Transcript Ch 17
Chapter 17 From Gene to Protein How an Organism’s Genotype Produces Its Phenotype 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 links between genotype and phenotype Gene expressionprocess by which DNA directs protein synthesis Transcription DNA Translation RNA • 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 Protein EXPERIMENT Growth: Wild-type cells growing and dividing Classes of Neurospora crassa No growth: Mutant cells cannot grow and divide Wild type Minimal medium (MM) (control) Minimal medium • Using crosses, they identified three classes of argininedeficient mutants, each lacking a different enzyme necessary for synthesizing arginine MM ornithine Condition • 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 Class I mutants Class II mutants Class III mutants MM citrulline MM arginine (control) Summary of results Gene (codes for enzyme) Gene A Gene B Gene C Can grow with or without any supplements Can grow on ornithine, citrulline, or arginine Can grow only on citrulline or arginine Require arginine to grow Wild type Class I mutants (mutation in gene A) Precursor 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 Class II mutants Class III mutants (mutation in (mutation in gene B) gene C) Beadle, Tatum, and colleagues one gene- one enzyme states that each gene dictates production of a specific enzyme one gene- one protein researchers revised since some proteins aren’t enzymes one gene–one polypeptide hypothesisstates that the function of an individual gene is to dictate the production of a specific polypeptide DNA TRANSCRIPTION mRNA Ribosome TRANSLATION Polypeptide (a) Bacterial cell DNA DNA TRANSCRIPTION TRANSCRIPTION mRNA mRNA Ribosome Ribosome TRANSLATION TRANSLATION Polypeptide Polypeptide In prokaryotes, mRNA produced by transcription is immediately translated without more processing Prokaryotic Prokaryotic cell cell Nuclear Nuclear envelope envelope DNA DNA TRANSCRIPTION TRANSCRIPTION Pre-mRNA RNA PROCESSING mRNA Ribosome TRANSLATION Polypeptide Eukaryotic Eukaryotic cell cell In a eukaryotic cell, the nuclear envelope separates transcription from translation primary transcript Eukaryotic RNA transcripts are modified through RNA processing to yield finished mRNA 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 • So how many bases correspond to an amino acid? • The flow of information from gene to protein is based on a triplet code a series of nonoverlapping, three-nucleotide words • • • During transcription, a DNA strand called the template strand provides a template for ordering the sequence of nucleotides in an RNA transcript The template strand is always the same strand for a given gene During translation, the mRNA base triplets, codons, DNA molecule Gene 2 Gene 1 Gene 3 DNA template strand “coding strand” TRANSCRIPTION are read in the 5 to 3 direction • Each codon specifies the amino acid to be placed at the corresponding position along a polypeptide mRNA Codon TRANSLATION Protein Amino acid Genetic Code • Second mRNA base 64 codons • redundant (>1 codon may specify a particular aa) but not ambiguous; no codon specifies more than 1 aa • codons must be read in the correct reading frame (correct groupings) in order for the specified polypeptide to be produced Third mRNA base (3 end) – includes 3 Stop codons Evolution of the Genetic Code • The genetic code is nearly universal, shared by the simplest bacteria to the most complex animals • Genes can be transcribed and translated after being transplanted from one species to another (a) Tobacco plant expressing a firefly gene (b) Pig expressing a jellyfish gene April 30, 2012 With apologies to the Smashing Pumpkins Announcements • Final exam: Friday, May 11 6-8 PM – Written study guide tomorrow – MC practice exam by Friday • Quiz over 16 and 17 due Tuesday May 1 • MB homework 8: Ch 18 due May 3 Ch 17: from gene to protein • Understanding the link between genes and polypeptides • The genetic code • Transcription • Translation • Mutations Molecular Components of Transcription • RNA synthesis – is catalyzed by RNA polymerase, which pries the DNA strands apart & hooks together RNA nucleotides – follows the same base-pairing rules as DNA, except uracil substitutes for thymine the DNA sequence where RNA polymerase attaches the stretch of DNA that is transcribed Promoter Transcription unit 5 3 Start point RNA polymerase 3 5 DNA 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. Initiation 5 3 3 5 RNA Template strand Unwound tran- of DNA DNA script The polymerase moves downstream, unwinding the DNA and elongating the RNA transcript 5 3. During transcription, the DNA strands re-form a double helix. Elongation Rewound DNA 5 3 3 5 3 5 RNA transcript Eventually, the RNA transcript is released, and the polymerase detaches from the DNA. Termination 5 3 3 5 5 Completed RNA transcript 3 Initiation Eukaryotic promoter Promoter • Promoters signal the initiation of RNA synthesis 5 3 TATA box – A promoter called a TATA box is crucial in forming the initiation complex in eukaryotes • Transcription factors (TFs; think cell communication) mediate the binding of RNA polymerase and initiation of transcription 3 5 Start point Template DNA strand Several transcription factors Transcription factors 5 3 3 5 Additional transcription factors RNA polymerase II • transcription initiation complexcompleted assembly of TFs & RNA polymerase II bound to a promoter 5 3 Transcription factors 3 5 5 RNA transcript Transcription initiation complex Elongation of the RNA Strand • As RNA polymerase moves along the DNA, it untwists the double helix, 10-20 bases at a time • RNA polymerase adds nucleotides to the 3’ end of the growing RNA molecule as it continues along the double helix • Transcription progresses at a rate of 40 nucleotides/ sec in eukaryotes Non-template strand of DNA Elongation RNA nucleotides RNA polymerase “coding strand” 3 3 end 5 5 Direction of transcription (“downstream”) Template strand of DNA Newly made RNA • A gene can be transcribed simultaneously by several RNA polymerases Termination of Transcription • The mechanisms of termination are different in prokaryotes and eukaryotes – In bacteria, the polymerase stops transcription at the end of the terminator and the mRNA can be translated without further modification – In eukaryotes, RNA polymerase II transcribes the polyadenylation signal sequence; the RNA transcript is released 10–35 nucleotides past this polyadenylation sequence Eukaryotic cells • Enzymes in the eukaryotic nucleus modify pre-mRNA before the genetic messages are dispatched to the cytoplasm • During RNA processing: 1. both ends of primary RNA transcript (pre-mRNA) are usually altered 2. usually some interior parts of the molecule are cut out, and the other parts spliced together Alteration of mRNA Ends Each end of a pre-mRNA molecule is modified in a particular way: 1. The 5 end receives a modified nucleotide cap 2. The 3 end gets a poly-A tail These modifications share several functions: 1. They seem to facilitate the export of mRNA 2. They protect mRNA from hydrolytic enzymes 3. They help ribosomes attach to the 5’ end Protein-coding segment Polyadenylation signal 5 5 Cap 5 UTR Start codon Stop codon 3 UTR Poly-A tail RNA splicing Most eukaryotic genes and their RNA transcripts have long noncoding stretches of nucleotides that lie between coding regions • intronsnoncoding regions, intervening sequences • exonsexpressed, usually translated into amino acid sequences RNA splicing removes introns and joins exons, creating an mRNA molecule w/ a continuous coding sequence 5 Exon Intron Pre-mRNA Exon Intron Exon 3 5Cap Poly-A tail 1 30 31 Coding segment 104 105 146 Introns cut out and exons spliced together Poly-A tail 5Cap 5 UTR 1 146 3 UTR • In some cases, RNA splicing is carried out by spliceosomes – recognize the splice sites – consist of: • small nuclear ribonucleoproteins (snRNPs) • a variety of proteins 5 RNA transcript (pre-mRNA) Exon 1 Intron Protein snRNA Exon 2 Other proteins snRNPs Spliceosome 5 Spliceosome components 5 mRNA Exon 1 Exon 2 Cut-out intron Ribozymes • Ribozymesare catalytic RNA molecules that function as enzymes and can splice RNA • The discovery of ribozymes rendered obsolete the belief that all biological catalysts were proteins The Functional & 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 • Proteins often have a modular architecture Gene DNA Exon 1 Intron Exon 2 Transcription RNA processing – consisting of discrete structural and functional regions called domains Intron Exon 3 Translation Domain 3 • In many casesdifferent exons code for the different domains in a protein Domain 2 Domain 1 Polypeptide Molecular Components of Translation A cell translates an mRNA message into protein with the help of transfer RNA (tRNA) Amino acids Polypeptide tRNA with amino acid attached tRNA carry 2 thingsone on each end: Ribosome 1. specific amino acid 2. an anticodon tRNA Anticodon The anticodon base-pairs with a 5 complementary codon on mRNA Codons mRNA 3 3 A tRNA molecule consists of a single RNA strand that is only about 80 nucleotides long Amino acid attachment site 5 Flattened into one plane to reveal its base pairing, a tRNA molecule looks like a cloverleaf Hydrogen bonds 3 5 Anticodon Two-dimensional structure Amino acid attachment site 5 3 Because of hydrogen bonds, tRNA actually twists and folds into a three-dimensional molecule Hydrogen bonds tRNA is roughly L-shaped Anticodon Three-dimensional structure 3 5 Anticodon Symbol used in this book Aminoacyl-tRNA synthetase (enzyme) Accurate translation requires 2 steps: Amino acid 1. a correct match between a tRNA and an amino acid, done by the enzyme Aminoacyl-tRNA aminoacyl-tRNA synthetase P Adenosine P P P Adenosine P Pi ATP Pi Active site binds the amino acid and ATP. Pi tRNA synthetase tRNA Appropriate tRNA covalently bonds to amino acid, displacing AMP. Amino acid P Adenosine AMP Computer model 2. a correct match between the tRNA anticodon & mRNA codon Aminoacyl tRNA (“charged tRNA”) Ribosomes • Ribosomes facilitate specific coupling of tRNA anticodons with mRNA codons in protein synthesis • The two ribosomal subunits (large and small) are made of proteins & ribosomal RNA (rRNA) tRNA molecules Growing polypeptide Exit tunnel note: tetracycline & streptomycin inactivate prokaryotic ribosomes Large subunit Small subunit 5 3 mRNA Computer model of functioning ribosome similar in prokaryotes & eukaryotes, but differences are medically significant • A ribosome has three binding sites for tRNA: 1. the A site holds the tRNA that carries the next amino acid to be added to the chain 2. the P site holds the tRNA that carries the growing polypeptide chain 3. the E site is the exit site, where discharged tRNAs leave the ribosome Exit tunnel P site (Peptidyl-tRNA binding site) A site (AminoacyltRNA binding site) E site (Exit site) E P A mRNA binding site (b) Schematic model showing binding sites Large subunit Small subunit Growing polypeptide Amino end Next amino acid to be added to polypeptide chain E tRNA mRNA 5 3 Codons 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 tRNA carrying the next amino acid to be added to the polypeptide chain. Discharged tRNA leaves via the E site. Building a Polypeptide • The three stages of translation: 1. Initiation 2. Elongation 3. Termination – All three stages require protein “factors” that aid in the process Ribosome Association & Initiation of Translation The initiation stage of translation brings together: 1. mRNA 2. a tRNA with the first amino acid 3. the two ribosomal subunits First, a small ribosomal subunit binds with mRNA and a special initiator tRNA Proteins called initiation factors bring in the large subunit so the initiator tRNA occupies the P site Then the small subunit moves along the mRNA until it reaches the start codon (AUG) Large ribosomal subunit P site Initiator tRNA GTP GDP E A the A site is available to the tRNA bearing the next amino acid mRNA 5 3 5 3 Start codon mRNA binding site Small ribosomal subunit Translation initiation complex Each addition involves proteins called elongation factors 1 Amino end of polypeptide Codon recognition E 3 mRNA Ribosome ready for next aminoacyl tRNA P A site site site 5 GTP GDP Elongation amino acids are added 1 by 1 to the preceding amino acid E P A P A 2 3 Translocation E Peptide bond formation GDP GTP E P A Termination of Translation • Termination occurs when a stop codon in the mRNA reaches the A site of the ribosome • The A site accepts a protein called a release factor – causes the addition of a water molecule instead of an amino acid • This reaction releases the polypeptide, and the translation assembly then comes apart Release factor Free polypeptide 5 3 3 3 5 5 Stop codon (UAG, UAA, or UGA) 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 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. The two ribosomal subunits and the other components of the assembly dissociate. • A number of ribosomes can translate a single mRNA simultaneously, forming a polyribosome (or polysome) Completed polypeptides Polyribosomes Growing polypeptides Incoming ribosomal subunits Start of mRNA (5 end) • Polyribosomes enable a cell to make many copies of a polypeptide very quickly End of mRNA (3 end) An mRNA molecule is generally translated simultaneously by several ribosomes in clusters called polyribosomes. Ribosomes mRNA 0.1 mm This micrograph shows a large polyribosome in a prokaryotic cell (TEM). Functional Protein • During and after synthesis, a polypeptide chain spontaneously coils and folds into its three-dimensional shape • Often translation is not sufficient to make a functional protein – Polypeptide chains are modified after translation • Proteins may require post-translational modifications – Some polypeptides are activated by enzymes that cleave them – Other polypeptides come together to form the subunits of a protein • Completed proteins are targeted to specific sites in the cell Ribosomes • Two populations of ribosomes are evident in cells: – free ribsomes (in the cytosol) – 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 • Polypeptide synthesis always begins in the cytosol • Synthesis finishes in the cytosol unless the polypeptide signals the ribosome to attach to the ER • Polypeptides destined for the ER or for secretion are marked by a signal peptide – A signal-recognition particle (SRP) binds to the signal peptide • The SRP brings the signal peptide and its ribosome to the ER 1 Ribosome 5 4 mRNA Signal peptide 3 SRP 2 ER LUMEN SRP receptor protein Translocation complex Signal peptide removed ER membrane Protein 6 CYTOSOL • Mutationsare changes in the genetic material of a cell or virus – Spontaneous mutations can occur during DNA replication, recombination, or repair – Mutagens are physical or chemical agents that can cause mutations • Point mutationsare chemical changes in just one base pair of a gene – can lead to the production of an abnormal protein Divided into 2 general categories: 1. Nucleotide-pair substitutions 2. One or more nucleotide-pair insertions or deletions Nucleotide-pair substitution • Silent mutationshave no effect on the amino acid produced by a codon because of redundancy in the genetic code • Missense mutationsstill code for an amino acid, but not necessarily the right amino acid • Nonsense mutationschange an amino acid codon into a stop codon, nearly always leading to a nonfunctional protein more common replaces one nucleotide and its partner with another pair of nucleotides Insertions and deletionsare additions or losses of nucleotide pairs in a gene These mutations have a disastrous effect on the resulting protein more often than substitutions do Insertion or deletion of nucleotides may alter the reading frame, producing a frameshift mutation Nucleotide-pair insertion/ deletion prokaryotes vs. eukaryotes • Prokaryotic cells lack a nuclear envelope, allowing translation to begin while transcription progresses • In a eukaryotic cell: – The nuclear envelope separates transcription from translation – Extensive RNA processing occurs in the nucleus 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 – In summary, a gene can be defined as a region of DNA that can be expressed to produce a final functional product, either a polypeptide or an RNA molecule