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The Flow of Genetic Information: DNA Transcription and Translation

This overview explores the process of DNA transcription and translation and how genetic information flows from DNA to proteins. It covers topics such as the role of RNA, transcription and translation in prokaryotic and eukaryotic cells, the genetic code, and the molecular components of transcription.

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The Flow of Genetic Information: DNA Transcription and Translation

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  1. Overview: The Flow of Genetic Information The information content of DNA Is in the form of specific sequences of nucleotides along the DNA strands Jan,09

  2. 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 Jan,09

  3. The ribosomeIs part of the cellular machinery for translation, polypeptide synthesis Jan,09

  4. Genes specify proteins via transcription and translation Jan,09

  5. Transcription • Is the synthesis of RNA under the direction of DNA • Produces messenger RNA (mRNA) • Translation • Is the actual synthesis of a polypeptide, which occurs under the direction of mRNA • Occurs on ribosomes Jan,09

  6. DNA TRANSCRIPTION mRNA Ribosome TRANSLATION Polypeptide (a) Prokaryotic cell. In a cell lacking a nucleus, mRNAproduced by transcription is immediately translatedwithout additional processing. prokaryotesTranscription and translation occur together Jan,09

  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 EukaryotesRNA transcripts are modified before becoming true mRNA Jan,09

  8. Cells are governed by a cellular chain of command • DNA RNA protein Jan,09

  9. Genetic information • Is encoded as a sequence of non overlapping base triplets, or codons Jan,09

  10. 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 3 mRNA Codon TRANSLATION Gly Phe Protein Trp Ser Figure 17.4 Amino acid The gene determines the sequence of bases along the length of an mRNA molecule Jan,09

  11. 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 GUG GCG GAG GGG G A codon in messenger RNAIs either translated into an amino acid or serves as a translational stop signal Jan,09

  12. Codons must be read in the correct reading frame • For the specified polypeptide to be produced Jan,09

  13. The genetic code • is nearly universal • Non-overlapp • Degenerate-- more than one codon specifies/ codes for a particular amino acid • -Shared by organisms from the simplest bacteria to the most complex animal Jan,09

  14. 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 Jan,09

  15. 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. 5 3 3 5 Template strand of DNA 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 DNA 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 Synhesis of an RNA Transcript • Initiation • Elongation • Termination Jan,09

  16. Non-template strand of DNA Elongation RNA nucleotides RNA polymerase T A C C A T A T U 3 C 3 end T G U A G G A E G A C A C C 5 A A T A G G T T Direction of transcription (“downstream”) 5 Template strand of DNA Newly made RNA Jan,09

  17. 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 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 Jan,09

  18. Elongation of the RNA Strand • It continues to untwist the double helix, exposing about 10 to 20 DNA bases at a time for pairing with RNA nucleotides Jan,09

  19. 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 Jan,09

  20. Termination of Transcription • The mechanisms of termination • Are different in prokaryotes and eukaryotes Jan,09

  21. 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 Jan,09

  22. 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 TRANSLATION Start codon Stop codon 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 Jan,09

  23. Eukaryotic mRNAs are modified. At the 5' end, a cap is added consisting of a modified GTP (guanosine triphosphate). This occurs at the beginning of transcription. The 5' cap is used as a recognition signal for ribosomes to bind to the mRNA.• At the 3' end, a poly(A) tail of 150 or more adenine nucleotides is added. The tail plays a role in the stability of the mRNA. Jan,09

  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 Split Genes and RNA Splicing • RNA splicing • Removes introns and joins exons Jan,09

  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 Jan,09

  26. Ribozymes • Ribozymes • Are catalytic RNA molecules that function as enzymes and can splice RNA Jan,09

  27. Translation is the RNA-directed synthesis of a polypeptide: • a closer look Jan,09

  28. Molecular Components of Translation • A cell translates an mRNA message into protein • With the help of transfer RNA (tRNA) Jan,09

  29. 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 Jan,09

  30. Molecules of tRNA are not all identical • Each carries a specific amino acid on one end • Each has an anticodon on the otherend Jan,09

  31. 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 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.) * G A C C G * C A G * U G * * G A G C Hydrogen bonds (a) G C U A G * A * A C * U A G A Anticodon The Structure and Function of Transfer • A tRNA molecule • Consists of a single RNA strand that is only about 80 nucleotides long • Is roughly L-shaped Jan,09

  32. 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 synthetaseJoins each amino acid to the correct tRNA Aminoacyl-tRNA synthetase (enzyme) 1. Active site binds the amino acid and ATP. Pyrophosphate Phosphates AMP Aminoacyl tRNA (an “activated amino acid”) Jan,09

  33. 1. Enzyme+ Amino acid+ ATP • Enzyme-Aa-AMP complex +2Pi • 2. Enzyme-Aa-AMP complex +tRNA • tRNA-Aa +AMP + Enzyme Jan,09

  34. Amino acid attachment site 5 3 Hydrogen bonds A A G 3 5 Anticodon Anticodon (c) Symbol used in the presentation (b) Three-dimensional structure Jan,09

  35. Ribosomes • Facilitate the specific coupling of tRNA anticodons with mRNA codons during protein synthesis Jan,09

  36. DNA TRANSCRIPTION mRNA Ribosome TRANSLATION Polypeptide Exit tunnel Growing polypeptide tRNA molecules Large subunit E P A Small subunit 5 3 mRNA 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 subunitsAre constructed of proteins and RNA molecules named ribosomal RNA or rRNA Jan,09

  37. P site (Peptidyl-tRNA binding site) A site (Aminoacyl- tRNA binding site) E site (Exit site) Large subunit mRNA binding site Small subunit 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. We can divide translation into three stagesInitiation, Elongation, Termination Jan,09

  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. Jan,09

  39. 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 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 Jan,09

  40. 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 Elongation of the Polypeptide Chain-Amino acids are added one by one to the preceding amino acid Jan,09

  41. 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 Termination of Translation-When the ribosome reaches a stop codon in the mRNA Jan,09

  42. 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). A number of ribosomes can translate a single mRNA molecule simultaneously- Polyribosome Jan,09

  43. Completing and Targeting the Functional Protein • Polypeptide chains • Undergo modifications after the translation process Jan,09

  44. Protein Folding and Post-Translational Modifications • After translation • Proteins may be modified in ways that affect their three-dimensional shape Jan,09

  45. 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 Jan,09

  46. Proteins destined for the endomembrane system or for secretion • Must be transported into the ER • Have signal peptides to which a signal-recognition particle (SRP) binds, enabling the translation ribosome to bind to the ER Jan,09

  47. 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 Jan,09

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