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Chapter 17

Chapter 17. From Gene to Protein. The Flow of Genetic Information. The DNA inherited by an organism Leads to specific traits by dictating the synthesis of proteins Gene expression is the process by which DNA directs protein synthesis DNA  RNA  Protein (polypeptide) Includes two stages:

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Chapter 17

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  1. Chapter 17 From Gene to Protein

  2. The Flow of Genetic Information • The DNA inherited by an organism • Leads to specific traits by dictating the synthesis of proteins • Gene expression is the process by which DNA directs protein synthesis • DNA  RNA  Protein (polypeptide) • Includes two stages: • Transcription (DNA  RNA) • Translation (RNA  Protein)

  3. Evidence from the Study of Metabolic Defects • In 1909, British physician Archibald Garrod • Was the first to suggest that genotypes (genes) dictate phenotypes (physical characteristics) through enzymes that catalyze specific chemical reactions within the cell

  4. “One Gene-One Enzyme” • In 1941, Beadle and Tatum developed the “one gene–one enzyme” hypothesis • Which states that the function of a gene is to dictate the production of a specific enzyme • Beadle and Tatum caused Neurospora, a bread mold, to mutate with X-rays (Fig 17.2) • Creating mutants that could not survive on minimal medium • Using genetic crosses • They determined that their mutants fell into three classes, each mutated in a different gene • Beadle and Tatum concluded that the inability to metabolize a particular amino acid is the result of an inability to produce necessary enzymes

  5. The Products of Gene Expression: A Developing Story • As researchers learned more about proteins • They made minor revisions to the one gene–one enzyme hypothesis • Genes code for polypeptide chains or for RNA molecules • Not all proteins are enzymes, nor are all made of a single peptide • “One Gene-One Polypeptide”

  6. Basic Principles of Transcription and Translation • Transcription is the synthesis of messenger RNA under the direction of DNA • players include: • DNA • RNA Polymerase • messenger RNA • Occurs in the nucleus (in eukaryotes) • Translation is the actual synthesis of a polypeptide under the direction of mRNA • players include: • Messenger RNA (mRNA) • Ribosomal RNA (rRNA) • Transfer RNA (tRNA) • Occurs on ribosomes in the cytoplasm

  7. DNA TRANSCRIPTION mRNA Ribosome TRANSLATION Polypeptide (a) Prokaryotic cell. In a cell lacking a nucleus, mRNAproduced by transcription is immediately translatedwithout additional processing. Prokaryotic Cell: Fig 17.3a • Prokaryotes lack a nucleus • Transcription and translation occur together Figure 17.3a

  8. Nuclear envelope DNA TRANSCRIPTION Pre-mRNA RNA PROCESSING mRNA Ribosome TRANSLATION 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. (b) Polypeptide Figure 17.3b Eukaryotic Cell: Fig 17.3b • In eukaryotes • RNA transcripts are modified before becoming true mRNA

  9. The Genetic Code • Only one strand of the double stranded DNA molecule is used as a template in transcription • The sequence of nucleotides in the DNA will determine the sequence of nucleotides in the mRNA molecule • The sequence of nucleotides in the mRNA will determine the sequence of amino acids in the polypeptide

  10. DNA molecule Gene 2 Gene 1 Gene 3 DNA strand (template) 5 3 A C C A A A C C G A G T TRANSCRIPTION G U G G U G C A U U U C 5 3 mRNA Codon TRANSLATION Gly Phe Protein Ser Trp Amino acid Figure 17.4 The triplet code

  11. Codons: Triplets of Bases • Genetic information • Is encoded as a sequence of nonoverlapping base triplets, or codons • A codon in messenger RNA • Is either translated into an amino acid or serves as a translational stop signal • Codons must be read in the correct reading frame • For the specified polypeptide to be produced

  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 GUG GCG GAG GGG G Figure 17.5 The dictionary of the genetic code Glu

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

  14. Transcription is the DNA-directed synthesis of RNA: a closer look • The stages of transcription are • Initiation • Elongation • Termination

  15. A U RNA/DNA pairing C G Molecular Components of Transcription • RNA synthesis • Is catalyzed by RNA polymerase, which pries the DNA strands apart and hooks together the RNA nucleotides • Follows the same base-pairing rules as DNA, except that in RNA, uracil substitutes for thymine

  16. 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 Initiation of Transcription • A specific sequence of bases in the DNA acts as a start signal (promotor) • RNA polymerase binds to the promoter • the DNA strands unwind, and • the polymerase initiates RNA synthesis at the start point on the template strand.

  17. 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 U C C A C A 5 A A T A G G T T Direction of transcription (“downstream) 5 Template strand of DNA Newly made RNA Elongation of the RNA Strand • As RNA polymerase moves along the DNA • It continues to untwist the double helix, exposing about 10 to 20 DNA bases at a time for pairing with RNA nucleotides

  18. Termination of Transcription • Eventually, a specific sequence of bases in the DNA acts as a stop signal (terminator) • The RNA transcript is released • The RNA polymerase detaches from the DNA • The mechanisms of termination • Are different in prokaryotes and eukaryotes

  19. 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

  20. 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 • The 5 end receives a modified nucleotide cap to prevent “unraveling” • The 3 end gets a poly-A tail to prevent unraveling, help ribosome attach, and to facilitate export from the nucleus Figure 17.9

  21. 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 (intervening sequences) and joins exons (expressed sequences) Figure 17.10

  22. RNA splicing • During RNA splicing • Small nuclear ribonucleicproteins (snRNP) recognize intron ends and together with proteins form a structure called a spliceosome • Spliceosomes remove introns while connecting exons together • Ribozymes (catalytic RNA molecules) may also catalyze this process in some organisms

  23. The Functional and Evolutionary Importance of Introns • Introns may regulate gene activity and the passage of mRNA into the cytoplasm • The presence of introns allows for alternative RNA splicing which may allow one gene to play roles in the synthesis of multiple proteins • enables a gene to be diverse in function • May increase recombination of genetic material (easier to cut and paste)

  24. Gene DNA Exon 1 Exon 2 Intron Exon 3 Intron Transcription RNA processing Translation Domain 3 Domain 2 Domain 1 Polypeptide The Functional Importance of Exons • In many cases • Different exons code for the different domains in a protein: Figure 17.12

  25. Translation is the RNA-directed synthesis of a polypeptide: a closer look • We can divide translation into three stages • Initiation • Elongation • Termination

  26. Molecular Components of Translation • A cell translates the messenger RNA (mRNA) message into a polypeptide with the help of transfer RNA (tRNA) and the ribosomes, which are made of ribosomal RNA (rRNA) and protein subunits

  27. The Structure and Function of Transfer RNA 3 • A tRNA molecule • Consists of a single RNA strand that is only about 80 nucleotides long • Molecules of tRNA are not all identical • Each carries a specific amino acid on one end • Each has an anticodon on the other end • The anticodon is complimentary to the codon on mRNA 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 G C U A G * A * A C * U A G A Anticodon

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

  29. Amino Acids attach to tRNA • Before a peptide can be assembled, tRNA must bind to the correct amino acid • This process involves the enzyme aminoacyl-tRNA synthetase and ATP

  30. Ribosomes • Ribosomes • Facilitate the specific coupling of tRNA anticodons with mRNA codons during protein synthesis • The ribosomal subunits • Are constructed of proteins and RNA molecules named ribosomal RNA or rRNA

  31. TRANSCRIPTION DNA 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. Figure 17.16 The anatomy of a functioning ribosome

  32. 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 5 Codons 3 mRNA Figure 17.13 Translation: the basic concept

  33. 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. Figure 17.16b Schematic model of a ribosome • The ribosome has three binding sites for tRNA • The P site • The A site • The E site E P A Figure 17.16b

  34. Initiation of Translation • The initiation stage of translation • Brings together mRNA, tRNA bearing the first amino acid of the polypeptide, and two subunits of a ribosome • Small ribosomal subunit attaches to mRNA near the 5’ cap • tRNA carrying the amino acid Methionine attaches to the start codon AUG (P site) • Large ribosomal subunit attaches

  35. Large ribosomal subunit P site 3 5 U C A Met Met 5 3 A G U Initiator tRNA GDP GTP E A mRNA 5 5 3 3 Start codon mRNA binding site Small ribosomal subunit Translation initiation complex 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). 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. 1 2 Figure 17.17 The initiation of translation

  36. Elongation of the Polypeptide Chain • In the elongation stage of translation • Amino acids are added one by one to the preceding amino acid • Codon recognition: Site A codon forms bonds with the anticodon of tRNA • Peptide bond formation: large subunit rRNA catalyzes the formation of a peptide bond between the amino acids of the tRNA’s at P & A sites • Translocation: rRNA shifts position by one codon with respect to mRNA. mRNA is read from 5’ to 3’.

  37. 1 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. 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 The elongation cycle of translation

  38. Termination of Translation • The final stage of translation is termination • When the ribosome reaches a stop codon (UAA, UAG, UGA) in the mRNA • Release factor protein binds to the stop codon, hydrolyzing it from the tRNA

  39. Release factor Free polypeptide 5 3 3 3 5 5 Stop codon (UAG, UAA, or UGA) The two ribosomal subunits and the other components of the assembly dissociate. 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 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. 1 2 3 Figure 17.19 The termination of translation

  40. 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 Polyribosomes • A number of ribosomes can translate a single mRNA molecule simultaneously • Forming a polyribosome

  41. Protein Folding and Post-Translational Modifications • Polypeptide chains • Undergo modifications after the translation process to achieve their three-dimensional shape and complete the functional protein

  42. Further modification of synthesized proteins • Further modification of synthesized proteins includes • attachment of sugar, lipids, functional groups • Removing amino acids from the leading end of protein (recall that all protein sequencing starts with amino acid methionine but not all finished proteins do) • Polypeptide chain may be divided up into smaller units • Protein may require several polypeptide chains

  43. Targeting Polypeptides to Specific Locations • Two populations of ribosomes are evident in cells • Free and bound • Free ribosomes in the cytosol • Initiate the synthesis of all proteins • Proteins destined for the endomembrane system or for secretion • Must be transported into the ER

  44. Point mutations can affect protein structure and function • Mutations • Are changes in the genetic material of a cell • Point mutations • Are changes in just one base pair of a gene • The change of a single nucleotide in the DNA’s template strand • Can lead to the production of an abnormal protein

  45. Wild-type hemoglobin DNA Mutant hemoglobin DNA In the DNA, the mutant template strand has an A where the wild-type template has a T. 3 5 3 5 T T C A T C mRNA mRNA The mutant mRNA has a U instead of an A in one codon. G A A U A G 5 3 5 3 Normal hemoglobin Sickle-cell hemoglobin The mutant (sickle-cell) hemoglobin has a valine (Val) instead of a glutamic acid (Glu). Val Glu Figure 17.23 The molecular basis of sickle-cell disease: a point mutation

  46. Types of Point Mutations • Point mutations within a gene can be divided into two general categories • Base-pair substitutions • Base-pair insertions or deletions

  47. Wild type A A G G G G A U U U C U A A U mRNA 5 3 Lys Protein Met Phe Gly Stop Amino end Carboxyl end Base-pair substitution No effect on amino acid sequence U instead of C A U G A A G U U U G G U U A A Lys Met Phe Gly Stop Missense A instead of G A A U G A A G U U U A G U U A Lys Met Phe Ser Stop Nonsense U instead of A G A A G G U U G A A U U U U C Met Stop Substitutions • A base-pair substitution • Is the replacement of one nucleotide and its partner with another pair of nucleotides • Silent mutation – the resulting protein has the correct number and type of amino acids (due to redundancy in the genetic code) • Missense – the resulting protein has the correct number of amino acids, but one incorrect amino acid • Nonsense – codes for stop instead of an amino acid, the resulting protein has fewer than the correct number of amino acids Figure 17.24

  48. Wild type A A A G G G A G U U U U C U A mRNA 3 5 Gly Met Lys Phe Protein Stop Amino end Carboxyl end Base-pair insertion or deletion Frameshift causing immediate nonsense Extra U A G A A G U U U U U G G C U A Met Stop Frameshift causing extensive missense Missing U A A A U A G U A G U U G G C Met Lys Ala Leu Insertion or deletion of 3 nucleotides: no frameshift but extra or missing amino acid Missing A A G A G G A A G U U U U U C Met Phe Gly Stop Insertions and Deletions • Insertions and deletions • Are additions or losses of nucleotide pairs in a gene • May produce frameshift mutations • Immediate nonsense • Extensive missense • Insertion or deletion of 3 base pairs, results in missing or extra amino acid(s) Figure 17.25

  49. Mutagens • Mutagens • Are physical or chemical agents that can cause mutations • Spontaneous mutations • Can occur during DNA replication, recombination, or repair

  50. DNA TRANSCRIPTION RNA is transcribed from a DNA template. 5 2 1 3 4 3 Poly-A RNA transcript RNA polymerase 5 Exon RNA PROCESSING In eukaryotes, the RNA transcript (pre- mRNA) is spliced and modified to produce mRNA, which moves from the nucleus to the cytoplasm. RNA transcript (pre-mRNA) Intron Aminoacyl-tRNA synthetase Cap NUCLEUS Amino acid FORMATION OF INITIATION COMPLEX AMINO ACID ACTIVATION tRNA CYTOPLASM After leaving the nucleus, mRNA attaches to the ribosome. Each amino acid attaches to its proper tRNA with the help of a specific enzyme and ATP. Growing polypeptide mRNA Activated amino acid Poly-A Poly-A Ribosomal subunits Cap 5 TRANSLATION C A succession of tRNAs add their amino acids to the polypeptide chain as the mRNA is moved through the ribosome one codon at a time. (When completed, the polypeptide is released from the ribosome.) C A U A E A C Anticodon A A A U G G U G U U U A Codon Ribosome Figure 17.26 A summary of transcription and translation in a eukaryotic cell

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