbone Sugar-Phosphate Backbone DNA • structure determined by Watson and Crick-1953 • discovered DNA is double stranded helix • composed of two strands • wrapped around each other in helical formation • core -bases of one DNA strand bonded to bases in other strand • if think of DNA molecule as ladder – sugar-phosphate backbone would be sides of ladder – paired bases would be rungs DNA • base pairing is specific • A-T • G-C • amount of A = amount of T • one strand is complementary to the other Replication • cells divide & reproduce daily • giving rise to 2 daughter cells • with same genetic makeup • Before cell can divide, DNA must duplicate • replication • uses template mechanism Replication • to replicate • strands of DNA must separate • double helix unwound by helicase – breaks H bonds between base pairs REPLICATION • unwinding takes place in a replication bubble • new strand of DNA is formed in both directions on both strands of DNA in bubble Replication • proceeds in both directions • DNA strand has 3’ end & 5’ end • at one end carbon 3 of sugar is attached to –OH group • at other end carbon 5 is attached to a phosphate group • DNA polymerase – enzyme that binds single nucleotides into new strand of DNA – works only in 3' to 5' direction • consequently DNA synthesis only occurs in 5' to 3' direction • means one daughter strand can be made as continuous strand – leading strand • other is made in short pieces • linked together with DNA ligase – lagging strand REPLICATION • each strand of DNA is used as template to make new, complementary strand • semi-conservative replication REPLICATION • at completion of process 2 DNA molecules have been formed each identical to original • one strand of each of new DNA molecules is strand of original DNA • other strand is complementary strand made during replication • semi conservative replication PHENOTYPIC EXPRESSION • small sections of chromosomes are genes • genetic makeup is genotype • expression of genes into specific traits is phenotype – result of proteins • one geneone protein • protein production is directed by DNA Expression of Genotype • protein production is dictated by DNA • information about specific proteins is transferred to another nucleic acid-RNA • RNA is translated into a protein Genetic Code • DNAmRNAproteins • Proteins are long strands of amino acids held by peptide bonds • each has unique amino acid sequence • language of DNA is chemical • must be translated into different chemical languagethat of polypeptides • DNA language is written in linear sequence of nucleotide bases that comprise itAACCDDGGGACAC • specific sequence of bases makes up a gene glu lys ser ala met phe leu glu Expression of Genotype • transfer of information from DNA to RNA and then to proteins takes place in two processes • Transcription • Translation Transcription • DNA directs ribonucleic acid synthesis • transfers genetic information from DNA to RNA RNA • made of monomers or nucleotides – ribonucleotides • same basic components as DNA • single strand • 5 C sugar-ribose • phosphate groups • nitrogenous bases – same as in DNA with one exception • RNA has Uracil (U) instead of T • base pairing rules are same • Uracil is substituted for thymine • U-A not T-A Types of RNA • Messenger – mRNA • Ribosomal – rRNA • Transfer – tRNA • all involved in translation Transcription • DNAmRNA • nucleic acid language of DNA is rewritten as sequence of RNA bases Transcription • process of transferring genetic information from DNA to RNA • similar to DNA replication • DNA is used as template to make RNA Transcription • stands of DNA must separate • only one serves as template • nucleotides take their places one at a time along template using same base pairing rules as replication except AU • 3 stages • Initiation • Elongation • Termination Initiation • RNA polymerase attaches to promoter – specific nucleotide sequence • RNA synthesis begins • RNA polymerase decides which strand to use as template • strand used- antisense strand • other stand-sense strand Elongation • RNA strand grows longer • RNA strand peels away from template allowing separated DNA strands to come back together • bases are added at 50/second • RNA strand formed is directly complementary to its DNA template • each time C is found in antisense strand of DNA template a G is paired with it Termination • RNA polymerase reaches special sequence of bases in templateterminator • ends transcription • RNA polymerase detaches Post-transcriptional Modifications • in prokaryotic cells RNA can function immediately • in eukaryotes RNA is processed before moving to cytoplasm for translation • post-transcriptional modifications • capping-tailing • splicing • ligation Capping-Tailing • nucleotides are added to either end of RNA • a “G” nucleotide might be added to one end • A nucleotides might be added to other • additions make RNA more stable • ends protect molecule from attack by enzymes • helps ribosomes recognize mRNA Splicing & Ligation • precursor mRNA contains exons & introns • exons – segments containing information for formation of proteins • Introns – internal non-coding regions • before mRNA can leave nucleusintrons must be removed from strand • Introns are spliced out • exons are ligated (or attached) together • RNA can now move to cytoplasm through nuclear membrane pores Translation • conversion of nucleic acid language into protein language • proteins are macromolecules-polymers of amino acids • 20 are common to all organisms • sequence of nucleotides in mRNA dictates sequence of amino acids in polypeptide • sequence of bases in a molecule of DNA is genetic code GENETIC CODE • DNA & RNA are made of 4 different nucleotides • there are 20 amino acids • if each nucleotide coded for one amino acidcould only be 4 amino acids • if each 2 coded for onecould be 16 amino acids • smallest number of bases that can code for 20 amino acids is 3 • particular triplet of nucleotides in mRNA is a codon – specific for a particular amino acid • 64 possible triplet codes • code is redundant – more than one codon for each amino acid Codons • 61 code for amino acids • some have regulatory purposes – start & stop translation • AUG-start codon – codes for METmethionine • UAA, UAG, UGA- stop codons – tell ribosomes to end polypeptide synthesis Genetic Code • highly conserved • same in all organisms • genes can be transcribed & translated even if transferred from one species into another • opened door for genetic recombinant technology & genetic engineering Translation • amino acids are not able to recognize codons of mRNA • requires an interpreter – intermediate that can understand language of one form & translate that message into another • tRNA (transfer RNA) is interpreter • pick s appropriate amino acid & recognizes appropriate codon in mRNA • converts 3 letter code of nucleic acids into amino acidsproteins tRNA • • • • • • • • • • • structure allows it to match correct amino acids to mRNA sequence tRNA is composed of one strand of RNA chain twists & folds on itself making some double stranded areas one end-special triplet of basesanticodon contains complementary sequence of bases to sequence of bases in mRNA recognizes bases in mRNA by applying standard base pairing rules other end is site where amino acid can attach enzyme recognizes both tRNA and its amino acid partner there are at least 32 different tRNA in eukaryotic cells anticodons are redundant there is at least one anticodon for each amino acid Translation • ribosomes coordinate process of translation • ribosomes are formed from 2 subunits each made of proteins & rRNA (ribosomal RNA) • completely assembled ribosome has binding site for mRNA on its small subunit & two binding sites for tRNA on its large subunit Translation Stages • Initiation • Elongation • Termination Initiation • mRNA molecule binds to small ribosomal subunit • special initiator tRNA binds to specific codon-AUG – start codon • anticodon is UAC • start codon also carries amino acid methionine • next large ribosomal subunit binds to small one creating functional ribosome • initiator tRNA fits into one of two tRNA binding sites on ribosome called P site • other tRNA binding site-A site is vacant • P site holds tRNA containing growing peptide chain • A site holds tRNA carrying next amino acid to be added to chain Elongation • amino acids are added one by one to first amino acid • each addition is composed of 3 steps • First • anticodon of incoming tRNA carrying an amino acid pairs with mRNA codon in A site of ribosome Elongation • next peptide bond forms between carboxyl group of one amino acid & amino group of next • to do this polypeptide leaves tRNA in P site & attaches to amino acid on tRNA in A site • attached by a peptide bond • ribosome catalyzes bond formation Elongation • last stage-translocation • P site tRNA leaves ribosome • ribosome moves or translocates tRNA in the A site with its attached polypeptide to P site • movement brings next mRNA codon to be translated into A site • process begins again • elongation continues until stop codon is reached Termination • UAA, UAG & UGA are stop codons • when one of these sequences is detetectedpeptid e released from last tRNA • Ribosome splits back into its separate subunits Polysomes • single mRNA has many ribosomes traveling along it • Polysomes • in various stages of synthesizing polypeptide Mutations • any change in nucleotide sequence of DNA • production of mutations is mutagenesis • some are spontaneous • Some due to mutagens • radiation, chemicals & viruses • two categories – base substitutions – insertions & deletions Base substitutions • Point mutation – replacement of one nucleotide for another • may go unnoticed • may cause significant issues • hemophilia • sickle cell anemia • Huntingtons Chorea • Tay Sachs disease Insertion & Deletion • mRNA is read as a series of triplet codons during translation • adding or deleting one base will change reading frame for tRNA • Frame-shift mutations have dramatic effects • all nucleotides downstream from insertion or deletion will be regrouped into different codons • result is usually nonfunctional protein