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Chapter 17 From Gene to Protein PowerPoint® Lecture Presentations for Biology Eighth Edition Neil Campbell and Jane Reece Lectures by Chris Romero, updated by Erin Barley with contributions from Joan Sharp Copyright © 2008 Pearson Education, Inc., publishing as Pearson Benjamin Cummings Overview: The Flow of Genetic Information • The information content of DNA is in the form of specific sequences of nucleotides – The DNA inherited by an organism leads to specific traits by dictating the synthesis of proteins • Proteins are the links between genotype and phenotype • Gene expression, the process by which DNA directs protein synthesis, includes two stages: transcription and translation Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Experiments Leading to Understanding the Link between Genes and Proteins • Nirenberg & Matthaei – determining the language of the genetic code. – http://bcs.whfreeman.com/thelifewire/content /chp12/1202002.html • Beadle & Tatum – One Gene…One Enzyme hypothesis. – http://www.dnalc.org/view/16360-Animation16-One-gene-makes-one-protein-.html Evidence from the Study of Metabolic Defects • In 1909, British physician Archibald Garrod first suggested that genes dictate phenotypes through enzymes that catalyze specific chemical reactions • He thought symptoms of an inherited disease reflect an inability to synthesize a certain enzyme • Linking genes to enzymes required understanding that cells synthesize and degrade molecules in a series of steps, a metabolic pathway Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Nutritional Mutants in Neurospora: Scientific Inquiry • George Beadle and Edward Tatum exposed bread mold to X-rays, creating mutants that were unable to survive on minimal medium as a result of inability to synthesize certain molecules • Using crosses, they identified three classes of arginine-deficient mutants, each lacking a different enzyme necessary for synthesizing arginine • They developed a one gene–one enzyme hypothesis, which states that each gene dictates production of a specific enzyme Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Fig. 17-2 EXPERIMENT No growth: Mutant cells cannot grow and divide Growth: Wild-type cells growing and dividing Minimal medium RESULTS Classes of Neurospora crassa Wild type Class I mutants Class II mutants Class III mutants Condition Minimal medium (MM) (control) MM + ornithine MM + citrulline MM + arginine (control) CONCLUSION Wild type Precursor Gene A Gene B Gene C Class I mutants Class II mutants Class III mutants (mutation in (mutation in (mutation in gene B) gene A) gene C) Precursor Precursor Precursor Enzyme A Enzyme A Enzyme A Enzyme A Ornithine Ornithine Ornithine Ornithine Enzyme B Enzyme B Enzyme B Enzyme B Citrulline Citrulline Citrulline Citrulline Enzyme C Enzyme C Enzyme C Enzyme C Arginine Arginine Arginine Arginine The Products of Gene Expression: A Developing Story • Some proteins aren’t enzymes, so researchers later revised the hypothesis: one gene–one protein • Many proteins are composed of several polypeptides, each of which has its own gene • Therefore, Beadle and Tatum’s hypothesis is now restated as the one gene–one polypeptide hypothesis • Note that it is common to refer to gene products as proteins rather than polypeptides Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Basic Principles of Transcription and Translation • RNA is the intermediate between genes and the proteins for which they code • Transcription is the synthesis of RNA under the direction of DNA – Transcription produces messenger RNA (mRNA) • Translation is the synthesis of a polypeptide, which occurs under the direction of mRNA – Ribosomes are the sites of translation • A primary transcript is the initial RNA transcript from any gene • The central dogma is the concept that cells are governed by a cellular chain of command: DNA RNA protein Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Overview of Gene Expression • Genes contain the blueprints for building proteins in cells. • RNA is the bridge between DNA and its protein. • The “genetic code” is the sequence of bases in DNA that will code for specific amino acids in a growing polypeptide (protein). – There are 20 possible amino acids. Types of RNA • • RNA is a nitrogenous base that consists of a ribose (5 carbon sugar). – In RNA, the base Uracil is substituted for Thymine in DNA. – The RNA nucleic acid is single stranded. In most cells, RNA molecules are involved in just one job – protein synthesis! – • The assembly of amino acids into proteins is controlled by RNA There are 3 main types of RNA: 1. Messenger RNA (mRNA) 2. Ribosomal RNA (rRNA) 3. Transfer RNA (tRNA) Protein Synthesis • Building a protein involves 2 stages: – (1) Transcription; (2) Translation • Transcription occurs in the nucleus – DNA mRNA • Making mRNA from a DNA template in the nucleus. • Translation occurs in the cytoplasm at a ribosome – mRNA protein (using tRNA and rRNA) • Translating instructions in mRNA into a growing polypeptide protein chain at the ribosome. Prokaryotic v. Eukaryotic Gene Expression http://highered.mcgraw-hill.com/olc/dl/120077/bio25.swf The Genetic Code • How are the instructions for assembling amino acids into proteins encoded into DNA? • There are 20 amino acids, but there are only four nucleotide bases in DNA • How many bases correspond to an amino acid? Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Codons: Triplets of Bases • The flow of information from gene to protein is based on a triplet code: a series of nonoverlapping, three-nucleotide words • These triplets are the smallest units of uniform length that can code for all the amino acids • Example: AGT at a particular position on a DNA strand results in the placement of the amino acid serine at the corresponding position of the polypeptide to be produced Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Codons: Triplets of Bases • During transcription, one of the two DNA strands called the template strand provides a template for ordering the sequence of nucleotides in an RNA transcript • During translation, the mRNA base triplets, called codons, are read in the 5 to 3 direction • Each codon specifies the amino acid to be placed at the corresponding position along a polypeptide • Codons along an mRNA molecule are read by translation machinery in the 5 to 3 direction • Each codon specifies the addition of one of 20 amino acids Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Fig. 17-4 DNA molecule Gene 2 Gene 1 Gene 3 DNA template strand TRANSCRIPTION mRNA Codon TRANSLATION Protein Amino acid Cracking the Code • All 64 codons were deciphered by the mid1960s • Of the 64 triplets, 61 code for amino acids; 3 triplets are “stop” signals to end translation • The genetic code is redundant but not ambiguous; no codon specifies more than one amino acid • Codons must be read in the correct reading frame (correct groupings) in order for the specified polypeptide to be produced Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Third mRNA base (3 end of codon) First mRNA base (5 end of codon) Fig. 17-5 Second mRNA base Fig. 17-6 (a) Tobacco plant expressing a firefly gene (b) Pig expressing a jellyfish gene Types of RNA http://www-class.unl.edu/biochem/gp2/m_biology/animation/gene/gene_a1.html rR NA Transcription Section 12-3 http://bcs.whfreeman.com/thelifewire/content/chp12/1202001.html Adenine (DNA and RNA) Cystosine (DNA and RNA) Guanine(DNA and RNA) Thymine (DNA only) Uracil (RNA only) RNA polymerase DNA RNA During transcription, RNA polymerase uses one strand of DNA as a template to assemble nucleotides into a strand of RNA. Transcription • Transcription, the first stage of gene expression, is the DNA-directed synthesis of mRNA • RNA synthesis is catalyzed by RNA polymerase, which pries the DNA strands apart and hooks together the RNA nucleotides • RNA synthesis follows the same base-pairing rules as DNA, except uracil substitutes for thymine • The DNA sequence where RNA polymerase attaches is called the promoter; in bacteria, the sequence signaling the end of transcription is called the terminator • The stretch of DNA that is transcribed is called a transcription unit Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Fig. 17-7 Promoter Transcription unit 5 3 Start point RNA polymerase 3 5 DNA 1 Initiation 5 3 RNA transcript RNA polymerase Template strand of DNA 3 2 Elongation Rewound DNA 5 3 RNA nucleotides 3 5 Unwound DNA 3 5 5 5 Direction of transcription (“downstream”) 3 Termination 3 5 5 3 5 3 end 5 3 RNA transcript Nontemplate strand of DNA Elongation Completed RNA transcript 3 Newly made RNA Template strand of DNA RNA Polymerase Binding and Initiation of Transcription • Promoters signal the initiation of RNA synthesis • Transcription factors mediate the binding of RNA polymerase and the initiation of transcription • The completed assembly of transcription factors and RNA polymerase II bound to a promoter is called a transcription initiation complex • A promoter called a TATA box is crucial in forming the initiation complex in eukaryotes Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Fig. 17-8 1 Promoter A eukaryotic promoter includes a TATA box Template 5 3 3 5 TATA box Start point Template DNA strand 2 Transcription factors Several transcription factors must bind to the DNA before RNA polymerase II can do so. 5 3 3 5 3 Additional transcription factors bind to the DNA along with RNA polymerase II, forming the transcription initiation complex. RNA polymerase II Transcription factors 5 3 3 5 5 RNA transcript Transcription initiation complex http://bcs.whfreeman.com/thelif ewire/content/chp14/1402002.ht ml Concept 17.3: Eukaryotic cells modify RNA after transcription • Enzymes in the eukaryotic nucleus modify premRNA before the genetic messages are dispatched to the cytoplasm • During RNA processing, both ends of the primary transcript are usually altered • Also, usually some interior parts of the molecule are cut out, and the other parts spliced together Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Alteration of mRNA Ends • Each end of a pre-mRNA molecule is modified in a particular way: – The 5 end receives a modified nucleotide 5 cap – The 3 end gets a poly-A tail • These modifications share several functions: – They seem to facilitate the export of mRNA – They protect mRNA from hydrolytic enzymes – They help ribosomes attach to the 5 end Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Fig. 17-9 50 to 250 adenine nucleotides added to the 3’ end created by cleavage downstream of the termination signal. A modified guanosine triphosphate added to the 5’ end. 5 G Protein-coding segment Polyadenylation signal 3 P P P 5 Cap AAUAAA 5 UTR Start codon Stop codon 3 UTR AAA…AAA Poly-A tail Split Genes and RNA Splicing • Most eukaryotic genes and their RNA transcripts have long noncoding stretches of nucleotides that lie between coding regions • These noncoding regions are called intervening sequences, or introns • The other regions are called exons because they are eventually expressed, usually translated into amino acid sequences • RNA splicing removes introns and joins exons, creating an mRNA molecule with a continuous coding sequence Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Fig. 17-10 5 Exon Intron Exon Exon Intron 3 Pre-mRNA 5 Cap Poly-A tail 1 30 31 Coding segment mRNA 5 Cap 1 5 UTR 104 105 146 Introns cut out and exons spliced together Poly-A tail 146 3 UTR Pre-RNA Splicing http://bcs.whfreeman.com/thelifewire/content/chp14/1402001.html • In some cases, RNA splicing is carried out by spliceosomes • Spliceosomes consist of a variety of proteins and several small nuclear ribonucleoproteins (snRNPs) that recognize the splice sites • snRNP’s : small nuclear ribonucleoproteins – these look for special sequences at ends of introns to know where to cut – made up of snRNA (small nuclear RNA) • Several different snRNP’s join with additional proteins to form the splicesome – this interacts with the splice sites at intron ends, and cuts them out – then joins the two exons together. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Figure 17.10 The roles of snRNPs and spliceosomes in mRNA splicing 1. Pre-mRNA containing exons and introns combines with small nuclear ribonuceloproteins (snRNPs) and other proteins to form a molecular complex call spliceosome. 2. Within the spliceosome, snRNA base-pairs with nucleotides at the ends of the intron. 3. The RNA transcript is cut to release the intron, and the exons are spliced together. The spliceosome then comes apart, releasing mRNA, which now contains only exons. The Functional and Evolutionary Importance of Introns • Some genes can encode more than one kind of polypeptide, depending on which segments are treated as exons during RNA splicing • Such variations are called alternative RNA splicing • Because of alternative splicing, the number of different proteins an organism can produce is much greater than its number of genes Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Translation • Translation is the RNA-directed synthesis of a polypeptide • Generally: is the reading of the codons on the mRNA strand and the sequencing of them into an amino acid sequence – polypeptide. • The Players: – mRNA: already processed within the nucleus via transcription, will be the template for the sequence of amino acids. – tRNA: transfers amino acids from the cytoplasm to the ribosome. – rRNA (ribosome): adds amino acids together from the tRNA and in the sequence of the mRNA. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Fig. 17-13 http://highered.mcgrawhill.com/olcweb/cgi/pluginpop.cgi?it=swf::535:: 535::/sites/dl/free/0072437316/120077/micro0 6.swf::Protein%20Synthesis Amino acids Polypeptide tRNA with amino acid attached Ribosome tRNA Anticodon Codons 5 mRNA 3 Fig. 17-14 3 Amino acid attachment site 5 Hydrogen bonds Anticodon (a) Two-dimensional structure Amino acid attachment site 5 3 Hydrogen bonds 3 Anticodon (b) Three-dimensional structure 5 Anticodon (c) Symbol used in this book Fig. 17-15-4 Aminoacyl-tRNA synthetase (enzyme) Amino acid P P P Adenosine ATP P P Pi Pi Adenosine tRNA Aminoacyl-tRNA synthetase Pi tRNA P Adenosine AMP Computer model Aminoacyl-tRNA (“charged tRNA”) Fig. 17-16 Growing polypeptide Exit tunnel tRNA molecules EP Large subunit A Small subunit 5 mRNA 3 (a) Computer model of functioning ribosome P site (Peptidyl-tRNA binding site) E site (Exit site) A site (AminoacyltRNA binding site) E P A mRNA binding site Large subunit Small subunit (b) Schematic model showing binding sites Growing polypeptide Amino end Next amino acid to be added to polypeptide chain E mRNA 5 tRNA 3 Codons (c) Schematic model with mRNA and tRNA http://highered.mcgrawhill.com/olc/dl/120077/mi cro06.swf Building a Polypeptide • The three stages of translation: – Initiation (requires energy “GTP”) – Elongation (requires energy “GTP”) – Termination • All three stages require protein “factors” that aid in the translation process Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Fig. 17-17 INITIATION OF TRANSLATION 3 U A C 5 5 A U G 3 Initiator tRNA Large ribosomal subunit P site GTP GDP E mRNA 5 Start codon mRNA binding site 3 Small ribosomal subunit 5 A 3 Translation initiation complex Fig. 17-18-4 ELONGATION OF TRANSLATION Amino end of polypeptide E 3 mRNA Ribosome ready for next aminoacyl tRNA P A site site 5 GTP GDP E E P A P A GDP GTP E P A Fig. 17-19-3 TERMINATION OF TRANSLATION Release factor Free polypeptide 5 3 5 5 Stop codon (UAG, UAA, or UGA) 3 2 GTP 2 GDP 3 Fig. 17-20 Growing polypeptides Completed polypeptide Incoming ribosomal subunits Start of mRNA (5 end) (a) End of mRNA (3 end) Ribosomes mRNA (b) 0.1 µm Completing and Targeting the Functional Protein • Often translation is not sufficient to make a functional protein…THEREFORE…polypeptide chains are modified after translation • Completed proteins are targeted to specific sites in the cell – During and after synthesis, a polypeptide chain spontaneously coils and folds into its three-dimensional shape – Proteins may also require post-translational modifications before doing their job – Some polypeptides are activated by enzymes that cleave them – Other polypeptides come together to form the subunits of a protein Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Targeting Polypeptides to Specific Locations • Two populations of ribosomes are evident in cells: free ribsomes (in the cytosol) and bound ribosomes (attached to the ER) • Free ribosomes mostly synthesize proteins that function in the cytosol • Bound ribosomes make proteins of the endomembrane system and proteins that are secreted from the cell • Ribosomes are identical and can switch from free to bound Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Review – PROTEIN SYNTHESIS • Transcription – DNA mRNA (in nucleus) – In eukaryotes will have RNA processing (in nucleus). • Translation – mRNA Polypeptide (at ribosome in cytoplasm) • Coiling & Folding – Three dimensional (in cytoplasm as translation is occurring) – Chaperone proteins involved Protein Synthesis Nucleus Messenger RNA Messenger RNA is transcribed in the nucleus. Phenylalanine tRNA The mRNA then enters the cytoplasm and attaches to a ribosome. Translation begins at AUG, the start codon. Each transfer RNA has an anticodon whose bases are complementary to a codon on the mRNA strand. The ribosome positions the start codon to attract its anticodon, which is part of the tRNA that binds methionine. The ribosome also binds the next codon and its anticodon. Ribosome Go to Section: mRNA Transfer RNA Methionine mRNA Lysine Start codon Protein Synthesis Continued Concept 17.5: Point mutations can affect protein structure and function • Mutations are changes in the genetic material of a cell or virus • Point mutations are chemical changes in just one base pair of a gene • The change of a single nucleotide in a DNA template strand can lead to the production of an abnormal protein Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Fig. 17-22 Wild-type hemoglobin DNA Mutant hemoglobin DNA C T T C A T 3 5 3 G T A 5 G A A 3 5 mRNA 5 5 3 mRNA G A A Normal hemoglobin Glu 3 5 G U A Sickle-cell hemoglobin Val 3 Types of Point Mutations • Point mutations within a gene can be divided into two general categories – Base-pair substitutions – Base-pair insertions or deletions Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Fig. 17-23 http://nortonbooks.com/col lege/biology/animations/ch 13a08.htm Wild-type DNA template strand 3 5 5 3 mRNA 5 3 Protein Stop Amino end Carboxyl end A instead of G 3 5 Extra A 5 3 3 5 3 5 U instead of C 5 5 3 Extra U 3 Stop Stop Silent (no effect on amino acid sequence) Frameshift causing immediate nonsense (1 base-pair insertion) T instead of C missing 3 5 5 3 3 5 3 5 5 3 A instead of G missing 5 3 Stop Missense Frameshift causing extensive missense (1 base-pair deletion) missing A instead of T 5 3 3 5 U instead of A 5 5 3 3 5 missing 3 5 Stop Stop Nonsense (a) Base-pair substitution 3 No frameshift, but one amino acid missing (3 base-pair deletion) (b) Base-pair insertion or deletion Mutagens • Spontaneous mutations can occur during DNA replication, recombination, or repair • Mutagens are physical or chemical agents that can cause mutations Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Comparing Gene Expression in Bacteria, Archaea, and Eukarya • Archaea are prokaryotes, but share many features of gene expression with eukaryotes • Bacteria and eukarya differ in their RNA polymerases, termination of transcription and ribosomes; archaea tend to resemble eukarya in these respects • Bacteria can simultaneously transcribe and translate the same gene • In eukarya, transcription and translation are separated by the nuclear envelope • In archaea, transcription and translation are likely coupled Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings What Is a Gene? Revisiting the Question • The idea of the gene itself is a unifying concept of life • We have considered a gene as: – A discrete unit of inheritance – A region of specific nucleotide sequence in a chromosome – A DNA sequence that codes for a specific polypeptide chain Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Fig. 17-25 DNA TRANSCRIPTION 3 RNA polymerase 5 RNA transcript RNA PROCESSING Exon RNA transcript (pre-mRNA) Intron Aminoacyl-tRNA synthetase NUCLEUS Amino acid CYTOPLASM AMINO ACID ACTIVATION tRNA mRNA Growing polypeptide 3 A Activated amino acid P E Ribosomal subunits 5 TRANSLATION E A Codon Ribosome Anticodon The Essay – IT’S A DOOZY! • A portion of a specific DNA molecule consists of the following sequence of nucleotide triplets: TAC • GAA CTT CGG TCC This DNA sequence codes for the following short polypeptide: methionine - leucine - glutamic acid - proline - arginine a) Describe the steps in the synthesis of this polypeptide. b) What would be the effect of a deletion or an addition in one of the DNA nucleotides? c) What would be the effect of a substitution in one of the nucleotides? d) Cells regulate both protein synthesis and protein activity. Discuss TWO specific mechanisms of protein regulation in eukaryotic cells. You should now be able to: 1. Describe the contributions made by Garrod, Beadle, and Tatum to our understanding of the relationship between genes and enzymes 2. Briefly explain how information flows from gene to protein 3. Compare transcription and translation in bacteria and eukaryotes 4. Explain what it means to say that the genetic code is redundant and unambiguous Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings 5. Include the following terms in a description of transcription: mRNA, RNA polymerase, the promoter, the terminator, the transcription unit, initiation, elongation, termination, and introns 6. Include the following terms in a description of translation: tRNA, wobble, ribosomes, initiation, elongation, and termination Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings