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From Gene to Protein

From Gene to Protein. Overview of Transcription & Translation. One Gene - One Polypeptide. Genes provide the instructions for making specific proteins Linear sequence of genes in DNA ultimately determines the linear sequence of amino acids in the polypeptide

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From Gene to Protein

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  1. From Gene to Protein Mader 2007-08

  2. Overview of Transcription & Translation Mader 2007-08

  3. One Gene - One Polypeptide • Genes provide the instructions for making specific proteins • Linear sequence of genes in DNA ultimately determines the linear sequence of amino acids in the polypeptide • DNA does not make proteins directly • Requires RNA Mader 2007-08

  4. Transcription • Synthesis of RNA using DNA as a template • mRNA carries message from nucleus to cytoplasm • Translation • Synthesis of a polypeptide under the direction of mRNA • Occurs in the cytoplasm Mader 2007-08

  5. Nucleotides Ribose Phosphate group A, U, G, C Single stranded mRNA, tRNA, rRNA Structure of RNA Mader 2007-08

  6. Function of Genes • Genes Specify Enzymes • Beadle and Tatum: • Experiments on fungus Neurospora crassa • Proposed that each gene specifies the synthesis of one enzyme • One-gene-one-enzyme hypothesis • Genes Specify a Polypeptide • A gene is a segment of DNA that specifies the sequence of amino acids in a polypeptide • Suggests that genetic mutations cause changes in the primary structure of a protein Mader 2007-08

  7. Structure of RNA Mader 2007-08

  8. Genetic Code • DNA and RNA made of sequence of nucleotides (4 nucleotides) • Polypeptides made of sequence of amino acids (20 amino acids) • Code required to translate the sequence of nucleotides into a sequence of amino acids • Triplet code of nucleotides as minimum code length to code for all 20 a.a. (43=64) Mader 2007-08

  9. The Genetic Code • The unit of a code consists of codons, each of which is a unique arrangement of symbols • Each of the 20 amino acids found in proteins is uniquely specified by one or more codons • The symbols used by the genetic code are the mRNA bases • Function as “letters” of the genetic alphabet • Genetic alphabet has only four “letters” (U, A, C, G) Mader 2007-08

  10. Genetic Code • Codons in the genetic code are all three bases (symbols) long • Codon = three-nucleotide sequence in mRNA that specifies an amino acid • Function as “words” of genetic information • Permutations: • There are 64 possible arrangements of four symbols taken three at a time • Often referred to as triplets • Genetic language only has 64 “words” • 61 of 64 triplets code for amino acids (3 stop codons) Mader 2007-08

  11. Genetic Code • Redundancy - more than 1 codon codes for each amino acid • No ambiguity - A codon only codes for 1 amino acid Mader 2007-08

  12. The Genetic Code (mRNA) Mader 2007-08

  13. Transcription of mRNA • A single chromosomes consists of one very long molecule encoding hundreds or thousands of genes • The genetic information in a gene describes the amino acid sequence of a protein • The information is in the base sequence of one side (the “sense” strand) of the DNA molecule • The gene is the functional equivalent of a “sentence” Mader 2007-08

  14. Transcription of mRNA • The segment of DNA corresponding to a gene is unzipped to expose the bases of the sense strand • The genetic information in the gene is transcribed (rewritten) into an mRNA molecule • The exposed bases in the DNA determine the sequence in which the RNA bases will be connected together • RNA polymerase connects the loose RNA nucleotides together • The completed transcript contains the information from the gene, but in a mirror image, or complementary form Mader 2007-08

  15. Transcription Mader 2007-08

  16. 1. Initiation 2. Elongation 3. Termination Mader 2007-08

  17. Promoter = region of DNA where RNA polymerase binds and where transcription begins • about 100 nucleotides long • Transcription factors = DNA-binding proteins which bind to specific DNA nucleotide sequences at the promoter and help RNA polymerase recognize and bind the promoter • TATA box • RNA polymerase unwinds the helix and begins transcription Mader 2007-08

  18. Elongation • RNA polymerase II • untwists the double helix, exposing 10-20 bases at a time • adds RNA nucleotides to the 3’ end of the elongating strand • follows base-pairing rules: A-U, G-C • mRNA grows about 30-60 nucleotides/second • Several molecules of RNA polymerase II can simultaneously transcribe the same gene • can produce proteins in large amounts Mader 2007-08

  19. Termination of Transcription • Transcription proceeds until RNA polymerase transcribes a terminator sequence which functions as a terminator signal • In prokaryotes, transcription stops right at end of termination signal • In eukaryotes, transcription proceeds about 10-35 nucleotides past termination signal • In eukaryotes, most common termination signal is AAUAAA Mader 2007-08

  20. Post-Transcriptional Modification • 5’ cap = modified guanine nucleotide (GPPP) added to 5’ end of mRNA after transcription begins • protects the growing mRNA from degradation by hydrolytic enzymes • helps ribosome recognize attachment site for translation • Poly-A tail = sequence of 30-200 As added to 3’ end before it exits nucleus • Inhibit degradation of mRNA in cytoplasm, facilitate attachment of ribosome, facilitate export from nucleus to cytoplasm Mader 2007-08

  21. mRNA Processing in Eukaryotes Mader 2007-08

  22. RNA Splicing • RNA splicing removes introns and joins exons to form functional mRNA • Introns = intervening sequences = noncoding sequences of DNA; are transcribed, but not translated • Exons = expressed sequences = coding sequences of a gene Mader 2007-08

  23. Functions of Introns • As organismal complexity increases; • Number of protein-coding genes does not keep pace • But the proportion of the genome that is introns increases • Humans: • Genome has only about 25,000 coding genes • Up to 95% of this DNA genes is introns • Possible functions of introns: • More bang for buck • Exons might combine in various combinations • Would allow different mRNAs to result from one segment of DNA • Introns might regulate gene expression • Exciting new picture of the genome is emerging Mader 2007-08

  24. Sliceosomes • Enzymes excise introns and join exons to form a mRNA with a continuous coding sequence • Short nucleotide sequences at the end of introns are recognized by snRNPs which form a spliceosome which cuts introns at splice sites and splices exons together Mader 2007-08

  25. Importance of Introns • May play regulatory role in cell • May control gene activity • May help regulate the export of mRNA to the cytoplasm • May allow a single gene to direct synthesis of different proteins • Single pre-mRNA is processed differently • Increase probability of recombination to increase genetic diversity Mader 2007-08

  26. Translation: Basic Concept • Proteins are synthesized according to genetic message of sequential codons along mRNA • tRNA is the interpreter • tRNA aligns appropriate a.a. to form new polypeptide by • transferring a.a. from cytoplasm’s a.a. pool to ribosome • recognizing the correct codons in mRNA • Molecules of tRNA are specific for only one a.a. Mader 2007-08

  27. tRNA • tRNA molecules come in 64 different kinds • All very similar except that • One end bears a specific triplet (of the 64 possible) called the anticodon • Other end binds with a specific amino acid type • tRNA synthetases attach correct amino acid to the correct tRNA molecule • All tRNA molecules with a specific anticodon will always bind with the same amino acid Mader 2007-08

  28. Structure of tRNA Mader 2007-08

  29. Structure & Function of tRNA Mader 2007-08

  30. Anticodon = nucleotide triplet in tRNA that base pairs with complementary codon in mRNA • Wobble = ability of one tRNA to recongnize two or three different mRNA codons; occurs when the third base of the tRNA can H bond with more than one kind of base in the third position of the codon • U can pair with A or G • some tRNAs contain modified base (I) that can H bond with U, C, or A Mader 2007-08

  31. Aminoacyl-tRNA Synthetase • Catalyzes attachment of tRNA to a.a. • 20 types of aminoacyl-tRNA synthetase • Active site fits only a specific combination of a.a. and tRNA • Endergonic reaction driven by hydrolysis of ATP Mader 2007-08

  32. Ribosomes • Ribosomal RNA (rRNA): • Produced from a DNA template in the nucleolus • Combined with proteins into large and small ribosomal subunits • A completed ribosome has three binding sites to facilitate pairing between tRNA and mRNA • The E (for exit) site • The P (for peptide) site, and • The A (for amino acid) site Mader 2007-08

  33. Ribosomal Structure and Function Mader 2007-08

  34. Large and small subunits separated when not involved in protein synthesis • About 60% rRNA and 40% protein • mRNA binding site + 3 tRNA binding sites • tRNA binding sites • A site = aminoacyl-tRNA binding site • P site = peptidyl-tRNA binding site • E site = exit site Mader 2007-08

  35. Steps in Translation:#1 - Initiation • Components necessary for initiation are: • Small ribosomal subunit • mRNA transcript • Initiator tRNA, and • Large ribosomal subunit • Initiation factors (special proteins that bring the above together) • Initiator tRNA: • Always has the UAC anticodon • Always carries the amino acid methionine • Capable of binding to the P site Mader 2007-08

  36. Initiation of Translation • Binding of small ribosomal subunit to mRNA and initiator tRNA • In prokaryotes, rRNA in small subunit base pairs with specific nucleotides in leader sequence of mRNA • In eukaryotes, the 5’ cap of the mRNA aids in the binding of leader sequence to small ribosomal subunit • Start codon is AUG = methionine Mader 2007-08

  37. Steps in Translation:#2 - Elongation • “Elongation” refers to the growth in length of the polypeptide • RNA molecules bring their amino acid fares to the ribosome • Ribosome reads a codon in the mRNA • Allows only one type of tRNA to bring its amino acid • Must have the anticodon complementary to the mRNA codon being read • Joins the ribosome at it’s A site • Methionine of initiator is connected to amino acid of 2nd tRNA by peptide bond Mader 2007-08

  38. Steps in Translation:#2 – Elongation (cont.) • Second tRNA moves to P site (translocation) • Spent initiator moves to E site and exits • Ribosome reads the next codon in the mRNA • Allows only one type of tRNA to bring its amino acid • Must have the anticodon complementary to the mRNA codon being read • Joins the ribosome at it’s A site • Dipeptide on 2nd amino acid is connected to amino acid of 3nd tRNA by peptide bond Mader 2007-08

  39. Elongation Cycle of Translation Energy from hydrolysis of GTP Energy from hydrolysis of GTP peptidyl transferase Mader 2007-08

  40. Steps in Translation:#2 - Elongation Mader 2007-08

  41. Termination of Translation • Translation continues until termination codon (stop codon) is reached • Stop codons are UAA, UAG, UGA; do not code for amino acids • Release factor binds to A site • Hydrolyzes bond between polypeptide and tRNA in P site • Polypeptide and tRNA released from ribosome • Translation complex dissociates Mader 2007-08

  42. Polyribosomes • Clusters of ribosomes that translate a mRNA at once, making many copies of the polypeptide • Found in both prokaryotes and eukaryotes Mader 2007-08

  43. Post-translational Modifications • Primary structure (a.a. sequence) determines secondary and tertiary structure (3D) • Chemical modification • Sugars, lipids, phosphate groups, … may be attached to some a.a. • Chain-length modification • One or more a.a. may be cleaved from leading end of polypeptide chain • Single polypeptide chains may be divided into two or more pieces • Two or more polypeptides may join as subunits of a protein that has quaternary structure Mader 2007-08

  44. Protein Destinations • Signal peptides target some polypeptides to specific destinations in the cell • Proteins made by free ribosomes function in cytosol • Proteins made by bound ribosomes (attached to ER) generally destined for: • Membrane inclusion (membrane-bound enzymes) • Secretion from cell (hormones) Mader 2007-08

  45. Polypeptides destined for endomembrane system or secretion marked by a signal peptide of 16-20 hydrophobic a.a. at leading end of growing polypeptide • Signal peptide recognized by SRP which binds signal peptide and then links to SRP receptor protein on ER membrane Mader 2007-08

  46. Transcription & Translation in Prokaryotes • One type of RNA polymerase • No transcription factors • No post-transcriptional modification • Ribosomes smaller with different molecular composition • Transcription and translation are simultaneous because no nucleus Mader 2007-08

  47. Molecular Basis of Sickle-Cell Disease Point mutation = mutation in one to a few base pais in gene Mader 2007-08

  48. Types of Point Mutations Mader 2007-08

  49. Summary of Transcription & Translation Mader 2007-08

  50. Summary of Gene Expression(Eukaryotes) Mader 2007-08

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