From Gene to Protein Chapter 17 Lecture adapted from Chris Romero, Erin Barley, and Joan Sharp by Emily Blake

The Flow of Genetic Information Genes (segments of DNA) code for proteins. 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

Albino deer in Germany led to an outcry in 2006 Fig. 17-1 Albino deer in Germany led to an outcry in 2006 Figure 17.1 How does a single faulty gene result in the dramatic appearance of an albino deer?

One Gene, One Polypeptide 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 This led to the development of the one gene–one polypeptide hypothesis Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

EXPERIMENT Growth: Wild-type cells growing and dividing No growth: Fig. 17-2a EXPERIMENT Growth: Wild-type cells growing and dividing No growth: Mutant cells cannot grow and divide Figure 17.2 Do individual genes specify the enzymes that function in a biochemical pathway? Minimal medium

Basic Principles of Transcription and Translation RNA is the intermediate between genes and the coded proteins Transcription is the synthesis of mRNA under the direction of DNA Translation is the synthesis of a polypeptide, which occurs under the direction of mRNA Ribosomes are the sites of translation Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Fig. 17-3 DNA TRANSCRIPTION mRNA Ribosome TRANSLATION Polypeptide (a) Bacterial cell 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

How many bases correspond to an amino acid? 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 During transcription, the DNA template strand provides the triplet code sequence of nucleotides that will be transcribed into mRNA (mRNA grows in a 5’ to 3’ direction) During translation, mRNA codons are read in the 5 to 3 direction Each codon specifies the precise amino acid (out of the 20) in a polypeptide chain 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

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

Cracking the Code Of the 64 codons, 61 code for amino acids; 3 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

(a) Tobacco plant expressing a firefly gene (b) Pig expressing a Fig. 17-6 Genes can be transcribed and translated after being transplanted from one species to another Figure 17.6 Expression of genes from different species (a) Tobacco plant expressing a firefly gene (b) Pig expressing a jellyfish gene

Molecular Components of Transcription RNA polymerase pries the DNA strands apart and adds RNA nucleotides RNA synthesis follows same base-pairing rules as DNA, except uracil substitutes for thymine The DNA sequence where RNA polymerase attaches is 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 Animation: Transcription Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

A gene can be transcribed simultaneously by several RNA polymerases Fig. 17-7 Promoter Transcription unit As RNA polymerase moves along the DNA, it untwists the double helix, 10 to 20 bases at a time 5 3 3 5 DNA Start point RNA polymerase 1 Initiation Elongation Nontemplate strand of DNA RNA nucleotides 5 3 RNA polymerase 3 5 RNA transcript Template strand of DNA Unwound DNA Elongation 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 Template strand of DNA 3 Termination Newly made RNA 5 3 3 5 A gene can be transcribed simultaneously by several RNA polymerases 5 3 Completed RNA transcript

Synthesis of an RNA Transcript The three stages of transcription: Initiation Elongation Termination Steps: 1) Promoters signal the start of RNA synthesis 2) Transcription factors assist the binding of RNA polymerase to the promoter 3) The whole complex 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

Several transcription factors must bind to the DNA before RNA Fig. 17-8 1 A eukaryotic promoter includes a TATA box Promoter Template 5 3 3 5 TATA box Start point Template DNA strand 2 Several transcription factors must bind to the DNA before RNA polymerase II can do so. Transcription factors 5 3 3 5 3 Additional transcription factors bind to the DNA along with RNA polymerase II, forming the transcription initiation complex. Figure 17.8 The initiation of transcription at a eukaryotic promoter RNA polymerase II Transcription factors 5 3 3 5 5 RNA transcript Transcription initiation complex

Termination of Transcription After RNA polymerase transcribes a terminator sequence in the DNA, the RNA transcript is released and the polymerase detaches. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Eukaryotic cells modify RNA after transcription Enzymes in the eukaryotic nucleus modify pre-mRNA before RNA goes to the cytoplasm Both ends of pre-mRNA are altered; some interior parts are cut out while some are spliced together It gets a cap and a tail, then gets spliced! Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

mRNA’s cap and tail Each end is modified in a particular way: Fig. 17-9 mRNA’s cap and tail Protein-coding segment Polyadenylation signal 5 3 G P P P AAUAAA AAA … AAA 5 Cap 5 UTR Start codon Stop codon 3 UTR Poly-A tail Each end 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 Figure 17.9 RNA processing: addition of the 5 cap and poly-A tail

Split Genes and RNA Splicing Eukaryotic DNA & RNA have long noncoding stretches of nucleotides that lie between coding regions 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…like electricians splice wires together to reconnect them Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

In some cases, RNA splicing is carried out by spliceosomes Fig. 17-10 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 mRNA 5 Cap Poly-A tail 1 146 5 UTR 3 UTR 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 Figure 17.10 RNA processing: RNA splicing

RNA transcript (pre-mRNA) 5 Exon 1 Intron Exon 2 Fig. 17-11-3 RNA transcript (pre-mRNA) 5 Exon 1 Intron Exon 2 Protein Other proteins snRNA snRNPs Spliceosome 5 Figure 17.11 The roles of snRNPs and spliceosomes in pre-mRNA splicing Spliceosome components Cut-out intron mRNA 5 Exon 1 Exon 2

Ribozymes Ribozymes are catalytic RNA molecules that splice RNA and act like enzymes (not all catalysts are proteins!) Three properties of RNA enable it to function as an enzyme It can form a three-dimensional structure because of its ability to base pair with itself Some bases in RNA contain functional groups RNA may hydrogen-bond with other nucleic acid molecules Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

The 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 We have fewer than 25,000 genes to make 100,000 polypeptides, which means the number of different proteins an organism can produce is greater than its number of genes Exon shuffling occurs, too…this could lead to evolution due to crossing over from introns. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Gene DNA Exon 1 Intron Exon 2 Intron Exon 3 Transcription Fig. 17-12 Gene DNA Exon 1 Intron Exon 2 Intron Exon 3 Transcription RNA processing Translation Domain 3 Figure 17.12 Correspondence between exons and protein domains Domain 2 Domain 1 Polypeptide

Molecular Components of Translation A cell translates an mRNA message into protein with the help of transfer RNA (tRNA) Molecules of tRNA are not identical: Each carries a specific amino acid on one end Each has an anticodon on the other end; the anticodon base-pairs with a complementary codon on mRNA BioFlix: Protein Synthesis Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Amino acids tRNA with amino acid attached Ribosome tRNA Anticodon 5 Fig. 17-13 Amino acids Polypeptide tRNA with amino acid attached Ribosome Trp Phe Gly Figure 17.13 Translation: the basic concept tRNA Anticodon 5 Codons 3 mRNA

Fig. 17-14 3 Amino acid attachment site 5 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 Hydrogen bonds Anticodon (a) Two-dimensional structure 5 Amino acid attachment site 3 Figure 17.14 The structure of transfer RNA (tRNA) Hydrogen bonds 3 5 Anticodon Anticodon (c) Symbol used in this book (b) Three-dimensional structure

Accurate translation requires two steps: First: a correct match between a tRNA and an amino acid, done by the enzyme aminoacyl-tRNA synthetase Second: a correct match between the tRNA anticodon and an mRNA codon Flexible pairing at the third base of a codon is called wobble and allows some tRNAs to bind to more than one codon Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Aminoacyl-tRNA Amino acid synthetase (enzyme) tRNA Aminoacyl-tRNA Fig. 17-15-4 Aminoacyl-tRNA synthetase (enzyme) Amino acid P P P Adenosine ATP P Adenosine tRNA P P i Aminoacyl-tRNA synthetase P i P i tRNA Figure 17.15 An aminoacyl-tRNA synthetase joining a specific amino acid to a tRNA P Adenosine AMP Computer model Aminoacyl-tRNA (“charged tRNA”)

Fig. 17-16 Growing polypeptide Exit tunnel tRNA molecules Ribosomes Large subunit E P A Small subunit Ribosomes couple tRNA anticodons with mRNA codons in protein synthesis The two ribosomal subunits (large and small) are made of proteins and ribosomal RNA (rRNA) 5 mRNA 3 (a) Computer model of functioning ribosome 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 Figure 17.16 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

A ribosome has three binding sites for tRNA: The P site holds the tRNA that carries the growing polypeptide chain The A site holds the tRNA that carries the next amino acid to be added to the chain The E site is the exit site, where discharged tRNAs leave the ribosome E PA Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Building Polypeptides: Initiation, Elongation, Termination The initiation stage of translation brings a tRNA with methionine to the start codon (AUG) of mRNA as well as the 2 ribosome subunits Large ribosomal subunit 3 U C 5 A P site Met 5 Met A 3 U G Initiator tRNA GTP GDP E A mRNA 5 5 3 3 Start codon Small ribosomal subunit mRNA binding site Translation initiation complex Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Fig. 17-18-2 Amino end of polypeptide E 3 mRNA P site A site 5 GTP During the elongation stage, amino acids are added one by one to the preceding amino acid in three steps: codon recognition, peptide bond formation, and translocation GDP E P A Figure 17.18 The elongation cycle of translation

Fig. 17-18-3 Amino end of polypeptide E 3 mRNA P site A site 5 GTP During the elongation stage, amino acids are added one by one to the preceding amino acid in three steps: codon recognition, peptide bond formation, and translocation GDP E P A Figure 17.18 The elongation cycle of translation E P A

GDP GDP Fig. 17-18-4 Amino end of polypeptide E 3 mRNA Ribosome ready for next aminoacyl tRNA P site A site 5 GTP GDP E E P A P A Figure 17.18 The elongation cycle of translation GDP GTP E P A

Animation: Translation Termination: a stop codon in mRNA reaches the A site of the ribosome A site accepts a “release factor” protein, which 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 Figure 17.19 The termination of translation 5 3 3 3 2 5 5 GTP Stop codon (UAG, UAA, or UGA) 2 GDP Fig. 17-19-1 Animation: Translation

Fig. 17-20 Completed polypeptide Growing polypeptides Many 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) (a) Ribosomes Figure 17.20 Polyribosomes mRNA (b) 0.1 µm

Protein Folding and Modifications After translation, a polypeptide chain spontaneously coils & folds into its 3-D shape Proteins may also require post-translational modifications before they are functional Two types of ribosomes exist: free (in the cytosol) and bound (attached to the ER) -Free ribosomes synthesize proteins that function in the cytosol -Bound ribosomes make proteins of the endomembrane system and proteins that are secreted from the cell (these are marked with a signal peptide, kind of like a zipcode -Ribosomes are identical and can switch from free to bound Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Ribosome mRNA Signal peptide ER membrane Signal peptide removed Fig. 17-21 Ribosome mRNA Signal peptide ER membrane Signal peptide removed Signal- recognition particle (SRP) Protein CYTOSOL Translocation complex Figure 17.21 The signal mechanism for targeting proteins to the ER ER LUMEN SRP receptor protein

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; they can lead to the production of an abnormal protein Two types: Base-pair substitutions Base-pair insertions or deletions Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Wild-type hemoglobin DNA Mutant hemoglobin DNA 3 C T T 5 3 C A T 5 Fig. 17-22 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 Figure 17.22 The molecular basis of sickle-cell disease: a point mutation Normal hemoglobin Sickle-cell hemoglobin Glu Val

Silent (no effect on amino acid sequence) Fig. 17-23a 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 17.23 Types of point mutations U instead of C 5 3 Stop Silent (no effect on amino acid sequence)

Wild type DNA template strand 3 5 5 3 mRNA 5 3 Protein Stop Fig. 17-23b 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 17.23 Types of point mutations A instead of G 5 3 Stop Missense

Wild type DNA template strand 3 5 5 3 mRNA 5 3 Protein Stop Fig. 17-23c 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 17.23 Types of point mutations U instead of A 5 3 Stop Nonsense

Frameshift causing immediate nonsense (1 base-pair insertion) Fig. 17-23d 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 17.23 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. 17-23e Wild type DNA template strand 3 5 5 3 mRNA 5 3 Protein Stop Amino end Carboxyl end missing 3 5 5 3 Figure 17.23 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. 17-23f Wild type DNA template strand 3 5 5 3 mRNA 5 3 Protein Stop Amino end Carboxyl end missing 3 5 5 3 Figure 17.23 Types of point mutations missing 5 3 Stop No frameshift, but one amino acid missing (3 base-pair deletion)

Substitutions A base-pair substitution replaces one nucleotide and its partner with another pair of nucleotides Silent mutations have no effect on the amino acid produced by a codon because of redundancy in the genetic code Point missense mutations still code for an amino acid, but not necessarily the right amino acid Point nonsense mutations change an amino acid codon into a stop codon, nearly always leading to a nonfunctional protein Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Insertions and Deletions 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 Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Mutagens Spontaneous mutations can occur during replication, recombination, or repair Mutagens are physical or chemical agents that can cause mutations Can you think of some examples of mutagens? Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Fig. 17-25 DNA TRANSCRIPTION 3 Poly-A RNA polymerase 5 RNA transcript RNA PROCESSING Exon RNA transcript (pre-mRNA) Intron Aminoacyl-tRNA synthetase Poly-A NUCLEUS Amino acid AMINO ACID ACTIVATION CYTOPLASM tRNA mRNA Growing polypeptide Cap 3 A Activated amino acid Poly-A P Ribosomal subunits E Figure 17.25 A summary of transcription and translation in a eukaryotic cell Cap 5 TRANSLATION E A Anticodon Codon Ribosome

Did you get it? How specific are the tRNA molecules? How does energy play a role in transcription and translation? In which direction do ribosomes travel down the mRNA strand? Hoe does translation begin and end? What happens to the mRNA strand? Complete the following charts: Transcription Translation Where? What is used as a template? What is used to synthesize the new strand? What is the new strand made of?

Complete the Chart Replication Protein Synthesis Where? When? Purpose? Starting Point? What is used to synthesize the new strand? Associated proteins? Nucleotides? Finishing processes? Where does the finished “product(s)” go?

You should now be able to: Describe the contributions made by Beadle and Tatum to our understanding of the relationship between genes and enzymes Briefly explain how information flows from gene to protein Compare transcription and translation in bacteria and eukaryotes Explain what it means to say that the genetic code is redundant and unambiguous Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

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

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