1 / 50

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

simeon
Télécharger la présentation

From Gene to Protein

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  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

More Related