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Dn ato protein Dn ato protein Presentation Transcript

  • The Molecular Basis of Inheritance
    • Overview: Life’s Operating Instructions
    • In 1953, James Watson and Francis Crick shook the world
      • With an elegant double-helical model for the structure of deoxyribonucleic acid, or DNA
    Figure 16.1
    • Hereditary information
      • Is encoded in the chemical language of DNA and reproduced in all the cells of your body
    • It is the DNA program
      • That directs the development of many different types of traits
  • Evidence That DNA Can Transform Bacteria
    • The role of DNA in heredity
      • Was first worked out by studying bacteria and the viruses that infect them
    • Frederick Griffith was studying Streptococcus pneumoniae
      • A bacterium that causes pneumonia in mammals
    • He worked with two strains of the bacterium
      • A pathogenic strain and a nonpathogenic strain
    • Griffith found that when he mixed heat-killed remains of the pathogenic strain
      • With living cells of the nonpathogenic strain, some of these living cells became pathogenic
    Bacteria of the “S” (smooth) strain of Streptococcus pneumoniae are pathogenic because they have a capsule that protects them from an animal’s defense system. Bacteria of the “R” (rough) strain lack a capsule and are nonpathogenic. Frederick Griffith injected mice with the two strains as shown below: Griffith concluded that the living R bacteria had been transformed into pathogenic S bacteria by an unknown, heritable substance from the dead S cells. EXPERIMENT RESULTS CONCLUSION Living S (control) cells Living R (control) cells Heat-killed (control) S cells Mixture of heat-killed S cells and living R cells Mouse dies Mouse healthy Mouse healthy Mouse dies Living S cells are found in blood sample. Figure 16.2
    • Griffith called the phenomenon transformation
    Bacteria of the “S” (smooth) strain of Streptococcus pneumoniae are pathogenic because they have a capsule that protects them from an animal’s defense system. Bacteria of the “R” (rough) strain lack a capsule and are nonpathogenic. Frederick Griffith injected mice with the two strains as shown below: Griffith concluded that the living R bacteria had been transformed into pathogenic S bacteria by an unknown, heritable substance from the dead S cells. EXPERIMENT RESULTS CONCLUSION Living S (control) cells Living R (control) cells Heat-killed (control) S cells Mixture of heat-killed S cells and living R cells Mouse dies Mouse healthy Mouse healthy Mouse dies Living S cells are found in blood sample. Figure 16.2
  • Evidence That Viral DNA Can Program Cells
    • Additional evidence for DNA as the genetic material
      • Came from studies of a virus that infects bacteria
    • Viruses that infect bacteria, bacteriophages
      • Are widely used as tools by researchers in molecular genetics
    Figure 16.3 Phage head Tail Tail fiber DNA Bacterial cell 100 nm
  • Experiments showing that DNA is the genetic material of a phage (T2)
    • The Hershey and Chase experiment
    Animation of experiment In their famous 1952 experiment, Alfred Hershey and Martha Chase used radioactive sulfur and phosphorus to trace the fates of the protein and DNA, respectively, of T2 phages that infected bacterial cells. Radioactivity (phage protein) in liquid Phage Bacterial cell Radioactive protein Empty protein shell Phage DNA DNA Centrifuge Pellet (bacterial cells and contents) Radioactive DNA Centrifuge Pellet Batch 1: Phages were grown with radioactive sulfur ( 35 S ), which was incorporated into phage protein (pink). Batch 2: Phages were grown with radioactive phosphorus ( 32 P ), which was incorporated into phage DNA (blue). 1 2 3 4 Agitated in a blender to separate phages outside the bacteria from the bacterial cells. Mixed radioactively labeled phages with bacteria. The phages infected the bacterial cells. Centrifuged the mixture so that bacteria formed a pellet at the bottom of the test tube. Measured the radioactivity in the pellet and the liquid Phage proteins remained outside the bacterial cells during infection, while phage DNA entered the cells. When cultured, bacterial cells with radioactive phage DNA released new phages with some radioactive phosphorus. Hershey and Chase concluded that DNA, not protein, functions as the T2 phage’s genetic material. RESULTS CONCLUSION EXPERIMENT Radioactivity (phage DNA) in pellet Figure 16.4
  • Additional Evidence That DNA Is the Genetic Materia
    • Prior to the 1950s, it was already known that DNA
      • Is a polymer of nucleotides, each consisting of three components: a nitrogenous base, a sugar, and a phosphate group
    Sugar-phosphate backbone Nitrogenous bases 5 end O – O P O CH 2 5 4 O – H H O H H H 3 1 H O CH 3 N O N H Thymine (T) O O P O O – CH 2 H H O H H H H N N N H N H H Adenine (A) O O P O O – CH 2 H H O H H H H H H H N N N O Cytosine (C) O O P O CH 2 5 4 O – H O H H 3 1 OH 2 H N N N H O N N H H H H Sugar (deoxyribose) 3 end Phosphate Guanine (G) DNA nucleotide 2 N Figure 16.5
    • Erwin Chargaff analyzed the base composition of DNA
      • From a number of different organisms
    • In 1947, Chargaff reported
      • That DNA composition varies from one species to the next
    • This evidence of molecular diversity among species
      • Made DNA a more credible candidate for the genetic material
  • Building a Structural Model of DNA: Scientific Inquiry
    • Maurice Wilkins and Rosalind Franklin
      • Were using a technique called X-ray crystallography to study molecular structure
    • Rosalind Franklin
      • Produced a picture of the DNA molecule using this technique
    (a) Rosalind Franklin Franklin’s X-ray diffraction Photograph of DNA (b) Figure 16.6 a, b
    • Watson and Crick deduced that DNA was a double helix
      • Through observations of the X-ray crystallographic images of DNA
    Figure 16.7a, c C T A A T C G G C A C G A T A T A T T A C T A 0.34 nm 3.4 nm (a) Key features of DNA structure G 1 nm G (c) Space-filling model T
    • Franklin had concluded that DNA
      • Was composed of two antiparallel sugar-phosphate backbones, with the nitrogenous bases paired in the molecule’s interior
    • The nitrogenous bases
      • Are paired in specific combinations: adenine with thymine, and cytosine with guanine
  • O – O O OH O – O O O H 2 C O – O O O H 2 C O – O O O OH O O O T A C G C A T O O O CH 2 O O – O O CH 2 CH 2 CH 2 5 end Hydrogen bond 3 end 3 end G P P P P O OH O – O O O P P O – O O O P O – O O O P (b) Partial chemical structure H 2 C 5 end Figure 16.7b O
    • Watson and Crick reasoned that there must be additional specificity of pairing
      • Dictated by the structure of the bases
    • Each base pair forms a different number of hydrogen bonds
      • Adenine and thymine form two bonds, cytosine and guanine form three bonds
  • N H O CH 3 N N O N N N N H Sugar Sugar Adenine (A) Thymine (T) N N N N Sugar O H N H N H N O H H N Sugar Guanine (G) Cytosine (C) Figure 16.8 H
    • Many proteins work together in DNA replication and repair (DNA-Protein like Chicken-Egg debate, which came first?)
    • Since the two strands of DNA are complementary
      • Each strand acts as a template for building a new strand in replication
    • In DNA replication
      • The parent molecule unwinds, and two new daughter strands are built based on base-pairing rules
    (a) The parent molecule has two complementary strands of DNA. Each base is paired by hydrogen bonding with its specific partner, A with T and G with C. (b) The first step in replication is separation of the two DNA strands. (c) Each parental strand now serves as a template that determines the order of nucleotides along a new, complementary strand. (d) The nucleotides are connected to form the sugar-phosphate backbones of the new strands. Each “daughter” DNA molecule consists of one parental strand and one new strand. A C T A G A C T A G A C T A G A C T A G T G A T C T G A T C A C T A G A C T A G T G A T C T G A T C T G A T C T G A T C Figure 16.9 a–d
    • DNA replication is semiconservative
      • Each of the two new daughter molecules will have one old strand, derived from the parent molecule, and one newly made strand
    (a) (b) (c) Figure 16.10 a–c Conservative model. The two parental strands reassociate after acting as templates for new strands, thus restoring the parental double helix. Semiconservative model. The two strands of the parental molecule separate, and each functions as a template for synthesis of a new, comple- mentary strand. Dispersive model. Each strand of both daughter mol- ecules contains a mixture of old and newly synthesized DNA. Parent cell First replication Second replication
  • DNA Replication: A Closer Look
    • The copying of DNA
      • Is remarkable in its speed and accuracy
    • More than a dozen enzymes and other proteins
      • Participate in DNA replication
    • The replication of a DNA molecule
      • Begins at special sites called origins of replication, where the two strands are separated
    • A eukaryotic chromosome
      • May have hundreds or even thousands of replication origins
    Replication begins at specific sites where the two parental strands separate and form replication bubbles. The bubbles expand laterally, as DNA replication proceeds in both directions. Eventually, the replication bubbles fuse, and synthesis of the daughter strands is complete. 1 2 3 Origin of replication Bubble Parental (template) strand Daughter (new) strand Replication fork Two daughter DNA molecules In eukaryotes, DNA replication begins at many sites along the giant DNA molecule of each chromosome. In this micrograph, three replication bubbles are visible along the DNA of a cultured Chinese hamster cell (TEM). (b) (a) 0.25 µm Figure 16.12 a, b
  • Elongating a New DNA Strand
    • Elongation of new DNA at a replication fork
      • Is catalyzed by enzymes called DNA polymerases , which add nucleotides to the 3  end of a growing strand
    Nucleoside triphosphate Figure 16.13 New strand Template strand 5 end 3 end Sugar A T Base C G G C A C T P P P OH P P 5 end 3 end 5 end 5 end A T C G G C A C T 3 end Pyrophosphate 2 P OH Phosphate
    • DNA polymerases add nucleotides
      • Only to the free 3  end of a growing strand
    • Along one template strand of DNA, the leading strand
      • DNA polymerase III can synthesize a complementary strand continuously, moving toward the replication fork
    • To elongate the other new strand of DNA, the lagging strand
      • DNA polymerase III must work in the direction away from the replication fork
    • The lagging strand
      • Is synthesized as a series of segments called Okazaki fragments, which are then joined together by DNA ligase
    • Synthesis of leading and lagging strands during DNA replication
    Parental DNA DNA pol Ill elongates DNA strands only in the 5 3 direction. 1 Okazaki fragments DNA pol III Template strand Lagging strand 3 2 Template strand DNA ligase Overall direction of replication One new strand, the leading strand, can elongate continuously 5 3 as the replication fork progresses. 2 The other new strand, the lagging strand must grow in an overall 3 5 direction by addition of short segments, Okazaki fragments, that grow 5 3 (numbered here in the order they were made). 3 DNA ligase joins Okazaki fragments by forming a bond between their free ends. This results in a continuous strand. 4 Figure 16.14 3 5 5 3 3 5 2 1 Leading strand 1
  • Priming DNA Synthesis
    • DNA polymerases cannot initiate the synthesis of a polynucleotide
      • They can only add nucleotides to the 3  end
    • The initial nucleotide strand
      • Is an RNA or DNA primer
    • Only one primer is needed for synthesis of the leading strand
      • But for synthesis of the lagging strand, each Okazaki fragment must be primed separately
  • Replication Animation**** Overall direction of replication 3 3 3 3 5 3 5 3 5 3 5 3 5 3 5 3 5 3 5 5 1 1 2 1 1 2 5 5 1 2 3 5 Template strand RNA primer Okazaki fragment Figure 16.15 Primase joins RNA nucleotides into a primer. 1 DNA pol III adds DNA nucleotides to the primer, forming an Okazaki fragment. 2 After reaching the next RNA primer (not shown), DNA pol III falls off. 3 After the second fragment is primed. DNA pol III adds DNA nucleotides until it reaches the first primer and falls off. 4 DNA pol 1 replaces the RNA with DNA, adding to the 3 end of fragment 2. 5 DNA ligase forms a bond between the newest DNA and the adjacent DNA of fragment 1. 6 The lagging strand in this region is now complete. 7
  • Other Proteins That Assist DNA Replication
    • Helicase, topoisomerase, single-strand binding protein
      • Are all proteins that assist DNA replication
    Table 16.1
  • Replication Animation #2 Recap
    • A summary of DNA replication
    Figure 16.16 Overall direction of replication Leading strand Lagging strand Lagging strand Leading strand OVERVIEW Leading strand Replication fork DNA pol III Primase Primer DNA pol III Lagging strand DNA pol I Parental DNA 5 3 4 3 2 Origin of replication DNA ligase 1 5 3 Helicase unwinds the parental double helix. 1 Molecules of single- strand binding protein stabilize the unwound template strands. 2 The leading strand is synthesized continuously in the 5 3 direction by DNA pol III. 3 Primase begins synthesis of RNA primer for fifth Okazaki fragment. 4 DNA pol III is completing synthesis of the fourth fragment, when it reaches the RNA primer on the third fragment, it will dissociate, move to the replication fork, and add DNA nucleotides to the 3 end of the fifth fragment primer. 5 DNA pol I removes the primer from the 5 end of the second fragment, replacing it with DNA nucleotides that it adds one by one to the 3 end of the third fragment. The replacement of the last RNA nucleotide with DNA leaves the sugar- phosphate backbone with a free 3 end. 6 DNA ligase bonds the 3 end of the second fragment to the 5 end of the first fragment. 7
  • The DNA Replication Machine as a Stationary Complex
    • The various proteins that participate in DNA replication
      • Form a single large complex, a DNA replication “machine”
    • The DNA replication machine
      • Is probably stationary during the replication process
  • Proofreading and Repairing DNA
    • DNA polymerases proofread newly made DNA
      • Replacing any incorrect nucleotides
    • In mismatch repair of DNA
      • Repair enzymes correct errors in base pairing
    • In nucleotide excision repair
      • Enzymes cut out and replace damaged stretches of DNA
    Figure 16.17 Nuclease DNA polymerase DNA ligase A nuclease enzyme cuts the damaged DNA strand at two points and the damaged section is removed. Repair synthesis by a DNA polymerase fills in the missing nucleotides. 3 DNA ligase seals the Free end of the new DNA To the old DNA, making the strand complete. A thymine dimer distorts the DNA molecule. 1 2 4
  • Replicating the Ends of DNA Molecules
    • The ends of eukaryotic chromosomal DNA
      • Get shorter with each round of replication
    Figure 16.18 End of parental DNA strands Leading strand Lagging strand Last fragment Previous fragment RNA primer Lagging strand Removal of primers and replacement with DNA where a 3 end is available Primer removed but cannot be replaced with DNA because no 3 end available for DNA polymerase Second round of replication New leading strand New lagging strand 5 Further rounds of replication Shorter and shorter daughter molecules 5 3 5 3 5 3 5 3 3
    • Eukaryotic chromosomal DNA molecules
      • Have at their ends nucleotide sequences, called telomeres , that postpone the erosion of genes near the ends of DNA molecules
    Figure 16.19 1 µm
    • If the chromosomes of germ cells became shorter in every cell cycle
      • Essential genes would eventually be missing from the gametes they produce
    • An enzyme called telomerase
      • Catalyzes the lengthening of telomeres in germ cells
  • From Gene to Protein
    • The DNA inherited by an organism
      • Leads to specific traits by dictating the synthesis of proteins
    • The process by which DNA directs protein synthesis, gene expression
      • Includes two stages, called transcription and translation
    • The ribosome
      • Is part of the cellular machinery for translation, polypeptide synthesis
    Figure 17.1
  • Genes specify proteins via transcription and translation
    • In 1909, British physician Archibald Garrod
      • Was the first to suggest that genes dictate phenotypes through enzymes that catalyze specific chemical reactions in the cell
    • Beadle and Tatum causes bread mold to mutate with X-rays
      • Creating mutants that could not survive on minimal medium
    • Using genetic crosses
      • They determined that their mutants fell into three classes, each mutated in a different gene
    Figure 17.2 Working with the mold Neurospora crassa , George Beadle and Edward Tatum had isolated mutants requiring arginine in their growth medium and had shown genetically that these mutants fell into three classes, each defective in a different gene. From other considerations, they suspected that the metabolic pathway of arginine biosynthesis included the precursors ornithine and citrulline. Their most famous experiment, shown here, tested both their one gene–one enzyme hypothesis and their postulated arginine pathway. In this experiment, they grew their three classes of mutants under the four different conditions shown in the Results section below. The wild-type strain required only the minimal medium for growth. The three classes of mutants had different growth requirements EXPERIMENT RESULTS Class I Mutants Class II Mutants Class III Mutants Wild type Minimal medium (MM) (control) MM + Ornithine MM + Citrulline MM + Arginine (control)
  • CONCLUSION From the growth patterns of the mutants, Beadle and Tatum deduced that each mutant was unable to carry out one step in the pathway for synthesizing arginine, presumably because it lacked the necessary enzyme. Because each of their mutants was mutated in a single gene, they concluded that each mutated gene must normally dictate the production of one enzyme. Their results supported the one gene–one enzyme hypothesis and also confirmed the arginine pathway. (Notice that a mutant can grow only if supplied with a compound made after the defective step.) Class I Mutants (mutation in gene A ) Class II Mutants (mutation in gene B ) Class III Mutants (mutation in gene C ) Wild type Gene A Gene B Gene C Precursor Precursor Precursor Precursor Ornithine Ornithine Ornithine Ornithine Citrulline Citrulline Citrulline Citrulline Arginine Arginine Arginine Arginine Enzyme A Enzyme B Enzyme C A A A B B B C C C
    • Beadle and Tatum developed the “one gene–one enzyme hypothesis”
      • Which states that the function of a gene is to dictate the production of a specific enzyme
    • As researchers learned more about proteins
      • They made minor revision to the one gene–one enzyme hypothesis
    • Genes code for polypeptide chains or for RNA molecules
  • Basic Principles of Transcription and Translation
    • 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
    http://vcell.ndsu.nodak.edu/animations/transcription/index.htm - animations
    • In prokaryotes
      • Transcription and translation occur together
    Figure 17.3a Prokaryotic cell. In a cell lacking a nucleus, mRNA produced by transcription is immediately translated without additional processing. (a) TRANSLATION TRANSCRIPTION DNA mRNA Ribosome Polypeptide
  • Prokaryote/Eukaryote differences animation
    • In eukaryotes
      • RNA transcripts are modified before becoming true mRNA
    Figure 17.3b Eukaryotic cell. The nucleus provides a separate compartment for transcription. The original RNA transcript, called pre-mRNA, is processed in various ways before leaving the nucleus as mRNA. (b) TRANSCRIPTION RNA PROCESSING TRANSLATION mRNA DNA Pre-mRNA Polypeptide Ribosome Nuclear envelope
    • Cells are governed by a cellular chain of command
      • DNA  RNA  protein
    • Genetic information
      • Is encoded as a sequence of nonoverlapping base triplets, or codons
    • During transcription
      • The gene determines the sequence of bases along the length of an mRNA molecule
    Figure 17.4 DNA molecule Gene 1 Gene 2 Gene 3 DNA strand (template) TRANSCRIPTION mRNA Protein TRANSLATION Amino acid A C C A A A C C G A G T U G G U U U G G C U C A Trp Phe Gly Ser Codon 3 5 3 5
  • Cracking the Code
    • A codon in messenger RNA
      • Is either translated into an amino acid or serves as a translational stop signal
    • Codons must be read in the correct reading frame
      • For the specified polypeptide to be produced
  • Molecular Components of Transcription
    • RNA synthesis
      • Is catalyzed by RNA polymerase , which pries the DNA strands apart and hooks together the RNA nucleotides
      • Follows the same base-pairing rules as DNA, except that in RNA, uracil substitutes for thymine
  • Synthesis of an RNA Transcript
    • The stages of transcription are
      • Initiation
      • Elongation
      • Termination
    Figure 17.7 Promoter Transcription unit RNA polymerase Start point 5 3 3 5 3 5 5 3 5 3 3 5 5 3 3 5 5 5 Rewound RNA RNA transcript 3 3 Completed RNA transcript Unwound DNA RNA transcript Template strand of DNA DNA 1 Initiation. 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. 2 Elongation. The polymerase moves downstream, unwinding the DNA and elongating the RNA transcript 5  3 . In the wake of transcription, the DNA strands re-form a double helix. 3 Termination. Eventually, the RNA transcript is released, and the polymerase detaches from the DNA.
  • Elongation RNA polymerase Non-template strand of DNA RNA nucleotides 3 end C A E G C A A U T A G G T T A A C G U A T C A T C C A A T T G G 3 5 5 Newly made RNA Direction of transcription (“downstream”) Template strand of DNA
  • RNA Polymerase Binding and Initiation of Transcription
    • Promoters signal the initiation of RNA synthesis
    • Transcription factors
      • Help eukaryotic RNA polymerase recognize promoter sequences
    Figure 17.8 Figure 17.8 TRANSCRIPTION RNA PROCESSING TRANSLATION DNA Pre-mRNA mRNA Ribosome Polypeptide T A T A A A A A T A T T T T TATA box Start point Template DNA strand 5 3 3 5 Transcription factors 5 3 3 5 Promoter 5 3 3 5 5 RNA polymerase II Transcription factors RNA transcript Transcription initiation complex Eukaryotic promoters 1 Several transcription factors 2 Additional transcription factors 3
  • Elongation of the RNA Strand
    • As RNA polymerase moves along the DNA
      • It continues to untwist the double helix, exposing about 10 to 20 DNA bases at a time for pairing with RNA nucleotides
    • The mechanisms of termination
      • Are different in prokaryotes and eukaryotes
    • Eukaryotic cells modify RNA after transcription
    • Enzymes in the eukaryotic nucleus
      • Modify pre-mRNA in specific ways before the genetic messages are dispatched to the cytoplasm
  • Alteration of mRNA Ends
    • Each end of a pre-mRNA molecule is modified in a particular way
      • The 5  end receives a modified nucleotide cap
      • The 3  end gets a poly-A tail
    Figure 17.9 Video clip A modified guanine nucleotide added to the 5 end 50 to 250 adenine nucleotides added to the 3 end Protein-coding segment Polyadenylation signal Poly-A tail 3 UTR Stop codon Start codon 5 Cap 5 UTR AAUAAA AAA…AAA TRANSCRIPTION RNA PROCESSING DNA Pre-mRNA mRNA TRANSLATION Ribosome Polypeptide G P P P 5 3
  • Split Genes and RNA Splicing
    • RNA splicing
      • Removes introns (supposed “Junk-DNA”) and joins exons
    Figure 17.10 TRANSCRIPTION RNA PROCESSING DNA Pre-mRNA mRNA TRANSLATION Ribosome Polypeptide 5 Cap Exon Intron 1 5 30 31 Exon Intron 104 105 146 Exon 3 Poly-A tail Poly-A tail Introns cut out and exons spliced together Coding segment 5 Cap 1 146 3 UTR 3 UTR Pre-mRNA mRNA
    • Is carried out by spliceosomes in some cases
    Figure 17.11 Animation
    • Ribozymes
      • Are catalytic RNA molecules that function as enzymes and can splice RNA
    RNA transcript (pre-mRNA) Exon 1 Intron Exon 2 Other proteins Protein snRNA snRNPs Spliceosome Spliceosome components Cut-out intron mRNA Exon 1 Exon 2 5 5 5 1 2 3
    • 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
    Figure 17.12 Gene DNA Exon 1 Intron Exon 2 Intron Exon 3 Transcription RNA processing Translation Domain 3 Domain 1 Domain 2 Polypeptide
    • A cell translates an mRNA message into protein
      • With the help of transfer RNA (tRNA)
    Figure 17.13 TRANSCRIPTION TRANSLATION DNA mRNA Ribosome Polypeptide Polypeptide Amino acids tRNA with amino acid attached Ribosome tRNA Anticodon mRNA Trp Phe Gly A G C A A A C C G U G G U U U G G C Codons 5 3
    • Molecules of tRNA are not all identical
      • Each carries a specific amino acid on one end
      • Each has an anticodon on the other end
    (b) Three-dimensional structure Symbol used in this book Amino acid attachment site Hydrogen bonds Anticodon Anticodon A A G 5 3 3 5 (c)
  • The Structure and Function of Transfer RNA
    • A tRNA molecule
      • Consists of a single RNA strand that is only about 80 nucleotides long
    A C C Figure 17.14a Two-dimensional structure. The four base-paired regions and three loops are characteristic of all tRNAs, as is the base sequence of the amino acid attachment site at the 3 end. The anticodon triplet is unique to each tRNA type. (The asterisks mark bases that have been chemically modified, a characteristic of tRNA.) (a) 3  C C A C G C U U A A G A C A C C U * G C * * G U G U * C U * G A G G U * * A * A A G U C A G A C C * C G A G A G G G * * G A C U C * A U U U A G G C G 5  Amino acid attachment site Hydrogen bonds Anticodon A
    • A specific enzyme called an aminoacyl-tRNA synthetase
      • Joins each amino acid to the correct tRNA
    Figure 17.15 Amino acid ATP Adenosine Pyrophosphate Adenosine Adenosine Phosphates tRNA P P P P P P i P i P i P AMP Aminoacyl tRNA (an “activated amino acid”) Aminoacyl-tRNA synthetase (enzyme) Active site binds the amino acid and ATP. 1 ATP loses two P groups and joins amino acid as AMP. 2 3 Appropriate tRNA covalently Bonds to amino Acid, displacing AMP. Activated amino acid is released by the enzyme. 4
  • Ribosomes
    • Ribosomes
      • Facilitate the specific coupling of tRNA anticodons with mRNA codons during protein synthesis
    • The ribosomal subunits
      • Are constructed of proteins and RNA molecules named ribosomal RNA or rRNA
    Figure 17.16a TRANSCRIPTION TRANSLATION DNA mRNA Ribosome Polypeptide Exit tunnel Growing polypeptide tRNA molecules E P A Large subunit Small subunit mRNA Computer model of functioning ribosome. This is a model of a bacterial ribosome, showing its overall shape. The eukaryotic ribosome is roughly similar. A ribosomal subunit is an aggregate of ribosomal RNA molecules and proteins. (a) 5 3
    • The ribosome has three binding sites for tRNA
      • The P site
      • The A site
      • The E site
    Figure 17.16b E P A P site ( P eptidyl-tRNA binding site) E site ( E xit site) mRNA binding site A site ( A minoacyl- tRNA binding site) Large subunit Small subunit Schematic model showing binding sites. A ribosome has an mRNA binding site and three tRNA binding sites, known as the A, P, and E sites. This schematic ribosome will appear in later diagrams. (b)
  • Figure 17.16c Amino end Growing polypeptide Next amino acid to be added to polypeptide chain tRNA mRNA Codons 3 5 Schematic model with mRNA and tRNA. 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 the tRNA carrying the next amino acid to be added to the polypeptide chain. Discharged tRNA leaves via the E site. (c)
  • Building a Polypeptide
    • We can divide translation into three stages
      • Initiation
      • Elongation
      • Termination
  • Ribosome Association and Initiation of Translation
    • The initiation stage of translation
      • Brings together mRNA, tRNA bearing the first amino acid of the polypeptide, and two subunits of a ribosome
    Large ribosomal subunit The arrival of a large ribosomal subunit completes the initiation complex. Proteins called initiation factors (not shown) are required to bring all the translation components together. GTP provides the energy for the assembly. The initiator tRNA is in the P site; the A site is available to the tRNA bearing the next amino acid. 2 Initiator tRNA mRNA mRNA binding site Small ribosomal subunit Translation initiation complex P site GDP GTP Start codon A small ribosomal subunit binds to a molecule of mRNA. In a prokaryotic cell, the mRNA binding site on this subunit recognizes a specific nucleotide sequence on the mRNA just upstream of the start codon. An initiator tRNA, with the anticodon UAC, base-pairs with the start codon, AUG. This tRNA carries the amino acid methionine (Met). 1 Met Met U A C A U G E A 3 5 5 3 3 5 3 5 Figure 17.17
  • Elongation of the Polypeptide Chain
    • In the elongation stage of translation
      • Amino acids are added one by one to the preceding amino acid
    Figure 17.18 Amino end of polypeptide mRNA Ribosome ready for next aminoacyl tRNA E P A E P A E P A E P A GDP GTP GTP GDP 2 2 site site 5 3 TRANSCRIPTION TRANSLATION DNA mRNA Ribosome Polypeptide Codon recognition. The anticodon of an incoming aminoacyl tRNA base-pairs with the complementary mRNA codon in the A site. Hydrolysis of GTP increases the accuracy and efficiency of this step. 1 Peptide bond formation. An rRNA molecule of the large subunit catalyzes the formation of a peptide bond between the new amino acid in the A site and the carboxyl end of the growing polypeptide in the P site. This step attaches the polypeptide to the tRNA in the A site. 2 Translocation. The ribosome translocates the tRNA in the A site to the P site. The empty tRNA in the P site is moved to the E site, where it is released. The mRNA moves along with its bound tRNAs, bringing the next codon to be translated into the A site. 3
  • Termination of Translation
    • The final stage of translation is termination
      • When the ribosome reaches a stop codon in the mRNA
    Protein Synthesis Animation Figure 17.19 Release factor Free polypeptide Stop codon (UAG, UAA, or UGA) 5 3 3 5 3 5 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. 1 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. 2 3 The two ribosomal subunits and the other components of the assembly dissociate.
  • Polyribosomes
    • A number of ribosomes can translate a single mRNA molecule simultaneously
      • Forming a polyribosome
    Figure 17.20a, b Growing polypeptides Completed polypeptide Incoming ribosomal subunits Start of mRNA (5 end) End of mRNA (3 end) Polyribosome An mRNA molecule is generally translated simultaneously by several ribosomes in clusters called polyribosomes. (a) Ribosomes mRNA This micrograph shows a large polyribosome in a prokaryotic cell (TEM). 0.1 µm (b)
  • Protein Folding and Post-Translational Modifications
    • After translation
      • Proteins may be modified in ways that affect their three-dimensional shape
  • Targeting Polypeptides to Specific Locations
    • Two populations of ribosomes are evident in cells
      • Free and bound
    • Free ribosomes in the cytosol
      • Initiate the synthesis of all proteins
    • Proteins destined for the endomembrane system or for secretion
      • Must be transported into the ER
      • Have signal peptides to which a signal-recognition particle (SRP) binds, enabling the translation ribosome to bind to the ER
    • The signal mechanism for targeting proteins to the ER
    Figure 17.21 Ribosome mRNA Signal peptide Signal- recognition particle (SRP) SRP receptor protein Translocation complex CYTOSOL Signal peptide removed ER membrane Protein ERLUMEN Polypeptide synthesis begins on a free ribosome in the cytosol. 1 An SRP binds to the signal peptide, halting synthesis momentarily. 2 The SRP binds to a receptor protein in the ER membrane. This receptor is part of a protein complex (a translocation complex) that has a membrane pore and a signal-cleaving enzyme. 3 The SRP leaves, and the polypeptide resumes growing, meanwhile translocating across the membrane. (The signal peptide stays attached to the membrane.) 4 The signal- cleaving enzyme cuts off the signal peptide. 5 The rest of the completed polypeptide leaves the ribosome and folds into its final conformation. 6
    • RNA plays multiple roles in the cell: a review
    • RNA
      • Can hydrogen-bond to other nucleic acid molecules
      • Can assume a specific three-dimensional shape
      • Has functional groups that allow it to act as a catalyst
    • Types of RNA in a Eukaryotic Cell
    Table 17.1
    • Comparing gene expression in prokaryotes and eukaryotes reveals key differences
    • Prokaryotic cells lack a nuclear envelope
      • Allowing translation to begin while transcription is still in progress
    Figure 17.22 DNA Polyribosome mRNA Direction of transcription 0.25 m RNA polymerase Polyribosome Ribosome DNA mRNA (5 end) RNA polymerase Polypeptide (amino end)
    • 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
    • A gene
      • Is a region of DNA whose final product is either a polypeptide or an RNA molecule
    • A summary of transcription and translation in a eukaryotic cell
    Figure 17.26 TRANSCRIPTION RNA is transcribed from a DNA template. DNA RNA polymerase RNA transcript RNA PROCESSING In eukaryotes, the RNA transcript (pre- mRNA) is spliced and modified to produce mRNA, which moves from the nucleus to the cytoplasm. Exon Poly-A RNA transcript (pre-mRNA) Intron NUCLEUS Cap FORMATION OF INITIATION COMPLEX After leaving the nucleus, mRNA attaches to the ribosome. CYTOPLASM mRNA Poly-A Growing polypeptide Ribosomal subunits Cap Aminoacyl-tRNA synthetase Amino acid tRNA AMINO ACID ACTIVATION Each amino acid attaches to its proper tRNA with the help of a specific enzyme and ATP. Activated amino acid TRANSLATION A succession of tRNAs add their amino acids to the polypeptide chain as the mRNA is moved through the ribosome one codon at a time. (When completed, the polypeptide is released from the ribosome.) Anticodon A C C A A A U G G U U U A U G U A C E A Ribosome 1 Poly-A 5 5 3 Codon 2 3 4 5
    • Point mutations can affect protein structure and function
    • Mutations
      • Are changes in the genetic material of a cell
    • Point mutations
      • Are changes in just one base pair of a gene
    • The change of a single nucleotide in the DNA’s template strand
      • Leads to the production of an abnormal protein
    Figure 17.23 In the DNA, the mutant template strand has an A where the wild-type template has a T. The mutant mRNA has a U instead of an A in one codon. The mutant (sickle-cell) hemoglobin has a valine (Val) instead of a glutamic acid (Glu). Mutant hemoglobin DNA Wild-type hemoglobin DNA mRNA mRNA Normal hemoglobin Sickle-cell hemoglobin Glu Val C T T C A T G A A G U A 3 5 3 5 5 3 5 3
  • Types of Point Mutations
    • Point mutations within a gene can be divided into two general categories
      • Base-pair substitutions
      • Base-pair insertions or deletions (indels)
  • Substitutions
    • A base-pair substitution
      • Is the replacement of one nucleotide and its partner with another pair of nucleotides
      • Can cause missense or nonsense
    Figure 17.24 Wild type A U G A A G U U U G G C U A A mRNA 5 Protein Met Lys Phe Gly Stop Carboxyl end Amino end 3 A U G A A G U U U G G U U A A Met Lys Phe Gly Base-pair substitution No effect on amino acid sequence U instead of C Stop A U G A A G U U U A G U U A A Met Lys Phe Ser Stop A U G U A G U U U G G C U A A Met Stop Missense A instead of G Nonsense U instead of A
  • Insertions and Deletions
    • Insertions and deletions
      • Are additions or losses of nucleotide pairs in a gene
      • May produce frameshift mutations
    Figure 17.25 mRNA Protein Wild type A U G A A G U U U G G C U A A 5 Met Lys Phe Gly Amino end Carboxyl end Stop Base-pair insertion or deletion Frameshift causing immediate nonsense A U G U A A G U U U G G C U A A U G A A G U U G G C U A A A U G U U U G G C U A A Met Stop U Met Lys Leu Ala Met Phe Gly Stop Missing A A G Missing Extra U Frameshift causing extensive missense Insertion or deletion of 3 nucleotides: no frameshift but extra or missing amino acid 3
  • Mutagens
    • Spontaneous mutations
      • Can occur during DNA replication, recombination, or repair
    • Mutagens
      • Are physical or chemical agents that can cause mutations