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Chapter 17 From Gene to Protein
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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
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process by which DNA directs protein synthesis
Gene expression- process by which DNA directs protein synthesis Transcription Translation DNA RNA Protein 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
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EXPERIMENT Classes of Neurospora crassa Growth: Wild-type cells growing and dividing No growth: Mutant cells cannot grow and divide Wild type Class I mutants Class II mutants Class III mutants Minimal medium (MM) (control) Minimal medium 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 MM ornithine Condition MM citrulline MM arginine (control) 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 Summary of results Figure 17.2 Inquiry: Do individual genes specify the enzymes that function in a biochemical pathway? Gene (codes for enzyme) Class I mutants (mutation in gene A) Class II mutants (mutation in gene B) Class III mutants (mutation in gene C) Wild type Precursor Precursor Precursor Precursor Gene A Enzyme A Enzyme A Enzyme A Enzyme A Ornithine Ornithine Ornithine Ornithine Gene B Enzyme B Enzyme B Enzyme B Enzyme B Citrulline Citrulline Citrulline Citrulline Gene C Enzyme C Enzyme C Enzyme C Enzyme C Arginine Arginine Arginine Arginine
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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 hypothesis- states that the function of an individual gene is to dictate the production of a specific polypeptide
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DNA DNA TRANSCRIPTION TRANSCRIPTION mRNA mRNA Ribosome TRANSLATION
Figure 17.3 Overview: the roles of transcription and translation in the flow of genetic information. Polypeptide (a) Bacterial cell (a) Bacterial cell
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DNA DNA DNA TRANSCRIPTION TRANSCRIPTION TRANSCRIPTION In prokaryotes, mRNA produced by transcription is immediately translated without more processing In a eukaryotic cell, the nuclear envelope separates transcription from translation Eukaryotic RNA transcripts are modified through RNA processing to yield finished mRNA mRNA mRNA mRNA Ribosome Ribosome Ribosome TRANSLATION TRANSLATION TRANSLATION Polypeptide Polypeptide Polypeptide Prokaryotic cell Prokaryotic cell Prokaryotic cell Nuclear envelope Nuclear envelope Nuclear envelope TRANSCRIPTION TRANSCRIPTION TRANSCRIPTION DNA DNA DNA primary transcript Pre-mRNA Pre-mRNA RNA PROCESSING RNA PROCESSING mRNA mRNA Ribosome TRANSLATION Polypeptide Eukaryotic cell Eukaryotic cell Eukaryotic cell
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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
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Gene 2 Gene 1 Gene 3 mRNA Codon TRANSLATION Protein Amino acid DNA
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, are read in the to 3 direction Each codon specifies the amino acid to be placed at the corresponding position along a polypeptide Gene 2 DNA molecule Gene 1 Gene 3 DNA template strand “coding strand” TRANSCRIPTION mRNA Codon TRANSLATION Protein Amino acid
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Genetic Code Second mRNA base 64 codons includes 3 Stop 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 First mRNA base (5¢ end) Third mRNA base (3¢ end)
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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 Tobacco plant expressing a firefly gene (b) Pig expressing a jellyfish gene
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With apologies to the Smashing Pumpkins
April 30, 2012 With apologies to the Smashing Pumpkins
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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
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Ch 17: from gene to protein
Understanding the link between genes and polypeptides The genetic code Transcription Translation Mutations
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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
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Initiation Elongation Termination
Promoter Transcription unit 5 3 3¢ 5¢ DNA Start point RNA polymerase 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 tran- script Template strand of DNA Unwound DNA 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¢ 3¢ 5¢ 5¢ RNA transcript Eventually, the RNA transcript is released, and the polymerase detaches from the DNA. Termination 5¢ 3¢ 3¢ 5¢ 5¢ 3¢ Completed RNA transcript
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Initiation Promoters signal the initiation of RNA synthesis
Eukaryotic promoter Promoter Promoters signal the initiation of RNA synthesis 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 transcription initiation complex- completed assembly of TFs & RNA polymerase II bound to a promoter 5¢ 3¢ 3¢ 5¢ TATA box Start point Template DNA strand Several transcription factors Transcription factors 5¢ 3¢ 3¢ 5¢ Additional transcription factors RNA polymerase II Transcription factors 5¢ 3¢ 3¢ 5¢ 5¢ RNA transcript Transcription initiation complex
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Elongation of the RNA Strand
As RNA polymerase moves along the DNA, it untwists the double helix, 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 A gene can be transcribed simultaneously by several RNA polymerases Elongation Non-template strand of DNA RNA nucleotides RNA polymerase “coding strand” 3¢ 3¢ end 5¢ 5¢ Direction of transcription (“downstream”) Template strand of DNA Newly made RNA
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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
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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
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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 UTR Start codon 5¢ Stop codon 3¢ UTR Poly-A tail
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RNA splicing Most eukaryotic genes and their RNA transcripts have long noncoding stretches of nucleotides that lie between coding regions introns- noncoding regions, intervening sequences exons- expressed, 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 Exon Intron Exon 3¢ Pre-mRNA 5¢Cap Poly-A tail 1 30 31 104 105 146 Introns cut out and exons spliced together Coding segment 5¢Cap Poly-A tail 1 146 5¢ UTR 3¢ UTR
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In some cases, RNA splicing is carried out by
RNA transcript (pre-mRNA) RNA transcript (pre-mRNA) RNA transcript (pre-mRNA) 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 5 5 Exon 1 Exon 1 Exon 1 Intron Intron Intron Exon 2 Exon 2 Exon 2 Protein Protein Protein Other proteins Other proteins Other proteins snRNA snRNA snRNA snRNPs snRNPs snRNPs Spliceosome Spliceosome 5 5 Figure The roles of snRNPs and spliceosomes in pre-mRNA splicing Spliceosome components Cut-out intron mRNA 5 Exon 1 Exon 2
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Ribozymes Ribozymes- are 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
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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 consisting of discrete structural and functional regions called domains In many cases different exons code for the different domains in a protein Gene DNA Exon 1 Intron Exon 2 Exon 3 Transcription RNA processing Translation Domain 3 Domain 1 Domain 2 Polypeptide
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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 things- one on each end: 1. specific amino acid 2. an anticodon Ribosome Trp Phe Gly Figure Translation: the basic concept tRNA Anticodon The anticodon base-pairs with a complementary codon on mRNA 5 Codons 3 mRNA
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tRNA is roughly L-shaped
3¢ A tRNA molecule consists of a single RNA strand that is only about 80 nucleotides long Flattened into one plane to reveal its base pairing, a tRNA molecule looks like a cloverleaf Amino acid attachment site 5¢ 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 tRNA is roughly L-shaped Hydrogen bonds 3¢ 5¢ Anticodon Anticodon Three-dimensional structure Symbol used in this book
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Aminoacyl-tRNA synthetase
Aminoacyl-tRNA synthetase (enzyme) Aminoacyl-tRNA synthetase (enzyme) Aminoacyl-tRNA synthetase (enzyme) Aminoacyl-tRNA synthetase (enzyme) Amino acid Amino acid Amino acid Amino acid Accurate translation requires 2 steps: 1. a correct match between a tRNA and an amino acid, done by the enzyme aminoacyl-tRNA synthetase 2. a correct match between the tRNA anticodon & mRNA codon P P P Adenosine Adenosine Adenosine P P P P P P P P P P P P Adenosine Adenosine Adenosine Adenosine P P P P P P i i i Aminoacyl-tRNA synthetase ATP ATP ATP ATP P P P i i i P P P tRNA tRNA i i i Active site binds the amino acid and ATP. tRNA tRNA Amino acid Amino acid Appropriate tRNA covalently bonds to amino acid, displacing AMP. Figure An aminoacyl-tRNA synthetase joining a specific amino acid to a tRNA. P P Adenosine Adenosine AMP AMP Computer model Aminoacyl tRNA (“charged tRNA”)
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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) similar in prokaryotes & eukaryotes, but differences are medically significant note: tetracycline & streptomycin inactivate prokaryotic ribosomes Exit tunnel Growing polypeptide tRNA molecules Large subunit Small subunit 5¢ 3¢ mRNA Computer model of functioning ribosome
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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 (Aminoacyl- tRNA binding site) E site (Exit site) E P A Large subunit mRNA binding site Small subunit (b) Schematic model showing binding sites
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Growing polypeptide Amino end Next amino acid to be added to polypeptide chain E tRNA mRNA 3 Codons 5 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.
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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
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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 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) First, a small ribosomal subunit binds with mRNA and a special initiator tRNA Large ribosomal subunit P site Met Met the A site is available to the tRNA bearing the next amino acid Initiator tRNA GTP GDP E A mRNA 5¢ 5¢ 3¢ 3¢ Start codon Small ribosomal subunit mRNA binding site Translation initiation complex
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amino acids are added 1 by 1 to the preceding amino acid
Each addition involves proteins called elongation factors 1 Amino end of polypeptide Amino end of polypeptide Amino end of polypeptide Amino end of polypeptide Codon recognition E E E E mRNA mRNA mRNA mRNA 3¢ 3¢ 3¢ 3¢ P site P site P site P site A site A site A site A site Ribosome ready for next aminoacyl tRNA 5¢ 5¢ 5¢ 5¢ GTP GTP GTP GDP GDP GDP Elongation amino acids are added 1 by 1 to the preceding amino acid E E E E P A P P P A A A 2 Figure The elongation cycle of translation 3 Peptide bond formation Translocation GDP GTP E E P P A A
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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 Stop codon (UAG, UAA, or UGA) 5¢ 3¢ Free polypeptide 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. 3¢ 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.
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Polyribosomes Completed polypeptides Growing polypeptides A number of ribosomes can translate a single mRNA simultaneously, forming a polyribosome (or polysome) Polyribosomes enable a cell to make many copies of a polypeptide very quickly Incoming ribosomal subunits Polyribosome Start of mRNA (5¢ end) 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).
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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
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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
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Signal peptide removed 3 SRP Protein 6 SRP receptor protein 2 CYTOSOL
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 ER membrane Signal peptide removed 3 SRP Protein 6 SRP receptor protein 2 CYTOSOL ER LUMEN Translocation complex
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are changes in the genetic material of a cell or virus
Mutations- are 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 mutations- are 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
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Nucleotide-pair substitution
Silent mutations- have no effect on the amino acid produced by a codon because of redundancy in the genetic code Missense mutations- still code for an amino acid, but not necessarily the right amino acid Nonsense mutations- change 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 Nucleotide-pair substitution
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Insertions and deletions-
are 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
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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
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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
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