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  1. Chapter 17 Gene to Protein

  2. Discussion • Overview: The Flow of Genetic Information • The information content of DNA • Is in the form of specific sequences of nucleotides along the DNA strands

  3. Discussion • The DNA inherited by an organism • Leads to specific traits by dictating the synthesis of proteins • The process by which DNA directs protein synthesis, gene expression • Includes two stages, called transcription and translation

  4. Discussion • The ribosome • Is part of the cellular machinery for translation, polypeptide synthesis Figure 17.1

  5. Secret of Life - Bozeman

  6. I. Transcription and Translation • Transcription • Synthesis of RNA (under the direction of DNA) • Produces messenger RNA (mRNA) • Translation • Synthesis of a polypeptide (under the direction of mRNA) • Occurs on ribosomes

  7. DNA TRANSCRIPTION mRNA Ribosome TRANSLATION Polypeptide (a) Prokaryotic cell. In a cell lacking a nucleus, mRNAproduced by transcription is immediately translatedwithout additional processing. • In prokaryotes • Transcription and translation occur together w/o additional processing Figure 17.3a

  8. 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 eukaryotes • RNA transcripts are modified before becoming true mRNA

  9. Cells are controlled by protein synthesis (DNA RNA protein)

  10. II. Genetic Code • Genetic information is encoded as a group of three bases (codons)

  11. 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 • Transcription • The gene determines the sequence of bases along the length of an mRNA molecule

  12. 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 III. Decoding RNA • Codons in messenger RNA are either translated into an amino acid or serves as a translational stop signal

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

  14. In laboratory experiments • Genes can be transcribed and translated after being transplanted from one species to another Tobacco plant expressing firefly gene Figure 17.6

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

  16. V. Molecular Components of Transcription • RNA synthesis • Catalyzed by RNA polymerase, which pries the DNA strands apart and hooks together the RNA nucleotides, 5’-3’ but does NOT need a primer • Follows same base-pairing rules as DNA, except that in RNA, uracil substitutes for thymine (A – U)

  17. 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 VI. Synthesis of an RNA Transcript • The stages of transcription are • Initiation • Elongation • Termination

  18. Eukaryotic promoters 1 TRANSCRIPTION DNA 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 Start point Template DNA strand 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 A. Initiation of Transcription • Promoters signal the initiation of RNA synthesis (where to bind and which DNA strand to use) • Transcription factors help eukaryotic RNA polymerase recognize promoter sequences TATA Box – TF bind, template DNA is the opposite strand (ATAT) TF allow for RNA Polymerase II to bind Transcription initiation complex is formed and DNA unwinds and gets transcribed Figure 17.8

  19. B. Elongation • RNA polymerase moves along the DNA untwisting the double helix, exposing about 10 to 20 DNA bases at a time for pairing with RNA nucleotides, builds 5’-3’ • Multiple RNAs can be made

  20. 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 C U 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

  21. C. Termination • Prokaryotes – have sequence of nucleotides (terminator) • Eukaryotes • RNA Polymerase II transcribes polyadenylation signal sequence in the pre-RNA (AAUAA) • 10-35 nucleotides later, proteins cut the RNA from the polymerase

  22. Discussion • Concept 17.3: Eukaryotic cells modify RNA after transcription • Enzymes in the eukaryotic nucleus • Modify pre-mRNA in specific ways before the genetic messages are dispatched to the cytoplasm

  23. 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 VII. Alteration of mRNA Ends • Each end of a pre-mRNA molecule is modified in a particular way • 5 end receives a modified guanine nucleotide cap • 3 end gets a poly-A tail (50-250 A bases) Figure 17.9

  24. 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 VIII. Split Genes and RNA Splicing • RNA splicing • Removes introns and joins exons • RNA Splicing Figure 17.10

  25. 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 • Is carried out by spliceosomes in some cases Figure 17.11

  26. A. Ribozymes • Ribozymes • Are catalytic RNA molecules that function as enzymes and can splice RNA

  27. B. The Functional and Evolutionary Importance of Introns • The presence of introns • Allows for alternative RNA splicing (genes leading to two or more proteins, based on which areas are treated as exons) • Allows for number of protein products to be much greater than the number of genes

  28. 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 and functional regions called domains • Different exons code for the different domains in a protein (active site vs attachment to membrane) Figure 17.12

  29. Concept 17.4: Translation is the RNA-directed synthesis of a polypeptide: a closer look

  30. IX. Molecular Components of Translation • A cell translates an mRNA message into protein • With the help of transfer RNA (tRNA)

  31. 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 • Translation: the basic concept Figure 17.13

  32. 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 A. Structure of tRNA • Consists of a single RNA strand that is only about 80 nucleotides long A C C

  33. 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

  34. 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 amino acid 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

  35. A. Ribosomes • Facilitate the specific coupling of tRNA anticodons with mRNA codons during protein synthesis

  36. 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. • Ribosomes are made of proteins and RNA molecules named ribosomal RNA (rRNA) Figure 17.16a

  37. 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. • The ribosome has three binding sites for tRNA • The A site • The P site • The E site E P A Figure 17.16b

  38. 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

  39. X. Building a Polypeptide • We can divide translation into three stages • Initiation • Elongation • Termination

  40. 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 A. Initiation of Translation • Brings together mRNA, tRNA w/ first amino acid of the polypeptide, and two subunits of a ribosome

  41. Codon recognition. The anticodon of an incoming aminoacyl tRNA base-pairs with the complementary mRNA codon in the A site. Hydrolysis of GTP increases the accuracy and efficiency of this step. 1 Amino end of polypeptide DNA TRANSCRIPTION mRNA Ribosome TRANSLATION Polypeptide E mRNA 3 Ribosome ready for next aminoacyl tRNA P A site site 5 2 GTP GDP 2 E E P A P A 2 Peptide bond formation. An rRNA molecule of the large subunit catalyzes the formation of a peptide bond between the new amino acid in the A site and the carboxyl end of the growing polypeptide in the P site. This step attaches the polypeptide to the tRNA in the A site. GDP Translocation. The ribosome translocates the tRNA in the A site to the P site. The empty tRNA in the P site is moved to the E site, where it is released. The mRNA moves along with its bound tRNAs, bringing the next codon to be translated into the A site. 3 GTP E P A Figure 17.18 B. Elongation of the Polypeptide Chain • Amino acids are added one by one to the preceding amino acid

  42. Release factor Free polypeptide 5 3 3 3 5 5 Stop codon (UAG, UAA, or UGA) 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. When a ribosome reaches a stop codon on mRNA, the A site of the ribosome accepts a protein called a release factor instead of tRNA. The two ribosomal subunits and the other components of the assembly dissociate. 2 1 3 Figure 17.19 C. Termination of Translation • When the ribosome reaches a stop codon in the mRNA, releasing factor binds and breaks ribosome away

  43. Completed polypeptide Growing polypeptides Incoming ribosomal subunits Start of mRNA (5 end) Polyribosome End of mRNA (3 end) (a) An mRNA molecule is generally translated simultaneously by several ribosomes in clusters called polyribosomes. Ribosomes mRNA 0.1 µm (b) This micrograph shows a large polyribosome in a prokaryotic cell (TEM). Figure 17.20a, b D. Polyribosomes • A number of ribosomes can translate a single mRNA molecule simultaneously • Forming a polyribosome

  44. DNA, RNA, Transcription and Translation Andersen

  45. XI. Completing the Functional Protein • Polypeptide chains • Undergo modifications after the translation process • Free ribosomes in the cytosol • Initiate the synthesis of all proteins • If ribosomes bind to ER then proteins are bound for endomembrane system or secretion

  46. An SRP binds to the signal peptide, halting synthesis momentarily. Polypeptide synthesis begins on a free ribosome in the cytosol. The SRP binds to a receptor protein in the ER membrane. This receptor is part of a protein complex (a translocation complex) that has a membrane pore and a signal-cleaving enzyme. The SRP leaves, and the polypeptide resumes growing, meanwhile translocating across the membrane. (The signal peptide stays attached to the membrane.) The rest of the completed polypeptide leaves the ribosome and folds into its final conformation. The signal- cleaving enzyme cuts off the signal peptide. 2 1 4 3 6 5 Ribosome mRNA Signal peptide ER membrane Signal peptide removed Signal- recognition particle (SRP) Protein SRP receptor protein CYTOSOL Translocation complex ERLUMEN Figure 17.21 • The signal mechanism for targeting proteins to the ER

  47. Types of RNA in a Eukaryotic Cell Table 17.1

  48. RNA polymerase DNA mRNA Polyribosome Direction of transcription 0.25 m RNA polymerase DNA Polyribosome Polypeptide (amino end) Ribosome mRNA (5 end) Discussion • Concept 17.6: Comparing gene expression in prokaryotes and eukaryotes reveals key differences • Prokaryotic cells lack a nuclear envelope • Allowing translation to begin while transcription is still in progress Figure 17.22

  49. Discussion • In a eukaryotic cell • The nuclear envelope separates transcription from translation • Extensive RNA processing occurs in the nucleus

  50. Discussion • Concept 17.7: Point mutations can affect protein structureandfunction • Mutations • Are changes in the genetic material of a cell • Point mutations • Are changes in just one base pair of a gene