No growth: Mutant cells cannot grow and divide Minimal medium. Classes of Neurospora crassa. Class I mutants Class II mutants Class III mutants

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1 EXPERIMENT Growth: Wild-type cells growing and dividing No growth: Mutant cells cannot grow and divide Minimal medium RESULTS Minimal medium (MM) (control) Wild type Classes of Neurospora crassa Class I mutants Class II mutants Class III mutants Condition MM + ornithine MM + citrulline MM + arginine (control) CONCLUSION 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 Enzyme A Enzyme A Enzyme A Ornithine Ornithine Ornithine Ornithine Enzyme B Enzyme B Enzyme B Enzyme B Citrulline Citrulline Citrulline Citrulline Enzyme C Enzyme C Enzyme C Enzyme C Arginine Arginine Arginine Arginine 1

2 TRANSCRIPTION TRANSLATION DNA Ribosome Polypeptide (a) Bacterial cell Nuclear envelope TRANSCRIPTION DNA RNA PROCESSING Pre- TRANSLATION Ribosome Polypeptide (b) Eukaryotic cell 2

3 DNA molecule Gene 1 Gene 2 Gene 3 DNA template strand TRANSCRIPTION Codon TRANSLATION Protein Amino acid 3

4 Second base First base ( end of codon) Third base ( end of codon) 4

5 (a) Tobacco plant expressing a firefly gene (b) Pig expressing a jellyfish gene 5

6 Promoter Transcription unit Start point RNA polymerase DNA 1 Initiation Elongation Nontemplate strand of DNA Unwound DNA RNA transcript Template strand of DNA 2 Elongation RNA polymerase end RNA nucleotides Rewound DNA RNA transcript 3 Termination Completed RNA transcript Newly made RNA Direction of transcription ( downstream ) Template strand of DNA 6

7 1 A eukaryotic promoter includes a TATA box Promoter Template TATA box Start point Template DNA strand Transcription factors 2 Several transcription factors must bind to the DNA before RNA polymerase II can do so. RNA polymerase II 3 Additional transcription factors bind to the DNA along with RNA polymerase II, forming the transcription initiation complex. Transcription factors RNA transcript Transcription initiation complex 7

8 G P Protein-coding segment Polyadenylation signal P P AAUAAA AAA AAA Cap UTR Start codon Stop codon UTR Poly-A tail 8

9 Pre- Cap Exon Intron Exon Intron Exon 146 Poly-A tail Coding segment Introns cut out and exons spliced together Cap UTR UTR Poly-A tail 9

10 RNA transcript (pre-) Exon 1 Intron Exon 2 Protein snrna snrnps Other proteins Spliceosome Spliceosome components Exon 1 Exon 2 Cut-out intron 10

11 DNA Gene Exon 1 Intron Exon 2 Intron Exon 3 Transcription RNA processing Translation Domain 3 Domain 2 Domain 1 Polypeptide 11

12 Polypeptide Amino acids Ribosome trna with amino acid attached Phe Gly trna Anticodon Codons 12

13 Amino acid attachment site Hydrogen bonds Anticodon (a) Two-dimensional structure Amino acid attachment site Hydrogen bonds Anticodon (b) Three-dimensional structure Anticodon (c) Symbol used in this book 13

14 Amino acid Aminoacyl-tRNA synthetase (enzyme) P P P Adenosine ATP P P i P Adenosine trna P i P i Aminoacyl-tRNA synthetase trna P Adenosine AMP Computer model Aminoacyl-tRNA ( charged trna ) 14

15 trna molecules Growing polypeptide E P A Exit tunnel Large subunit Small subunit (a) Computer model of functioning ribosome P site (Peptidyl-tRNA binding site) E site (Exit site) binding site E P A A site (AminoacyltRNA binding site) Large subunit Small subunit (b) Schematic model showing binding sites Amino end E Growing polypeptide Next amino acid to be added to polypeptide chain trna Codons (c) Schematic model with and trna 15

16 Met U A A U C G P site Met Large ribosomal subunit Initiator trna Start codon binding site Small ribosomal subunit GTP GDP E A Translation initiation complex 16

17 Amino end of polypeptide E Ribosome ready for next aminoacyl trna P site A site GTP GDP E E P A P A GDP GTP E P A 17

18 Release factor Free polypeptide Stop codon (UAG, UAA, or UGA) 2 GTP 2 GDP 18

19 Growing polypeptides Completed polypeptide Incoming ribosomal subunits (a) Start of ( end) Polyribosome End of ( end) Ribosomes (b) 0.1 µm 19

20 Ribosome Signal peptide Signalrecognition particle (SRP) CYTOSOL ER LUMEN SRP receptor protein Translocation complex Signal peptide removed ER membrane Protein 20

21 Wild-type hemoglobin DNA C T T G A A Mutant hemoglobin DNA C A T G T A G A A G U A Normal hemoglobin Glu Sickle-cell hemoglobin Val 21

22 Wild-type DNA template strand Protein Amino end Stop Carboxyl end A instead of G Extra A U instead of C Extra U Stop Stop Silent (no effect on amino acid sequence) Frameshift causing immediate nonsense (1 base-pair insertion) T instead of C missing A instead of G missing Stop Missense Frameshift causing extensive missense (1 base-pair deletion) A instead of T missing U instead of A missing Nonsense Stop Stop No frameshift, but one amino acid missing (3 base-pair deletion) (a) Base-pair substitution (b) Base-pair insertion or deletion 22

23 RNA polymerase DNA Polyribosome RNA polymerase Direction of transcription 0.25 µm DNA Polyribosome Polypeptide (amino end) Ribosome ( end) 23

24 TRANSCRIPTION DNA RNA transcript RNA PROCESSING Exon RNA polymerase RNA transcript (pre-) Intron NUCLEUS Aminoacyl-tRNA synthetase CYTOPLASM Amino acid trna AMINO ACID ACTIVATION E A P Ribosomal subunits Growing polypeptide Activated amino acid TRANSLATION E A Anticodon Codon Ribosome 24