The Central Dogma. DNA makes RNA makes Proteins

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2 The Central Dogma DNA makes RNA makes Proteins

3 TRANSCRIPTION DNA RNA transcript RNA polymerase RNA PROCESSING Exon RNA transcript (pre-) Intron Aminoacyl-tRNA synthetase NUCLEUS CYTOPLASM FORMATION OF INITIATION COMPLEX Amino acid trna AMINO ACID ACTIVATION A Growing polypeptide Activated amino acid E P Ribosomal subunits TRANSLATION E A Anticodon Codon Ribosome

4 The Central Dogma DNA makes RNA makes Proteins

5 Transcription DNA --> RNA requires RNA polymerase enzyme three types (in Euk) [IB: you don t need to know the differences] RNAp II makes RNAp extends new RNA in 5-3 (other RNA types in mito, chloro) reads template strand DNA 3 -> 5 adds nucleotides to the 3 end of growing RNA Initiation, Elongation, Termination...

6 MiniQuiz DNA sketch a molecule of DNA include and label phosphates, sugars, and bases (specific molecular structures need not be shown) indicate 5 and 3 ends label H-bonds and covalent bonds

7 Transcription Initiation In euk, initiation factors mediate RNAp binding after RNAp binds: transcription initiation complex How does the RNAp know which strand to copy? RNAp attaches to a promoter region Elongation dbl helix re-bonds upstream of RNAp multiple RNAp mols can operate on same gene at same time Termination RNAp stops at end of terminator

8 LE 17-7b Elongation RNA polymerase Non-template strand of DNA RNA nucleotides end Direction of transcription ( downstream ) Template strand of DNA Newly made RNA

9 LE 17-7a-1 Promoter Transcription unit Start point DNA RNA polymerase

10 LE 17-7a-2 Promoter Transcription unit Start point RNA polymerase DNA Initiation Unwound DNA RNA transcript Template strand of DNA

11 LE 17-7a-3 Promoter Transcription unit Start point RNA polymerase DNA Initiation 5 Unwound DNA RNA transcript Template strand of DNA Elongation 5 Rewound DNA 5 RNA transcript

12 LE 17-7a-4 Promoter Transcription unit 5 Start point RNA polymerase DNA Initiation 3 Unwound DNA RNA Template strand tran- of DNA script Elongation Rewound DNA RNA transcript Termination Completed RNA transcript

13

14 RNA editing in prok: transcript is translated directly (except trna, rrna) in euk: modifications in nucleus 5 cap added (modified G) 3 end gets poly-a-tail A-nucs

15 RNA editing, cont d in cytoplasm: RNA splicing avg transcript = 10,000bp. Avg prot = 400aa. (8800bp cut!) introns cut out, exons joined cut & splice by: spliceosome = prot + small nuclear ribonucleoproteins (snrnps) ( snurps ) Protein RNA transcript (pre-) Exon 1 Intron Exon 2 snrna snrnps Spliceosome Other proteins Why are introns a good thing? alternative splicing regulatory introns (mirna) Spliceosome components Exon 1 Exon 2 Cut-out intron

16 LE DNA Gene Exon 1 Intron Exon 2 Intron Exon 3 Transcription RNA processing Translation Domain 3 Domain 2 Domain 1 Polypeptide

17 Translation trna is the translator process mediated by ribosome large, small subunits proteins + rrna (most abundant RNA) trna deposits a.a., codon by codon ribosome joins a.a. s into polypeptide 45 different trna s; why not 64? wobble third base in codon doesn t have to match ex: U match with A or G thus: more than one codon can code for same a.a ex: ACA, ACU, ACC, ACG all code for Thr

18 Ribosomes during initiation: start codon attracts initiator trna w/ Met sm subunit attaches to sm subunit moves downstream lg subunit attaches three sites: A: holds trna w/ next a.a for growing chain P: hold trna carrying the growing chain E: ejection seat for trna ribosome = big ribozyme hydrolysis of GTP supplies E continues until stop codon reached

19 Translate this: AUG,GAG,GAA,AUA,GAU,UGA AUG,GUG,GAA,AUA,GAU,UGA AUG,GGG,AAA,UAG,AUU,GA AUG,GCA,GGA,AAU,AGA,UUG,A

20 TRANSCRIPTION DNA RNA transcript RNA polymerase RNA PROCESSING Exon RNA transcript (pre-) Intron Aminoacyl-tRNA synthetase NUCLEUS CYTOPLASM FORMATION OF INITIATION COMPLEX Amino acid trna AMINO ACID ACTIVATION A Growing polypeptide Activated amino acid E P Ribosomal subunits TRANSLATION E A Anticodon Codon Ribosome

21 Ribozymes RNA: more than DNA message H-bond to other nucleic acids form 3-D structure by self-bonding contains functional catalytic groups Ribozymes: def = RNA molecules catalyzing a chem rxn ribozymes catalyze their own cleavage, and even their own synthesis RNA can store, transmit, and duplicate genetic info also regulate gene expression snrna, sirna, mirna RNA nucleotides have been made spontaneously in lab Key to origin of life?

22 The End

23 DNA Review

24 DNA/RNA Review Scientists DNA structure bases nucleotides diagram DNA replication = semi-conservative RNA vs DNA RNA functions (r,t,m) transcription sense strand regulation of transcription promoter causes RNA polymerase to bind prokaryotes: introns introns/exons reverse transcriptase (Ch 18; HIV, cdna & mol bio) translation initiation, elongation, termination

25 LE Polypeptide Amino acids Ribosome trna with amino acid attached trna Anticodon Codons

26 LE 17-14a Amino acid attachment site Hydrogen bonds Anticodon Two-dimensional structure Amino acid attachment site Hydrogen bonds Anticodon Three-dimensional structure Anticodon Symbol used in this book

27 LE 17-14b Amino acid attachment site Hydrogen bonds Anticodon Anticodon Three-dimensional structure Symbol used in this book

28 LE Amino acid Aminoacyl-tRNA synthetase (enzyme) Pyrophosphate Phosphates trna AMP Aminoacyl trna (an activated amino acid )

29 LE trna molecules Exit tunnel Growing polypeptide E P A Large subunit Small subunit Computer model of functioning ribosome P site (Peptidyl-tRNA binding site) E site (Exit site) binding site E P A Schematic model showing binding sites A site (AminoacyltRNA binding site) Large subunit Small subunit Amino end Growing polypeptide Next amino acid to be added to polypeptide chain E trna Codons Schematic model with and trna

30 trna molecules Exit tunnel Growing polypeptide E P A Large subunit Small subunit 5! 3! Computer model of functioning ribosome P site (Peptidyl-tRNA binding site) E site (Exit site) binding site E P A Schematic model showing binding sites A site (AminoacyltRNA binding site) Large subunit Small subunit

31 LE 17-16c Amino end Growing polypeptide Next amino acid to be added to polypeptide chain E trna Codons Schematic model with and trna

32 LE Met P site Met Large ribosomal subunit Initiator trna GTP GDP E A Start codon binding site Small ribosomal subunit Translation initiation complex

33 LE Amino end of polypeptide E Ribosome ready for next aminoacyl trna P site site A 2 GTP 2 GDP E E P A P A GDP GTP E P A

34 LE Release factor Free polypeptide Stop codon (UAG, UAA, or UGA) When a ribosome reaches a stop codon on, the A site of the ribosome accepts a protein called a release factor instead of trna. The release factor hydrolyzes the bond between the trna in the P site and the last amino acid of the polypeptide chain. The polypeptide is thus freed from the ribosome. The two ribosomal subunits and the other components of the assembly dissociate.

35 LE Growing polypeptides Completed polypeptides Incoming ribosomal subunits Start of ( end) Polyribosome End of ( end) An molecule is generally translated simultaneously by several ribosomes in clusters called polyribosomes. Ribosomes 0.1 µm This micrograph shows a large polyribosome in a prokaryotic cell (TEM).

36 LE 17-20a Growing polypeptides Completed polypeptide Incoming ribosomal subunits Start of ( end) Polyribosome End of ( end) An molecule is generally translated simultaneously by several ribosomes in clusters called polyribosomes.

37 LE 17-20b Ribosomes 0.1 µm This micrograph shows a large polyribosome in a prokaryotic cell (TEM).

38 LE Ribosomes Signalrecognition particle (SRP) CYTOSOL Signal peptide SRP receptor protein Signal peptide removed ER membrane Protein ER LUMEN Translocation complex

39 LE RNA polymerase DNA Polyribosome RNA polymerase Direction of transcription 0.25 µ m DNA Polyribosome Polypeptide (amino end) Ribosome ( end)

40 LE Wild-type hemoglobin DNA Mutant hemoglobin DNA Normal hemoglobin Sickle-cell hemoglobin

41 LE Wild type Protein Stop Amino end Carboxyl end Base-pair substitution No effect on amino acid sequence U instead of C Stop Missense A instead of G Stop Nonsense U instead of A Stop

42 LE 17-24a Wild-type Protein Stop Amino end Carboxyl end

43 LE 17-24b Base-pair substitution No effect on amino acid sequence U instead of C Stop Missense A instead of G Stop Nonsense U instead of A Stop

44 LE Wild type Protein Amino end Stop Carboxyl end Base-pair insertion or deletion Frameshift causing immediate nonsense Extra U Stop Frameshift causing extensive missense Missing Insertion or deletion of 3 nucleotides: no frameshift but extra or missing amino acid Missing Stop

45 LE 17-25a Wild type Protein Amino end Stop Carboxyl end

46 LE 17-25b Base-pair insertion or deletion Frameshift causing immediate nonsense Extra U Stop Frameshift causing extensive missense Missing Insertion or deletion of 3 nucleotides: no frameshift but extra or missing amino acid Missing Stop

47 LE TRANSCRIPTION DNA RNA transcript RNA polymerase RNA PROCESSING Exon RNA transcript (pre-) Intron Aminoacyl-tRNA synthetase NUCLEUS CYTOPLASM FORMATION OF INITIATION COMPLEX Amino acid trna AMINO ACID ACTIVATION A Growing polypeptide Activated amino acid E P Ribosomal subunits TRANSLATION E A Anticodon Codon Ribosome

48 LE 17-2 Wild type Class I Mutants Class II Mutants Class III Mutants Minimal Medium (MM) (control) MM + Ornithine MM + Citrulline MM + arginine (control) Wild type Class I Mutants (mutation In gene A) Class II Mutants (mutation In gene B) Class III Mutants (mutation In gene C) Gene A Gene B Gene C Precursor Enzyme A Ornithine Enzyme B Citrulline Enzyme C Arginine Precursor Precursor Precursor A A Ornithine Ornithine Ornithine B B B Citrulline Citrulline Citrulline C C C Arginine Arginine Arginine A

49 LE 17-2a Wild type Class I Mutants Class II Mutants Class III Mutants Minimal medium (MM) (control) MM + Ornithine MM + Citrulline MM + Arginine (control)

50 LE 17-2b Wild type Class I Mutants (mutation In gene A) Class II Mutants (mutation In gene B) Class III Mutants (mutation In gene C) Gene A Gene B Gene C Precursor Precursor Precursor Precursor Enzyme A A Ornithine Ornithine Ornithine Ornithine Enzyme B B Citrulline Citrulline Citrulline Citrulline Enzyme C C Arginine Arginine Arginine Arginine A B C A B C

51 LE 17-3 TRANSCRIPTION DNA TRANSLATION Polypeptide Ribosome Prokaryotic cell Nuclear envelope TRANSCRIPTION DNA RNA PROCESSING Pre- TRANSLATION Ribosome Polypeptide Eukaryotic cell

52 LE TRANSCRIPTION DNA Prokaryotic cell

53 LE TRANSCRIPTION DNA Prokaryotic cell Polypeptide Ribosome Prokaryotic cell

54 LE TRANSCRIPTION DNA TRANSLATION Polypeptide Ribosome Prokaryotic cell Nuclear envelope TRANSCRIPTION DNA Eukaryotic cell

55 LE TRANSCRIPTION DNA TRANSLATION Polypeptide Ribosome Prokaryotic cell Nuclear envelope TRANSCRIPTION DNA RNA PROCESSING Pre- Eukaryotic cell

56 LE TRANSCRIPTION DNA TRANSLATION Polypeptide Ribosome Prokaryotic cell Nuclear envelope TRANSCRIPTION DNA RNA PROCESSING Pre- TRANSLATION Ribosome Polypeptide Eukaryotic cell

57 LE 17-4 DNA molecule Gene 1 Gene 2 Gene 3 DNA strand (template) TRANSCRIPTION Codon TRANSLATION Protein Amino acid

58 LE 17-5 Second base First base ( end) Third base ( end)

59 Figure 17-06

60 LE 17-9 Protein-coding segment Polyadenylation signal Cap UTR Start codon Stop codon UTR Poly-A tail

61 LE Pre- Exon Intron Exon Intron Exon Cap Poly-A tail Coding segment Introns cut out and exons spliced together Cap Poly-A tail UTR UTR

62 LE Exon 1 RNA transcript (pre-) Intron Exon 2 Protein snrna Other proteins snrnps Spliceosome

63 LE Spliceosome Spliceosome components Cut-out intron Exon 1 Exon 2

64 LE 17-8 Promoter Eukaryotic promoters TATA box Start point Template DNA strand Several transcription factors Transcription factors Additional transcription factors RNA polymerase II Transcription factors RNA transcript Transcription initiation complex

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