Chapter 17 From Gene to Protein.

Chapter 17 From Gene to Protein

Word Roots anti- = opposite exo- = out, outside, without
anticodon: specialized base triplet on one end of a tRNA molecule that recognizes particular complementary codon on mRNA molecule exo- = out, outside, without exon: coding region of eukaryotic gene that is expressed intro- = within intron: noncoding, intervening sequence within eukaryotic gene muta- = change; -gen = producing mutagen: physical or chemical agent that causes mutations poly- = many poly-A-tail: modified end of 3’ end of mRNA molecule consisting of addition of adenine nucleotides trans- = across; -script = write transcription: synthesis of RNA on DNA template

Overview: The Flow of Genetic Information
In eukaryotes, DNA instructions transcribed in nucleus to mRNA (processed)  complexed w/ribosomes to translate mRNA to sequence of amino acids in polypeptide in cytoplasm as tRNA match anticodons to mRNA codons Information content of DNA in form of specific sequences of nucleotides DNA inherited by organism leads to specific traits by dictating synthesis of proteins Proteins are links between genotype/phenotype Gene expression (process by which DNA directs protein synthesis) includes two stages: transcription and translation Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Concept 17.1: Genes specify proteins via transcription and translation/Evidence from the Study of Metabolic Defects How was fundamental relationship between genes and proteins discovered? In 1909, British physician Archibald Garrod first suggested that genes dictate phenotypes through enzymes that catalyze specific chemical reactions Symptoms of inherited disease reflect inability to synthesize certain enzyme (“inborn errors of metabolism”) Linking genes to enzymes required understanding that cells synthesize and degrade molecules in series of steps (metabolic pathway) Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Nutritional Mutants in Neurospora: Scientific Inquiry
Beadle/Tatum exposed bread mold to X-rays, creating mutants 1st grew nutritional mutants on complete growth medium (minimal medium w/all 20 amino acids/few other nutrients) that could support any mutant that couldn’t synthesize one of the supplements Transferred samples to various combinations of minimal medium (agar mixed only with inorganic salts, glucose, and vitamin biotin that wild-type mold could survive on) + one added nutrient to identify specific metabolic defect for each mutant Three classes of Neurospora mutants unable to synthesize arginine identified by supplementing different precursors of the pathway Reasoned that metabolic pathway of each class blocked at different step because mutant in that class lacked enzyme that catalyzes blocked step Formulated one gene–one enzyme hypothesis, which states that each gene dictates production of specific enzyme Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

EXPERIMENT RESULTS CONCLUSION Fig. 17-2
Growth: Wild-type cells growing and dividing No growth: Mutant cells cannot grow and divide Minimal medium RESULTS Classes of Neurospora crassa Wild type grow w/or without supplements Class I mutants can grow on ornithine, citrulline, or arginine Class II mutants can grown only on citruilline or arginine Class III mutants absolutely require arginine to grow Minimal medium (MM) (control) MM + ornithine Condition MM + citrulline MM + arginine (control) Figure 17.2 Do individual genes specify the enzymes that function in a biochemical pathway? CONCLUSION 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

The Products of Gene Expression: A Developing Story
Some proteins aren’t enzymes (but still gene products), so researchers later revised hypothesis: one gene–one protein Many proteins are composed of several polypeptides, each of which has its own gene Beadle/Tatum’s hypothesis now restated as one gene–one polypeptide hypothesis Note that it is common to refer to gene products as proteins rather than polypeptides Still not completely accurate Many eukaryotic genes can code for set of closely related polypeptides (alternative splicing) Quite a few genes code for RNA molecules Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Basic Principles of Transcription and Translation
In order for information in DNA to direct cellular processes, information must be transcribed (DNA RNA) and in many cases, translated (RNA protein)  products play important role in determining metabolism (cellular activities/phenotypes) RNA is intermediate between genes/proteins for which they code DNA does not make proteins directly/directs synthesis of RNA and copies of itself Name three ways in which RNA differs from DNA? Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Transcription (general term for synthesis of any kind of RNA on DNA template): synthesis of mRNA (messenger RNA) from nucleotide to nucleotide transfer of information under direction of DNA as template (both use same language) Translation: synthesis of a polypeptide, which occurs under direction of mRNA (change in language: cell must translate nucleotide base sequence of mRNA molecule into amino acid sequence of polypeptide) Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Ribosomes (complex particles that facilitate orderly linking of amino acids into polypeptide chain): sites of translation In prokaryotes, mRNA produced by transcription is immediately translated without more processing (ribosomes not separated from DNA by membrane) In eukaryotic cell, nuclear envelope separates transcription from translation (which occurs in cytoplasm) Eukaryotic initial RNA transcripts from any gene (pre-mRNA or primary transcript) are modified through RNA processing to yield finished mRNA Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Fig. 17-3 http://highered.mcgraw-hill.com/olc/dl/120077/bio25.swf
DNA TRANSCRIPTION mRNA Ribosome TRANSLATION Polypeptide Bacterial cell (lacks nucleus so mRNA produce by transcription immediately translated without additional processing) Nuclear envelope DNA TRANSCRIPTION Pre-mRNA RNA PROCESSING Figure 17.3 Overview: the roles of transcription and translation in the flow of genetic information mRNA TRANSLATION Ribosome Polypeptide (b) Eukaryotic cell (nucleus provides separate compartment for transcription; original RNA transcript, called pre-RNA, processed in various ways before leaving nucleus as RNA)

The Genetic Code/Codon: /Triplet of Bases
How are instructions for assembling amino acids into proteins encoded into DNA? 20 amino acids but only four nucleotide bases in DNA How many bases correspond to amino acid? Flow of information from gene to protein based on triplet code (series of nonoverlapping, three-nucleotide words) 43 = 64 combinations Smallest units of uniform length that can code for all amino acids Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

During transcription, template DNA strand directs ordering sequence of nucleotides in RNA transcript mRNA is complementary (not identical) to DNA template mRNA synthesized antiparallel to DNA template/identical in sequence to nontemplate strand of DNA except U for T Base triplet ACC along DNA (3’-ACC-5’) provides template for 5’-UGG-3” in mRNA molecule During translation, codons decoded (translated) into sequence of amino acids making up polypeptide chain Sequence of nucleotides on mRNA read in triplets (codons) read in 5 to 3 direction Each codon specifies one of 20 amino acids placed at corresponding position along polypeptide Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Gene 2 Gene 1 Gene 3 DNA template strand mRNA Codon TRANSLATION
Fig. 17-4 Gene 2 DNA molecule Gene 1 Gene 3 DNA template strand TRANSCRIPTION Figure 17.4 The triplet code mRNA Codon TRANSLATION Protein Amino acid

Cracking the Code Of 64 triplets (deciphered by mid-1960s) , 61 code for amino acids; 3 triplets are “stop” signals to end translation (UAA, UAG, UGA) and one for “start” (AUG) Genetic code redundancy (more than one triplet codes for amino acid) but no ambiguity (no codon specifies more than one amino acid) Codons must be read in correct reading frame (correct groupings) in order for specified polypeptide to be produced (“The red dog ate the bug” would be gibberish if read incorrectly “her edd oga tet heb ug”) Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

First mRNA base (5 end of codon) Third mRNA base (3 end of codon)
Fig. 17-5 Second mRNA base First mRNA base (5 end of codon) Third mRNA base (3 end of codon) Figure 17.5 The dictionary of the genetic code

Evolution of the Genetic Code
Genetic code is nearly universal, shared by simplest bacteria to most complex animals but few exceptions to universality of genetic code include translation systems in which few codon differ from standard ones Slight variations in certain unicellular eukaryotes and in organelle genes of some species Also, stop codons can be translated into one of two amino acids (pyrrolysine found in archae and selenocystein is component of some bacterial proteins and some human enzymes) Genes can be transcribed and translated after being transplanted from one species to another Bacteria can be programmed by insertion of human genes to synthesize certain human proteins for medical use (insulin) Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Concept 17.2: Transcription is the DNA-directed synthesis of RNA: a closer look/Molecular Components of Transcription Promoter is DNA sequence where RNA polymerase attaches and initiates transcription (decides where transcription begins) Don’t need primer (like DNA) Transcription unit is stretch of DNA that is transcribed May code for polypeptide, RNA (tRNA, rRNA) Separates two DNA strands/connects RNA nucleotides as they base-pair along DNA template strand Follows same base-pairing rules as DNA (U substitutes for T) Adds only in 5’  3’ direction (reads DNA molecule in 3’  5’ direction/ makes complementary mRNA molecule that determines order of amino acids in polypeptide) Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Synthesis of an RNA Transcript
Three stages of transcription Initiation/Elongation/Termination Prokaryotes have 1/Eukaryotes have 3 RNA polymerase RNA polymerase I produces ribosomal RNA RNA polymerase II transcribes genes RNA polymerase III produces tRNA Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

signals initiation of RNA synthesis
Fig The initiation of transcription at a eukaryotic promoter 1 Eukaryotic promoter includes TATA box (~25 nucleotides upstream from transcriptional start point) which is crucial in forming initiation complex (signals initiation of RNA synthesis) Promoter signals initiation of RNA synthesis Template 5 3 3 5 TATA box Start point Template DNA strand Several transcription factors (collection of proteins on recognizing TATA box) must bind to DNA before RNA polymerase II can do so and transcription can begin 2 Transcription factors RNA Polymerase Binding and Initiation of Transcription 5 3 3 5 3 Additional transcription factors bind to DNA along with RNA polymerase II, forming the transcription initiation complex. DNA helix unwinds, RNA synthesis begins at start point on template strand Figure 17.8 RNA polymerase II Transcription factors 5 3 3 5 5 RNA transcript Transcription initiation complex

Elongation of the RNA Strand Termination of Transcription
As RNA polymerase moves along DNA, it untwists double helix, 10 to 20 bases at time, adding nucleotides to 3’ end Rate of 40 nucleotides/second in eukaryotes Gene can be transcribed simultaneously by several RNA polymerases DNA helix reforms when RNA molecule peels away Mechanisms of termination different in bacteria/eukaryotes In bacteria, transcription stops at terminator (signals end of sequence in bacteria), detaches from DNA, releases transcript In eukaryotes, polymerase continues transcription after pre-mRNA is cleaved from growing RNA chain; polymerase eventually falls off DNA Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Initiation (RNA polymerase binds to promoter, DNA unwinds,
Fig. 17-7 Promoter Transcription unit 5 3 3 5 DNA Start point RNA polymerase Initiation (RNA polymerase binds to promoter, DNA unwinds, polymerase initiates RNA synthesis at start point on template strand) 1 Elongation Nontemplate strand of DNA RNA nucleotides 5 3 RNA polymerase 3 5 RNA transcript Template strand of DNA Unwound DNA Elongation (polymerase moves downstream, unwinding DNA, elongating RNA transcript 5’  3’, DNA reforms double helix) 3 2 3 end Rewound DNA 5 5 3 3 3 5 5 Figure 17.7 The stages of transcription: initiation, elongation, and termination 5 Direction of transcription (“downstream”) RNA transcript Termination (RNA transcript released and polymerase detaches from DNA) Template strand of DNA 3 Newly made RNA 5 3 3 5 5 3 Completed RNA transcript

Concept 17.3: Eukaryotic cells modify RNA after transcription/Alteration of mRNA Ends
During RNA processing, both ends of primary transcript (UTRs, untranslated regions) are usually altered in series of enzyme-regulated modifications before going to cytoplasm 5 end receives modified guanine nucleotide 5 cap (-P-P-P-G-5’) 3 end gets poly-A tail ( more adenine added) These modifications share several functions Seem to facilitate export of mRNA Protect mRNA from hydrolytic enzymes Help ribosomes attach to 5 end Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Split Genes and RNA Splicing
RNA splicing removes introns/joins exons, creating mRNA molecule (hundreds of nucleotides long) w/continuous coding sequence Introns (noncoding stretches of nucleotides between coding regions) found in most eukaryotic genes Exons eventually expressed, usually translated into amino acid sequences Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Signals for RNA splicing are sets of few nucleotides at either end of each intron Small nuclear ribonucleoproteins (snRNPs, composed of proteins + small nuclear RNA, snRNA, ~150 nucleotides long)  splicesome play major role in catalyzing excision of introns and joining of exons Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Ribozymes Ribozymes: catalytic RNA molecules that function as enzymes/splice RNA Discovery rendered obsolete belief that all biological catalysts were proteins Properties enabling some RNA molecules to function as enzymes (RNA catalyzes its own splicing) Single-stranded and can base-pair w/itself, forming specific 3-D structure Some of its functional groups can participate in catalysis Can hydrogen bond w/other nuclei acid molecules, allowing it to precisely locate slicing regions Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

The Functional and Evolutionary Importance of Introns
Some introns involved in regulating gene activity Splicing necessary for export of mRNA from nucleus Alternative RNA splicing allows some genes to produce different polypeptides since some can encode more than one kind of polypeptide, depending on which segments are treated as exons during RNA splicing # different proteins produced much greater than # genes One gene can often make more than one polypeptide Realize now we have only about ,000 genes to make ~100, polypeptides Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Exon shuffling can result in novel proteins/evolution of new proteins
Exons may code for polypeptide domains (functional segments of protein) In many cases, different exons code for different domains in protein One may include active site/another attach protein to membrane Introns may facilitate recombination of exons between different alleles or even between different genes Exon shuffling can result in novel proteins/evolution of new proteins May allow for more crossing over between exons of alleles or for mixing/matching of exons between nonallelic genes Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Gene DNA Exon 1 Intron Exon 2 Intron Exon 3 Transcription
Fig Correspondence between exons and protein domains Gene DNA Exon 1 Intron Exon 2 Intron Exon 3 Transcription RNA processing Translation Domain 3 Figure 17.12 Domain 2 Domain 1 Polypeptide

Translation of mRNA to protein occurs in cytoplasm on ribosome
Concept 17.4: Translation is the RNA-directed synthesis of a polypeptide: a closer look/Molecular Components of Translation Translation of mRNA to protein occurs in cytoplasm on ribosome tRNA molecules not identical Each has anticodon triplet which base-pairs with complementary codon on mRNA Each carries specific amino acid on one end Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Amino acids tRNA with amino acid attached Ribosome tRNA Anticodon 5
Fig Translation: the basic concept Amino acids Polypeptide tRNA with amino acid attached Ribosome Trp Phe Gly Figure 17.13 tRNA Anticodon 5 Codons 3 mRNA

The Structure and Function of Transfer RNA
tRNA molecules bind specific amino acids/allow information in mRNA to be translated to linear peptide sequence Transcribed from DNA templates in nucleus, used repeatedly, picking up designated amino acid in cytosol, depositing it onto polypeptide chain at ribosome, then leaving to pick up another one tRNA molecule consists of single RNA strand that is only ~80 nucleotides long Because of hydrogen bonds, tRNA actually twists and folds into 3-D molecule that is roughly L-shaped Flattened into one plane to reveal its base pairing, tRNA molecule looks like cloverleaf A C C For the Cell Biology Video A Stick and Ribbon Rendering of a tRNA, go to Animation and Video Files. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

3 end is attachment site for amino acid
Figure The structure of transfer RNA (tRNA) Form fits function 3 end is attachment site for amino acid Amino acid attachment site 5 Hydrogen bonds Anticodon written 3’  5’ to align properly w/codons written 5’  3’ (for base pairing, RNA strands must be antiparallel, like DNA. For Example, anticodon 3’-AAG-5” pairs w/mRNA codon 5’-UUC-3’ (a) Two-dimensional structure 5 Amino acid attachment site 3 Hydrogen bonds 3 5 Anticodon Anticodon (b) Three-dimensional structure (c) Symbol used in this book

Accurate translation requires two steps
Accurate translation requires two steps Correct match between tRNA and amino acid, done by 1of 20 enzyme aminoacyl-tRNA synthetase (1 for each amino acid) Correct match between tRNA anticodon and mRNA codon Only 45 tRNAs (some bind to more than one codon) Flexible pairing at third base of codon (wobble) allows this to happen Explains why synonymous codons for given amino acid can differ in 3rd base, but not usually others Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Figure An aminoacyl-tRNA synthetase joining a specific amino acid to a tRNA driven by hydrolysis of ATP Aminoacyl-tRNA synthetase (enzyme) Active site binds amino acid w/ATP ATP loses two P groups/joins amino acid as AMP Amino acid P P P Adenosine ATP P Adenosine tRNA P P i Aminoacyl-tRNA synthetase P i P i tRNA Appropriate tRNA covalently bonds to amino acid, displacing AMP P Adenosine AMP Computer model tRNA charged w/amino acid is release by enzyme Aminoacyl-tRNA (“charged tRNA”)

Ribosomes Ribosomes facilitate specific coupling of tRNA anticodons with mRNA codons in protein synthesis Two ribosomal subunits (large and small) are made of proteins and ribosomal RNA (rRNA-functional building blocks of ribosomes) Made in nucleolus in eukaryotes, transcribed from DNA, then processed and assembled with proteins imported into cytoplasm Subunits exported via nuclear pores to cytoplasm Subunits only join to form functional ribosome when they attach to mRNA molecule Eukaryotic ribosomes larger than bacterial and are different, giving ability for antibiotic drugs to inactivate those of bacteria without inhibiting eukaryotic ribosomes from making proteins Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Ribosome has three binding sites for tRNA
Fig Growing polypeptide Exit tunnel tRNA molecules Large subunit E P A Small subunit 5 mRNA 3 (a) Computer model of functioning ribosome P site (Peptidyl-tRNA binding site) holds tRNA that carries growing polypeptide chain A site (Aminoacyl-tRNA binding site) holds tRNA that carries next amino acid to be added to chain E site (Exit site) where discharged tRNAs leave ribosome E P A Large subunit mRNA binding site Small subunit Ribosome has three binding sites for tRNA (b) Schematic model showing binding sites Figure The anatomy of a functioning ribosome Amino end Growing polypeptide Next amino acid to be added to polypeptide chain E tRNA mRNA 3 5 Codons (c) Schematic model with mRNA and tRNA

Building a Polypeptide
Stages of translation occur in cytoplasm on ribosome Initiation/Elongation/Termination All three stages require protein “factors” that aid in translation process Energy required for chain initiation and elongation Provided by hydrolysis of GTP (guanosine triphosphate) Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Ribosome Association and Initiation of Translation
Initiation stage of translation brings together mRNA/tRNA w/1st amino acid/two ribosomal subunits (rRNA) First, small ribosomal subunit binds w/mRNA and special initiator tRNA carrying methionine (N-terminus) Then small subunit moves along mRNA until it reaches start codon (AUG)/binds to it using hydrogen bond Proteins (initiation factors) bring in large subunit that completes translation initiation complex GTP provides energy for assembly Initiator tRNA is in P site; A site available to tRNA bearing next amino acid Continues in one direction to final amino acid at carboxyl end (C-terminus)

Elongation of the Polypeptide Chain
During elongation stage, amino acids are added one by one to preceding amino acid Each addition involves proteins (elongation factors) and occurs in three steps: codon recognition, peptide bond formation, and translocation Energy needed in 1st/3rd steps (2 GTPs) mRNA moved through ribosome in one direction only, 5’ end first (equivalent to ribosome moving 5’  3’ on mRNA) Takes less than 1/10 second in bacteria Formation of peptide bonds between amino acids catalyzed by rRNA Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

2. Peptide bond formation: rRNA molecule of large subunit
Fig Amino end Codon recognition: anticodon of polypeptide of incoming aminoacyl tRNA base-pairs w/complementary mRNA codon in A site. Hydrolysis of GTP increases accuracy/efficiency of step. E 3 mRNA Ribosome ready for next aminoacyl tRNA 3. Translocation: Ribosome translocates tRNA in A site to P site. At same time, empty tRNA in P site moved to E site, where it is released. mRNA moves along w/its bound tRNAs, bringing next codon to be translated into A site. P site A site 5 GTP GDP 2. Peptide bond formation: rRNA molecule of large subunit catalyzes formation of peptide bond between new amino acid in A site and carboxyl end of growing polypeptide in P site. This removes polypeptide from tRNA in P site and attaches it to amino acid on tRNA in A site. E E P A P A Figure The elongation cycle of translation GDP GTP E P A

Termination of Translation
Fig Termination of Translation Release factor Free polypeptide 5 3 3 3 2 5 5 GTP 2 GDP Release factor causes addition of water molecule instead of amino acid/ promotes hydrolysis of bond between tRNA in P site and last amino acid of polypeptide, freeing polypeptide from ribosome Stop codon (UAG, UAA, or UGA) reached  A site accepts “release factor,” protein shaped like tRNA, instead of aminoacyl tRNA Figure The termination of translation Two ribosomal subunits/other components of translation assembly dissociate (requires 2 GTPs)

Polyribosomes Single ribosome can make average-sized polypeptide in less than minute Number of ribosomes can translate single mRNA simultaneously, forming polyribosome (or polysome) Enable cell to make many copies of polypeptide very quickly Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Completing and Targeting the Functional Protein/ Protein Folding and Post-Translational Modifications Often translation is not sufficient to make functional protein (polypeptide chains modified/completed proteins are targeted to specific sites in cell) During/after synthesis, polypeptide chain spontaneously coils (consequence of amino acid sequence, 10 structure) and folds into its 3-D shape (20/30 structure) Gene determines primary structure which in turn determines shape Chaperone protein (chaperonin) may help polypeptide fold correctly Proteins may also require post-translational modifications before doing their job Certain amino acids may be chemically modified by attachment of sugars, lipids, phosphate groups, other additions Some polypeptides are activated by enzymes that cleave them Other polypeptides come together to form subunits of 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 ribosomes (in cytosol) mostly synthesize proteins that function in cytosol Bound ribosomes (attached to cytoplasmic side of ER or to nuclear envelope) make proteins of endomembrane system (nuclear envelope, ER, Golgi, lysosomes, vacuoles, plasma membrane)/proteins secreted from cell (insulin) Ribosomes are identical/can switch from free to bound Those destined to be part of endomembrane system or for secretion transported into ER Those not part of endomembrane system are completed in cytosol before polypeptide imported into organelle (mitochondria, chloroplasts, interior of nucleus, others) Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Ribosome mRNA Signal peptide ER membrane Signal peptide removed
Fig 2. SRP (signal-recognition particle) binds to signal peptide, halting synthesis momentarily, cues ribosome to attach to ER. Synthesis finishes in cytosol unless polypeptide signals ribosome to attach to ER/polypeptides destined for ER or for secretion marked by signal peptide (~20 amino acids at/near leading end). 4. SRP leaves, polypeptide synthesis resumes w/simultaneous translocation across membrane (signal peptide stays attached to translocation complex) 1. Polypeptide synthesis always begins on free ribosome in cytosol Ribosome mRNA Signal peptide ER membrane Signal peptide removed Signal- recognition particle (SRP) Protein CYTOSOL Translocation complex Figure The signal mechanism for targeting proteins to the ER ER LUMEN SRP receptor protein 5. Signal-cleaving enzyme cuts off signal peptide 6. Rest of completed polypeptide leaves ribosome and folds into final conformation. It may become part of ER membrane or be exported from cell. 3. SRP binds to receptor protein in ER membrane. Receptor part of protein translocation complex that has membrane pore and signal-cleaving enzyme.

A Science Odyssey: You Try It: DNA Workshop
Justify the role of DNA replication being the starting point toward the goal of protein synthesis. Manipulate online models to create representations of DNA replication, transcription, and translation. Use construction paper, markers, and scissors to construct a model of DNA using at least 24 nucleotides. Use the model to distinguish between DNA and RNA; to model and explain the processes of replication, transcription, and translation; and to predict (with justification) the effects of change (mutation) on the original nucleotide sequence.

Concept 17.5: Point mutations can affect protein structure and function
Genetic information is set of instructions necessary for survival, growth and reproduction of organism For information to be useful, needs to be processed by cell Includes replication, decoding, transfer of information When genetic information changes, either through natural processes or genetic engineering, results may be observable changes in organism At the molecular level, these changes may be result of mutations in genetic material, effects of which may be seen when information is processed to yield nucleic acid or polypeptide Processes of transcription, mRNA processing and translation are imperfect, and errors can occur and may, in certain cases, alter phenotype Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Mutations: any changes in genetic material of cell or virus not due to segregation or to normal recombination of genetic material Point mutations: chemical changes in just one base pair of gene Change of single nucleotide, if present in protein-coding region in DNA template strand, can change amino acid sequence of polypeptide and can lead to production of abnormal or nonfunctioning protein Mutations can alter levels of gene expression/be silent If in gamete or cell giving rise to gamete, can be transmitted to offspring (genetic disorder or heredity disorder if mutation has adverse effect on phenotype)

Wild-type hemoglobin DNA Mutant hemoglobin DNA 3 C T T 5 3 C A T 5
Figure The molecular basis of sickle-cell disease: a point mutation Wild-type hemoglobin DNA Mutant hemoglobin DNA 3 C T T 5 3 C A T 5 5 G A A 3 5 G T A 3 mRNA mRNA 5 G A A 3 5 G U A 3 Normal hemoglobin Sickle-cell hemoglobin Glu Val

Types of Point Mutations
Point mutations within gene can be divided into two general categories Base-pair substitutions: replacement of one nucleotide and its complementary partner w/another pair of nucleotides (missense or nonsense mutations) Base-pair insertions or deletions: additions or losses of nucleotide pairs in gene Have disastrous effect on resulting protein more often than substitutions May alter reading frame of genetic message (frameshift mutation) Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Silent (no effect on amino acid sequence owing to redundancy)
Fig a Base-pair substitution Wild type DNA template strand 3 5 5 3 mRNA 5 3 Protein Stop Amino end Carboxyl end A instead of G 3 5 5 3 Figure Types of point mutations U instead of C 5 3 Stop Silent (no effect on amino acid sequence owing to redundancy)

Wild type DNA template strand 3 5 5 3 mRNA 5 3 Protein Stop
Fig b Base-pair substitution Wild type DNA template strand 3 5 5 3 mRNA 5 3 Protein Stop Amino end Carboxyl end T instead of C 3 5 5 3 Figure Types of point mutations A instead of G 5 3 Stop Missense (Substitutions that change one amino acid to another. May have little effect on protein if new amino acid has properties similar to those of amino acid it replaces, or it may be in region of protein where exact sequence of amino acids not essential to protein’s function.)

Nonsense (Change codon for amino acid into stop codon.
Fig c Base-pair substitution Wild type DNA template strand 3 5 5 3 mRNA 5 3 Protein Stop Amino end Carboxyl end A instead of T 3 5 5 3 Figure Types of point mutations U instead of A 5 3 Stop Nonsense (Change codon for amino acid into stop codon. Translation terminated prematurely resulting in polypeptide that will be shorter. Nearly all lead to nonfunctioning proteins.)

Frameshift causing immediate nonsense (1 base-pair insertion)
Fig d Base-pair insertion or deletion Wild type DNA template strand 3 5 5 3 mRNA 5 3 Protein Stop Amino end Carboxyl end Extra A 3 5 5 3 Figure Types of point mutations Extra U 5 3 Stop Frameshift causing immediate nonsense (1 base-pair insertion)

Frameshift causing extensive missense (1 base-pair deletion)
Fig e Base-pair insertion or deletion Wild type DNA template strand 3 5 5 3 mRNA 5 3 Protein Stop Amino end Carboxyl end missing 3 5 5 3 Figure Types of point mutations missing 5 3 Frameshift causing extensive missense (1 base-pair deletion)

Wild type DNA template strand 3 5 5 3 mRNA 5 3 Protein Stop
Fig f Base-pair insertion or deletion Wild type DNA template strand 3 5 5 3 mRNA 5 3 Protein Stop Amino end Carboxyl end missing 3 5 5 3 Figure Types of point mutations missing 5 3 Stop No frameshift, but one amino acid missing (3 base-pair deletion)

Mutagens Spontaneous mutations can occur during DNA replication, recombination, or repair Mutagens: physical/chemical agents that can cause DNA damage/alter genes X-rays/other high-energy radiation (UV light causes thymine dimers) Chemical mutagens include Base analogs (chemicals similar to normal DNA bases but pair incorrectly during DNA replication) Chemicals that interfere with correct DNA replication by inserting themselves into DNA/distorting helix Chemicals causing changes in bases that change their pairing properties Ames test used to measure chemicals that can be carcinogens Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Concept 17.6: While gene expression differs among the domains of life, the concept of a gene is universal/Comparing Gene Expression in Bacteria, Archaea, and Eukarya Archaea are prokaryotes, but share many features of gene expression with eukaryotes Characteristic Archaeal DNA Bacterial DNA Eukaryotic DNA RNA polymerases Resemble Eukarya Different from Eukarya Resemble Archaea Use complex set of transcription factors Use Don’t use Initiation of translation Most similar to bacteria Most similar to archaea Termination of transcription Ribosomes Size of bacterial but sensitivity to chemical inhibitors closely matches eukaryotic Size of Archaeal but sensitive to chemical inhibitors Larger and more complex Transcription and translation Likely coupled Simultaneously Separated by nuclear membrane Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Figure 17.24 Coupled transcription and translation in bacteria
RNA polymerase DNA mRNA Polyribosome Direction of transcription 0.25 µm RNA polymerase DNA Polyribosome Polypeptide (amino end) Ribosome mRNA (5 end)

What Is a Gene? Revisiting the Question
Idea of gene itself is unifying concept of life We have considered gene as Discrete unit of inheritance (Mendel) Region of specific nucleotide sequence in a chromosome (Morgan) DNA sequence that codes for specific polypeptide chain Final definition: Gene is region of DNA that can be expressed to produce final functional product that is either a polypeptide or RNA molecule Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Control-start and stop
Transcription Translation Template DNA RNA Location Nucleus (cytoplasm in prokaryotes) Cytoplasm; ribosomes can be free or attached to ER Molecules involved RNA nucleotides, DNA template strand, RNA polymerase, transcription factor Amino acids; tRNA, mRNA; ribosomes; ATP; GTP; enzymes; initiation, elongation and release factors Enzymes involved RNA polymerases, spliceosomes (ribozymes) Aminoacyl-tRNA synthetase; ribosomal enzymes (ribozymes) Control-start and stop Transcription factors locate promoter region w/TATA box, polyadenylation signal sequence Initiation factors, initiation sequence (AUG), stop codons, release factor Product Primary transcript (pre-mRNA) Polypeptide Product processing RNA processing: 5’ cap and poly-A tail, splicing of pre-mRNA (introns removed by snRNPs in spliceosomes) Spontaneous folding, disulfide bridges, signal peptide removed, cleaving, quaternary structure, modifications w/sugars, etc. Energy source Ribonucleoside triphosphate ATP and GTP

You should now be able to:
Describe the contributions made by Garrod, Beadle, and Tatum to our understanding of the relationship between genes and enzymes Briefly explain how information flows from gene to protein (Explain how one gene-one polypeptide came about) Compare and contrast RNA and DNA and three different types of ribosomes Compare transcription and translation in bacteria and eukaryotes Explain what it means to say that the genetic code is redundant and unambiguous Include the following terms in a description of transcription: mRNA, RNA polymerase, the promoter, the terminator, the transcription unit, initiation, elongation, termination, and introns 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

Chapter 17 From Gene to Protein.

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Chapter 17 From Gene to Protein.

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Presentation on theme: "Chapter 17 From Gene to Protein."— Presentation transcript:

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