Chapter 12 The genetic code and transcription


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Chapter 12 The genetic code and transcription

  1. 1. 1 Chapter 12 The genetic code and transcription Flow of genetic information (Fig.12.1) 1. RNA 1) Characteristics of genetic code (mRNA) (1) Written in linear form with complementary nucleotide bases of DNA (2) Three ribonucleotides compose a 'codon', specifies one amino acid (a) 64 (43) possible codons (b) T4 mutation experiment (Fig.12.2) No frame shift from deletion/insertion of three nucleotides (3) One codon specifies only a single amino acid (4) The codon is degenerate – one amino acid can be specified by more than one codon (a) There is no blank codon except termination codons (b) Usually the last base is different (5) There are codons for start (initiate) and stop (terminate) translation (6) Commaless –no internal punctuation (no blank codon) (7) The code is nonoverlapping (a) If overlapping is true, adjacent amino acids would be limited (b) A point mutation affects only one amino acid (c) Ribosome has two sites for t-RNA (8) The code is nearly universal (exceptions: Table 12.5) 2) Study of codon (1) In vitro protein synthesis by Nirenberg et al. (1961) (a) mRNA synthesis (Fig.12.3) Ingredients: rNDPs and polynucleotide phosphorylase *Didn't use RNA polymerase *Don't need DNA template Product: mRNA (random sequence) (b) Protein synthesis from homopolymers Ingredients: Ribosomes, tRNAs, mRNA, and amino acids Produced homopolymers (UUUU..., AAAA..... GGGG.... CCCC....) UUU: Phenylalanine AAA: Lysine CCC: Proline *Didn't get result from GGG sequence (c) Protein synthesis from copolymers Production of copolymer with different ratio of rNDPs Predict different ratio of amino acid incorporation into protein sequence (Example) 1A : 5C ratio (Fig.12.4) (d) Outcome They were able to interpret the result up to a ratio level, but not specific sequence (2) Triplet binding assay by Nirenberg et al. (1964) (a) Able to produce a known triplet RNA sequence (b) Experimental method (Fig.12.5) Incubate ribosomes, triplets, and charged t-RNAs with nitrocellulose filter *Aamino acids were labeled radioactively
  2. 2. 2 Large complex of ribosome-triplet-tRNA would bind to nitrocellulose with high affinity Wash off unbound small molecules Exposure filter to film Radioactivity indicates formation (of triplet-tRNA complex) (c) Results (Table 12.2) 26 codons were assigned to 9 amino acids (d) Outcome Genetic code is degenerate Codon is specific for specific amino acid (3) Repeating copolymers by Khorana (a) Synthesis of repeating polymers (Fig.12.6) Repeating sequences of di-, tri-, and tetranucleotides (b) Results (Table 12.3) 18 more codons were assigned with corresponding amino acids Finding of possible termination codons 2. Coding dictionary (Fig.12.7) 1) General 61 codons are responsible for amino acids synthesis 1 codon is responsible for initiation of translation: AUG (methionine) 3 codons are responsible for termination of translation: UAA, UAG, and UGA 2) Wobble hypothesis by Crick (1966) (1) First two bases are more critical than the third one of codon (2) Some of the first base of anticodons (tRNA) have alternative choices (Table 12.4) 3) Special codons (1) Initiation codon 'AUG' Initial amino acid would be methionine from codon AUG In bacteria, it puts N-formylmethionine (fmet) When protein synthesis is completed, fmet or formyl group will be removed In human methionine is not formylated (2) Termination/ stop codons UAA, UAG, and UGA No corresponding tRNAs Mutation may create unexpected stop codon (nonsense mutation) *Suppressor mutation (Suppressor mutation) -Mutation on t-RNA gene's anticodon sequence 'corrects' nonsense mutation of mRNA 4) Genetic code is nearly universal (1) Bacteriophage MS2 (1972) Three coat protein genes were studied Predicted relationship between DNA sequence and sequence of amino acid Same initiation codon and stop codons were found (2) Exceptions to universal code (Table 12.5) Human mitochondria Other organisms: mycoplasma, paramecium... 5) Overlapping genes (Fig.12.8)
  3. 3. 3 Found from viral genome Reported presence of overlapping genes from human and mouse genome (paper) Advantage -Conserve the size of genome Disadvantages -Single mutation may affect multiple genes 3. Transcription in prokaryotes -Synthesis of RNA molecules by using DNA as a template 1) RNA is an intermediate molecule in the protein synthesis (1) DNA is located in the nucleus, and translation occurs in the cytoplasm (2) RNA is synthesized from the nucleus where DNA is located, and chemically similar to DNA (3) After produced, RNA migrates into cytoplasm where proteins are synthesized (4) The amount of RNA is generally proportional to the amount of protein in a cell *mRNA as an intermediate molecule in microbial genetics (1) Preexisting radioactively labeled bacterial ribosomes were taken by infected phage RNA -Non-discriminatory manner -'Ribosomes are not protein specific': Simply "workbench" -RNA should be the 'messenger' from DNA (2) Infected bacteriophage synthesized RNA to produce viral protein -Labeled RNA from infected bacteria hybridized only with viral DNA -RNA should be the 'messenger' from DNA 2) Process of transcription (bacterial trascription1,2) (1) Initiation (a) RNA polymerase initiates RNA synthesis Use ribonucleoside triphosphates (NTPs) as substrates Does not require primer to initiate synthesis (NTP)n ----(DNA , enzyme)---- (NMP)n + (PPi)n (NMP)n + NTP ----(DNA, enzyme)---- (NMP)n+1 +PPi Subunits of prokaryotic RNA polymerase -b and b’ –catalytic basis and active site for transcription -s(sigma) - Initiation of RNA transcription; variations in s subunit is recognized -Active form is abb’s *Only one RNA polymerase in prokaryote RNA polymerase s subunit binds to promoter region (Fig.12.9a) *Promoter region -Located at upstream region -Two consensus sequences TATAAT sequence (-10 region) TTGACA sequence (-35 region) -Governs efficiency of initiation of transcription (different level of gene expression) -Directs the orientation of RNA polymerase (b) Binding of RNA polymerase on promoter initiates transcription (Fig.12.9b) Insert the first complementary ribonucleoside triphosphate at the start site
  4. 4. 4 No primer is required for initiation (2) Elongation (Fig.12.9c) Elongation by continuous insertion of complementary NTPs to make DNA-RNA duplex After a few NTP additions, s subunit dissociates from the active polymerase Rest of the elongation process directed by RNA polymerase core enzyme (3) Termination Elongation continues until meet the termination sequence -Includes stop codon -About 40bps Sometimes termination factor r(rho) is involved Release of transcribed RNA molecule from DNA template Dissociation of RNA polymerase core enzyme *Polycistronic mRNA (polycistronic) Related genes are clustered together -producing 'polycistronic mRNA Termination sequence at the end only Translate all proteins at the same time 4. Transcription in eukaryotes 1) Uniqueness in eukaryotic transcription (1) Transcription from nucleus (2) RNA polymerase has three different forms (Table 12.7) RNA polymerase I - produces rRNA RNA polymerase II - produces mRNA RNA polymerase III - produces tRNA (3) Transcribed mRNA need to move out to the cytoplasm for translation (4) Enhancer regulates transcription Position of enhancer is not universal (5) Processing: maturation of primary RNA transcript to mRNA (a) Addition of 5’-cap and 3’-tail to primary transcript (pre-mRNA) *Heterogeneous nuclear RNA (hnRNA) (b) Split and Splicing of genes Cut out RNA from intron, then splice remainders (exons) 2) Transcription process in eukaryotes (1) Initiation of transcription (initiation complex1,2) Promoter has TATA box (-30 region) and CAAT box (-80 region) Transcription factors help binding and initiation of RNA polymerase II to DNA template -TFIID and TATA-binding protein (TBP) bind to TATA box :TFIID changes DNA conformation -Other transcription factors bind sequentially to TFIID, forming 'pre-initiation complex' :TFIIA, TFIIB, TFIIE, and TFIIH (opens double helix) RNA polymerase II binds on pre-initiation complex Enhancer also has role on efficiency of promoter (enhancer) *cis-acting elements and trans-acting factors *Structure of RNA polymerase (RNA polymerase)
  5. 5. 5 (2) hnRNA capping (Fig.12.10a,b) 7-methylguanosine (7mG) cap on 5’-end of the hnRNA (capping) -To protect RNA from nuclease attack (5'-5' linkage) -The cap involves in mRNA transport from the nucleus to the cytoplasm (Transport1,2) (3) Addition of poly-A tail on 3’-end (PolyA tail1,2) Tail protects mRNA from degradation 3'-end cleavage by sequence recognition proteins -Recognition sequence is AAUAAA (by CPSF) :Sequence is 10-30nucleotides upstream from cleavage site (3-cleavage) -CstF (Cleavage stimulation factor F) -CPSF (Cleavage and polyadenylation specificity factor) Poly-A-polymerase adds about 200 As. -ATP is source for As -No template is needed Poly-A-binding proteins bind on the tail -Involved in mRNA transport to cytoplasm -Also involved in translation process (4) Splicing of mRNA (Fig.12.10a,b) Remove portions of hnRNA from intron and rejoin remaining hnRNA Split genes: genes containing introns (Fig.12.12) Intron: A noncoding sequence of DNA within a gene (introns and exons) Exon: The sequences of the primary RNA transcript (or the DNA that encodes them) Exits the nucleus as part of an mRNA molecule Most of the eukaryotic genes have introns: exceptions on histone and interferon genes Splicing mechanisms (a) Spliceosome formation (Fig.12.14a,b; spliceosome1,2) Prevalent way of splicing Usually related with longer introns Begins at 5’-end GU sequence and ends at 3’-end AG sequence The GU sequence in intron attracts molecules for splicing to form spliceosome Small nuclear RNAs (snRNAs) form snurps or snRNPs (small nuclear ribonucleoproteins) with proteins –U1, U2, … U6 (*uridine-rich) U1 binds to GU sequence of 5’-end Binding of U1 initiates the formation of the spliceosome (U2, U4, U5, and U6) 2’-OH from adenine residue on the 'branch point' cuts the 5’-splice site –form 'lariat' *Lariat formation OH from 3’-end of exon I cuts 3-end of intron Ligation of exons upon presence of U5 (b) Alternative splicing (splice variant) Different combinations of exons on mature mRNA Produces splice variants (c) Autocatalytic RNAs (ribozyme) No other involved proteins are known
  6. 6. 6 Intron has enzymatic activity necessary for its own removal Three demensional intron folding is important for enzymatic function Two times sequential transesterification reaction (i) Group1 (Fig.12.13a,b;GroupI) Guanosine is involved as a cofactor on removal of intron (ii) Group II (GroupII) Intron has autoreactive adenine residue(A) within the sequence Forms intermediate lariat Group II does not need Guanosine on self-splitting (5) RNA editing Posttranscriptional RNA processing Frequent from mitochondrial and chloroplast DNA gRNA (guide RNA) works as a template in trypanosomes Function of ADAR (adenosine deaminase acting on RNA) gene on glutamine receptor channel in mammals Substitution Insertion/ deletion *Comparisons between prokaryotic and eukaryotic transcription Prokaryotic transcription Eukaryotic transcription