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

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

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

Second mRNA base First mRNA base (5 end of codon) Third mRNA base (3 end of codon) 4

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

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

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

Protein-coding segment Polyadenylation signal 3 3 UTR5 UTR 5 5 Cap Start codon Stop codon Poly-A tail G PPPAAUAAA AAA … 8

Pre-mRNA mRNA Coding segment Introns cut out and exons spliced together 5 Cap Exon Intron ExonIntron 105 Exon Poly-A tail 5 Cap 5 UTR3 UTR

RNA transcript (pre-mRNA) Exon 1Exon 2Intron Protein snRNA snRNPs Other proteins 5 5 Spliceosome components Cut-out intron mRNA Exon 1 Exon

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

Polypeptide Ribosome Amino acids tRNA with amino acid attached tRNA Anticodon Trp Phe Gly Codons 3 5 mRNA 12

Amino acid attachment site 3 5 Hydrogen bonds Anticodon (a) Two-dimensional structure Amino acid attachment site 5 3 Hydrogen bonds 3 5 Anticodon (c) Symbol used in this book (b) Three-dimensional structure 13

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

Growing polypeptide Exit tunnel Large subunit Small subunit tRNA molecules E P A mRNA 5 3 (a) Computer model of functioning ribosome P site (Peptidyl-tRNA binding site) E site (Exit site) A site (Aminoacyl- tRNA binding site) E P A Large subunit mRNA binding site Small subunit (b) Schematic model showing binding sites Amino end Growing polypeptide Next amino acid to be added to polypeptide chain mRNA tRNA E 3 5 Codons (c) Schematic model with mRNA and tRNA 15

U U A A C G Met GTP GDP Initiator tRNA mRNA 5 3 Start codon mRNA binding site Small ribosomal subunit 5 P site Translation initiation complex 3 EA Met Large ribosomal subunit 16

Amino end of polypeptide mRNA 5 3 E P site A site GTP GDP E P A E PA GTP Ribosome ready for next aminoacyl tRNA E P A 17

Release factor 3 5 Stop codon (UAG, UAA, or UGA) Free polypeptide 2 GDP GTP

Growing polypeptides Completed polypeptide Incoming ribosomal subunits Start of mRNA (5 end) Polyribosome End of mRNA (3 end) (a) Ribosomes mRNA (b) 0.1 µm 19

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

Wild-type hemoglobin DNA mRNA Mutant hemoglobin DNA mRNA CCTT T T G G A A A A AA A GG U Normal hemoglobinSickle-cell hemoglobin Glu Val 21

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

RNA polymerase DNA Polyribosome mRNA 0.25 µm Direction of transcription DNA RNA polymerase Polyribosome Polypeptide (amino end) Ribosome mRNA (5 end) 23

TRANSCRIPTION RNA PROCESSING DNA RNA transcript 3 5 RNA polymerase Poly-A RNA transcript (pre-mRNA) Intron Exon NUCLEUS Aminoacyl-tRNA synthetase AMINO ACID ACTIVATION Amino acid tRNA CYTOPLASM Poly-A Growing polypeptide 3 Activated amino acid mRNA TRANSLATION Cap Ribosomal subunits Cap 5 E P A A Anticodon Ribosome Codon E 24