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Chapter 17

Chapter 17. From Gene to Protein. Transcription & Translation. DNA  RNA  protein The process by which DNA directs protein synthesis (gene expression) Includes two stages 1. transcription (DNA to RNA, occurs in the nucleus)

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Chapter 17

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  1. Chapter 17 From Gene to Protein

  2. Transcription & Translation • DNA RNA protein • The process by which DNA directs protein synthesis (gene expression) • Includes two stages • 1. transcription (DNA to RNA, occurs in the nucleus) • 2. translation (RNA to protein, occurs in the cytoplasm at the RIBOSOMES)

  3. 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 • During transcription • The gene determines the sequence of bases along the length of an mRNA molecule

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

  5. Transcription Genes (contain DNA) provide the instructions for proteins But a gene does not build a protein directly RNA is the messenger molecule between DNA & proteins • Transcription Def – process of copying a portion of DNA into RNA In most cases, the DNA copied is for a single gene that encodes a single protein

  6. 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 Figure 17.3a

  7. 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

  8. Structure of a Single Gene • Promoter – function as an on/off switch for a gene • Coding region – contains the actual “recipe” for a protein written in GENETIC CODE • Terminator (termination sequence) – contain signals that END transcription

  9. 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 • The stages of transcription are • Initiation • Elongation • Termination

  10. Steps involved in Transcription • Initiation a. Occurs @ the promoter b. w/in the promoter there is a series of T’s & A’s called the TATA box c. Enzyme RNA polymerase enters the promoter @ the TATA box & latched onto the coding strand. Like DNA polymerase, RNA polymerase can only manufacture new nucleic acids in 5’ to 3’ direction

  11. 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 RNA Polymerase Binding and Initiation of Transcription • Promoters signal the initiation of RNA synthesis • Transcription factors • Help eukaryotic RNA polymerase recognize promoter sequences Figure 17.8

  12. Steps of Transcription • Elongation • RNA polymerase enzyme moves along the coding strand. RNA pol adds ribonucleotide to align with the DNA template (T is replaced with U) b. RNA polymerase then catalyzes the formation of covalent phosphodiester linkages between the ribonucleotide to form the sugar phosphate backbone of the new RNA chain

  13. 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

  14. Steps of Transcription • Termination • Begins as RNA polymerase reaches the TERMINATION sequence (terminator) • Mechanism for removing RNA pol from DNA Rho independent release Involves formation of a fold or hairpin loop in new RNA The hairpin loop destabilizes the RNA pol/DNA/RNA complex Causes DNA to reclose, RNA released, and RNA pol recycled

  15. Steps of Transcription • Post transcriptional modification a. Only occurs in EUKARYOTIC cells (have a nucleus) b. 3 types of modifications to PRE messenger RNA 1. methylguanine “cap” is added to the 5’ end of RNA (thought to stabilize RNA) 2. a poly AAA tail is add to the 3’ end of the RNA 3. splicing of the mid region of the RNA occurs EXONS (coding region that make the proteins) INTRONS (spaces btwn the coding region)

  16. 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 • The 3 end gets a poly-A tail Figure 17.9

  17. 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 and joins exons Figure 17.10

  18. 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

  19. 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 • In many cases • Different exons code for the different domains in a protein Figure 17.12

  20. Translation (RNA to Protein) • Def – manufacture of protein from info on mRNA (occurs in the cytoplasm) • Sub cell players 1. mRNA (single stranded & made in nucleus) – organized into 3 letter words called CODONS. Each CODON (triplet) – specifies one amino acid.

  21. 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

  22. 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 messenger RNA • Is either translated into an amino acid or serves as a translational stop signal

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

  24. Translation 2. Ribosomes a. Participate in the “reading” of the codons in the mRNA (couple tRNA anticodons & mRNA codons) b. Consists of 2 sub units (one large & one small) Each SUB UNIT is composed of rRNA & protein rRNA – RIBOSOMAL RNA (made in the nucleolus in the nucleus)

  25. 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. • The ribosomal subunits • Are constructed of proteins and RNA molecules named ribosomal RNA or rRNA Figure 17.16a

  26. 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 P site • The A site • The E site E P A Figure 17.16b

  27. Translation • Transfer RNA (tRNA) a. Small RNA molecules that carry amino acids on one end & have an ANTICODON on the other end ANTICODON is complementary to a codon on the mRNA tRNA are NOT identical b/c they have anticodons for different proteins

  28. 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 • A tRNA molecule • Consists of a single RNA strand that is only about 80 nucleotides long • Is roughly L-shaped A C C

  29. 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

  30. 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

  31. 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

  32. Steps in TRANSLATION • We can divide translation into three stages 1. Initiation 2. Elongation 3. Termination

  33. Steps in TRANSLATION • Initiation • Small ribosomal sub unit slides along the mRNA until it reaches the first AUG codon (known as the start codon) • Initiation tRNA hydrogen bonds to the START CODON via its ANTICODON (AUG codes for METIONINE) • Large subunit of the RIBOSOME then wraps around the SMALL sub unit & the initiation tRNA

  34. Steps in TRANSLATION • Large subunit has 3 slots w/in A site (arrival)- new tRNA’s (and attached amino acids) enter P site (peptide bonding) – protein chain is held E site (exit) – exit site for empty tRNA’s • Large subunit places the initiation tRNA into the P slot Initiation complete!

  35. 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 • The initiation stage of translation • Brings together mRNA, tRNA bearing the first amino acid of the polypeptide, and two subunits of a ribosome

  36. Steps of Translation • Elongation (Protein chain grows by sequential addition of amino acids) a. New amino acids are brought into the A slot by their tRNA’s. • The tRNA bearing the appropriate anticodon hydrogen bonds to the CODON in the A slot • Amino acid(s) in the P slot are snipped from the tRNA & transferred onto the AMINO ACIDS that is linked to the tRNA in the A slot • TRANSLOCATION occurs (tRNA with a.acids move to P slot, empty tRNA moves to the E slot, leaving A site for next tRNA w/amino acids)

  37. 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 Elongation of the Polypeptide Chain • In the elongation stage of translation • Amino acids are added one by one to the preceding amino acid

  38. Steps of TRANSLATION • Termination • Begins when a stop codon enters the A slot of the large subunit (UAA, UAG, UGA) • There are no tRNA that can H bond to any of the 3 stop codons • An enzyme HYDROLASE enters A slot & catalyzes the hydrolytic cleavage of the last amino acid (carboxyl terminal) from tRNA When the protein is released, the ribosomal sub units fall apart the mRNA is released.

  39. 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 Termination of Translation • The final stage of translation is termination • When the ribosome reaches a stop codon in the mRNA

  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. Types of RNA in a Eukaryotic Cell Table 17.1

  42. RNA polymerase DNA mRNA Polyribosome Direction of transcription 0.25 m RNA polymerase DNA Polyribosome Polypeptide (amino end) Ribosome mRNA (5 end) Prokaryote vs. Eukaryote • Prokaryotic cells lack a nuclear envelope • Allowing translation to begin while transcription is still in progress Figure 17.22

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

  44. 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

  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 • The change of a single nucleotide in the DNA’s template strand • Leads to the production of an abnormal protein Figure 17.23

  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 • Can cause missense or nonsense 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 Figure 17.25

  49. Mutagens • Spontaneous mutations • Can occur during DNA replication, recombination, or repair

  50. Mutagens • Are physical or chemical agents that can cause mutations

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