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Chapter 17

Chapter 17. From Gene to Protein. Overview: The Flow of Genetic Information The information content of DNA Is in the form of specific seq’s of nt’s along the DNA strands. The DNA inherited by an organism Leads to specific traits by dictating the synthesis of proteins

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Chapter 17

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  1. Chapter 17 From Gene to Protein

  2. Overview: The Flow of Genetic Information • The information content of DNA • Is in the form of specific seq’s of nt’s along the DNA strands

  3. The DNA inherited by an organism • Leads to specific traits by dictating the synthesis of proteins • DNA directs protein synthesis/gene expression • 2 stages • transcription (txn) & • Translation (tsln)

  4. ribosome • cell machine for tsln, pp synthesis Figure 17.1

  5. Concept 17.1: Genes specify proteins via txn & tsln

  6. Evidence from the Study of Metabolic Defects • In 1909, British physician Archibald Garrod • Was the first to suggest that genes dictate phenotypes through enzymes that catalyze specific chemical reactions in the cell

  7. Nutritional Mutants in Neurospora: Scientific Inquiry • Beadle and Tatum causes bread mold to mutate with X-rays • Creating mutants that could not survive on minimal medium

  8. EXPERIMENT RESULTS Class I Mutants Class II Mutants Class III Mutants Wild type Minimal medium (MM) (control) MM + Ornithine MM + Citrulline MM + Arginine (control) • Using genetic crosses • They determined that their mutants fell into three classes, each mutated in a different gene Working with the mold Neurospora crassa, George Beadle and Edward Tatum had isolated mutants requiring arginine in their growth medium and had shown genetically that these mutants fell into three classes, each defective in a different gene. From other considerations, they suspected that the metabolic pathway of arginine biosynthesis included the precursors ornithine and citrulline. Their most famous experiment, shown here, tested both their one gene–one enzyme hypothesis and their postulated arginine pathway. In this experiment, they grew their three classes of mutants under the four different conditions shown in the Results section below. The wild-type strain required only the minimal medium for growth. The three classes of mutants had different growth requirements Figure 17.2

  9. CONCLUSION From the growth patterns of the mutants, Beadle and Tatum deduced that each mutant was unable to carry out one step in the pathway for synthesizing arginine, presumably because it lacked the necessary enzyme. Because each of their mutants was mutated in a single gene, they concluded that each mutated gene must normally dictate the production of one enzyme. Their results supported the one gene–one enzyme hypothesis and also confirmed the arginine pathway. (Notice that a mutant can grow only if supplied with a compound made after the defective step.) Class I Mutants (mutation in gene A) Class II Mutants (mutation in gene B) Class III Mutants (mutation in gene C) Wild type Precursor Precursor Precursor Precursor Enzyme A Gene A A A A Ornithine Ornithine Ornithine Ornithine Enzyme B Gene B B B B Citrulline Citrulline Citrulline Citrulline Enzyme C Gene C C C C Arginine Arginine Arginine Arginine

  10. Beadle & Tatum: “1 gene–1 enzyme hypothesis” • states the function of a gene is to dictate the production of a specific enzyme

  11. The Products of Gene Expression: A Developing Story • As researchers learned more about proteins • The made minor revision to the one gene–one enzyme hypothesis • Genes code for pp chains or RNA

  12. Basic Principles of Transcription and Translation • Txn • synthesis of mRNA under the direction of DNA • Produces messenger RNA (mRNA) • Tsln • actual synthesis of a pp, which occurs under the direction of mRNA • Occurs on ribosomes

  13. DNA TRANSCRIPTION mRNA Ribosome TRANSLATION Polypeptide (a) Prokaryotic cell. In a cell lacking a nucleus, mRNAproduced by transcription is immediately translatedwithout additional processing. • In prok’s • Txn & tsln occur together Figure 17.3a

  14. Nuclear envelope DNA TRANSCRIPTION Pre-mRNA RNA PROCESSING mRNA Ribosome TRANSLATION (b) Eukaryotic cell. The nucleus provides a separatecompartment for transcription. The original RNAtranscript, called pre-mRNA, is processed in various ways before leaving the nucleus as mRNA. Polypeptide Figure 17.3b • In euk’s • RNA transcripts are modified before becoming true mRNA

  15. LE 17-3-5 DNA TRANSCRIPTION mRNA Ribosome TRANSLATION Polypeptide Prokaryotic cell Nuclear envelope DNA TRANSCRIPTION Pre-mRNA RNA PROCESSING mRNA Ribosome TRANSLATION Polypeptide Eukaryotic cell

  16. Cells are governed by a cellular chain of command • DNA RNA protein

  17. The Genetic Code • How many bases correspond to an amino acid?

  18. Codons: Triplets of Bases • Genetic info • encoded as a seq of nonoverlapping base triplets, or codons

  19. Gene 2 DNA molecule Gene 1 Gene 3 DNA strand (template) 5 3 A C C T A A A C C G A G TRANSCRIPTION A U C G C U G G G U U U 5 mRNA 3 Codon TRANSLATION Gly Phe Protein Trp Ser Figure 17.4 Amino acid • txn • gene determines the sequence of bases along the length of an mRNA molecule

  20. Second mRNA base U C A G U UAU UUU UCU UGU Tyr Cys Phe UAC UUC UCC UGC C U Ser UUA UCA UAA Stop Stop UGA A Leu UAG UUG UCG Stop UGG Trp G CUU CCU U CAU CGU His CUC CCC CAC CGC C C Arg Pro Leu CUA CCA CAA CGA A Gln CUG CCG CAG CGG G Third mRNA base (3 end) First mRNA base (5 end) U AUU ACU AAU AGU Asn Ser C lle AUC ACC AAC AGC A Thr A AUA ACA AAA AGA Lys Arg Met or start G AUG ACG AAG AGG U GUU GCU GAU GGU Asp C GUC GCC GAC GGC G Val Ala Gly GUA GCA GAA GGA A Glu Figure 17.5 GUG GCG GAG GGG G Cracking the Code • A codon in mRNA • either translated into an aa or as a tsln stop signal

  21. Codons must be read in the correct reading frame • For correct pp to be made

  22. Evolution of the Genetic Code • genetic code is nearly universal • Shared by organisms from the simplest bacteria to the most complex animals

  23. In lab experiments • Genes can be transcribed & translated after being transplanted from one species to another Figure 17.6

  24. Concept 17.2: Transcription is the DNA-directed synthesis of RNA: a closer look

  25. Molecular Components of Transcription • RNA synthesis • catalyzed by RNA polymerase, which pries the DNA strands apart & hooks together the RNA nt’s - same base-pairing rules as DNA, except in RNA, Uracil subs for thymine

  26. 3 1 2 Promoter Transcription unit 5 3 3 5 Start point DNA RNA polymerase Initiation. After RNA polymerase binds to the promoter, the DNA strands unwind, and the polymerase initiates RNA synthesis at the start point on the template strand. Template strand of DNA 5 3 3 5 Unwound DNA RNA transcript Elongation. The polymerase moves downstream, unwinding the DNA and elongating the RNA transcript 5  3 . In the wake of transcription, the DNA strands re-form a double helix. Rewound RNA 5 3 3 5 3 RNA transcript 5 Termination. Eventually, the RNA transcript is released, and the polymerase detaches from the DNA. 5 3 3 5 3 5 Completed RNA transcript Figure 17.7 Synthesis of an RNA Transcript • stages of txn: • Initiation • Elongation • Termination

  27. Non-template strand of DNA Elongation RNA nucleotides RNA polymerase T A C C A T A T C 3 U 3 end T G A U G G A G E A C C C A 5 A A T A G G T T Direction of transcription (“downstream”) 5 Template strand of DNA Newly made RNA

  28. Eukaryotic promoters 1 DNA TRANSCRIPTION Pre-mRNA RNA PROCESSING mRNA Ribosome TRANSLATION Polypeptide Promoter 5 3 A T A T A A A 3 5 A T A T T T T TATA box Template DNA strand Start point Several transcription factors 2 Transcription factors 5 3 3 5 Additional transcription factors 3 RNA polymerase II Transcription factors 3 5 5 3 5 RNA transcript Figure 17.8 Transcription initiation complex RNA Polymerase Binding & Initiation of Txn • Initiation • Promoters signal initiation of RNA synthesis • Txn factors • Help euk RNA pol recognize promoter seq’s Figure 17.8

  29. Elongation of the RNA Strand • Elongation • RNA pol moves along the DNA • untwisting the double helix, exposing about 10 to 20 DNA bases at a time for pairing w/ RNA nt’s

  30. Termination of Transcription • termination • Is diff in pro’s & euk’s

  31. Concept 17.3: Euk cells modify RNA after txn • Enzymes in the euk nucleus • Modify pre-mRNA in specific ways before the genetic messages are dispatched to the cytoplasm

  32. A modified guanine nucleotide added to the 5 end 50 to 250 adenine nucleotides added to the 3 end TRANSCRIPTION DNA Polyadenylation signal Protein-coding segment Pre-mRNA RNA PROCESSING 5 3 mRNA G P P AAA…AAA P AAUAAA Ribosome Start codon Stop codon TRANSLATION Poly-A tail 5 Cap 5 UTR 3 UTR Polypeptide Alteration of mRNA Ends • Each end of a pre-mRNA molecule is modified in a particular way • 5 end-- modified nt cap • 3 end-- poly-A tail Figure 17.9

  33. Intron Exon 5 Exon Intron Exon 3 5 Cap Poly-A tail Pre-mRNA TRANSCRIPTION DNA 30 31 104 105 146 1 Pre-mRNA RNA PROCESSING Introns cut out and exons spliced together Coding segment mRNA Ribosome TRANSLATION 5 Cap Poly-A tail mRNA Polypeptide 1 146 3 UTR 3 UTR Split Genes and RNA Splicing • RNA splicing • Removes introns & joins exons Figure 17.10

  34. 3 1 2 RNA transcript (pre-mRNA) 5 Intron Exon 1 Exon 2 Protein Other proteins snRNA snRNPs Spliceosome 5 Spliceosome components Cut-out intron mRNA 5 Exon 1 Exon 2 • carried out by spliceosomes in some cases Figure 17.11

  35. Ribozymes • Ribozymes • catalytic RNA molecules that function as enzymes & can splice RNA

  36. The Functional and Evolutionary Importance of Introns • introns • Allow for alternative RNA splicing

  37. Gene DNA Exon 1 Exon 2 Intron Exon 3 Intron Transcription RNA processing Translation Domain 3 Domain 2 Domain 1 Polypeptide • Proteins often have a modular architecture • Consisting of discrete structural & functional regions called domains • In many cases • Diff exons code for the diff domains in a protein Figure 17.12

  38. Concept 17.4: Tsln is the RNA-directed synthesis of a pp: a closer look

  39. Molecular Components of Translation • A cell translates an mRNA message into protein • W/ help of transfer RNA (tRNA)

  40. DNA TRANSCRIPTION mRNA Ribosome TRANSLATION Polypeptide Amino acids Polypeptide tRNA with amino acid attached Ribosome Trp Phe Gly tRNA C C C G G Anticodon A A A A G G G U G U U U C Codons 5 3 mRNA • Tsln: the basic concept Figure 17.13

  41. Molecules of tRNA are not all identical • Each carries a specific aa on 1 end • Each has an anticodon on the other end

  42. 3 A Amino acid attachment site C C 5 A C G C G C G U G U A A U U A U C G * G U A C A C A * A U C C * G * U G U G G * G A C C G * C A G * U G * * G A G C Hydrogen bonds (a) G Two-dimensional structure. The four base-paired regions and three loops are characteristic of all tRNAs, as is the base sequence of the amino acid attachment site at the 3 end. The anticodon triplet is unique to each tRNA type. (The asterisks mark bases that have been chemically modified, a characteristic of tRNA.) C U A G * A * A C * U A G A Anticodon Figure 17.14a The Structure and Function of Transfer RNA • tRNA molecule • a single RNA strand only ~ 80 nt’s long • Is roughly L-shaped A C C

  43. Amino acid attachment site 5 3 Hydrogen bonds A A G 3 5 Anticodon Anticodon (c) Symbol used in this book (b) Three-dimensional structure Figure 17.14b

  44. ATP loses two P groups and joins amino acid as AMP. 2 3 Appropriate tRNA covalently Bonds to amino Acid, displacing AMP. 4 Activated amino acid is released by the enzyme. • A specific enzyme called an aminoacyl-tRNA synthetase • Joins each aa to the correct tRNA Amino acid Aminoacyl-tRNA synthetase (enzyme) Active site binds the amino acid and ATP. 1 Adenosine P P P ATP Adenosine P Pyrophosphate P Pi Pi Pi Phosphates tRNA Adenosine P AMP Aminoacyl tRNA (an “activated amino acid”) Figure 17.15

  45. Ribosomes • Ribosomes • Facilitate the specific coupling of tRNA anticodons w/ mRNA codons during protein synthesis

  46. DNA TRANSCRIPTION mRNA Ribosome TRANSLATION Polypeptide Exit tunnel Growing polypeptide tRNA molecules Large subunit E P A Small subunit 5 3 mRNA (a) Computer model of functioning ribosome. This is a model of a bacterial ribosome, showing its overall shape. The eukaryotic ribosome is roughly similar. A ribosomal subunit is an aggregate of ribosomal RNA molecules and proteins. • ribosomal subunits • Made of proteins & RNA molecules - ribosomal RNA (rRNA) Figure 17.16a

  47. P site (Peptidyl-tRNA binding site) A site (Aminoacyl- tRNA binding site) E site (Exit site) Large subunit mRNA binding site Small subunit (b) Schematic model showing binding sites. A ribosome has an mRNA binding site and three tRNA binding sites, known as the A, P, and E sites. This schematic ribosome will appear in later diagrams. -- ribosome has 3 binding sites for tRNA • P site • A site • E site E P A Figure 17.16b

  48. Growing polypeptide Amino end Next amino acid to be added to polypeptide chain tRNA 3 mRNA Codons 5 (c) Schematic model with mRNA and tRNA. A tRNA fits into a binding site when its anticodon base-pairs with an mRNA codon. The P site holds the tRNA attached to the growing polypeptide. The A site holds the tRNA carrying the next amino acid to be added to the polypeptide chain. Discharged tRNA leaves via the E site. Figure 17.16c

  49. Building a Polypeptide • tsln has 3 stages • Initiation • Elongation • Termination

  50. Large ribosomal subunit P site 5 3 U C A Met Met 3 5 A G U Initiator tRNA GDP GTP E A mRNA 5 5 3 3 Start codon Small ribosomal subunit mRNA binding site Translation initiation complex The arrival of a large ribosomal subunit completes the initiation complex. Proteins called initiation factors (not shown) are required to bring all the translation components together. GTP provides the energy for the assembly. The initiator tRNA is in the P site; the A site is available to the tRNA bearing the next amino acid. A small ribosomal subunit binds to a molecule of mRNA. In a prokaryotic cell, the mRNA binding site on this subunit recognizes a specific nucleotide sequence on the mRNA just upstream of the start codon. An initiator tRNA, with the anticodon UAC, base-pairs with the start codon, AUG. This tRNA carries the amino acid methionine (Met). 2 1 Figure 17.17 Ribosome Association and Initiation of Translation • initiation of tsln • Brings together mRNA, tRNA bearing the 1st aa of the pp, & 2 subunits of a ribosome

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