Chapter 17: From Gene to Protein

This is going to be a very long journey, but it is crucial to your understanding of biology. Work on this chapter a single concept at a time, and expect to spend at least 6 hours to truly master the material. To give you an idea of the depth and time required, we have spent over 5 hours writing this Reading Guide! You will need even longer to complete it and learn the information. Good luck, and take your time. Overview 1. What is gene expression? Gene expression is the process by which DNA directs the synthesis of proteins (or, in some cases, RNAs). Concept 17.1 Genes specify proteins via transcription and translation 2. What situation did Archibald Garrod suggest caused inborn errors of metabolism? Garrod postulated that the symptoms of an inherited disease reflect a persons inability to make a particular enzyme. 3. Describe one example Garrod used to illustrate his hypothesis. Garrod used one example as the hereditary condition called alkptonuria. This condition causes urine to become black because it contains the chemical alkapton, which darkens upon exposure to air. Garrod reasoned that most people have an enzyme that metabolizes alkapton, whereas people with alkptonuria have inherited an inability to make that enzyme. 4. State the hypothesis formulated by George Beadle while studying eye color mutations in Drosophila. George Beadle hypothesized that in Drosophila, each of the various mutations affecting eye color blocks pigment synthesis of at a specific step by preventing production of the enzyme that catalyzes that step. 5. What strategy did Beadle and Tatum adopt to test this hypothesis? Beadle and Tatum studied the genetics of a bread mold called Neurospora Crasa. When the radiated spores failed to produce the essential proteins, the one gene- one polypeptide hypothesis was born. 6. Which organism did Beadle and Tatum use in their research? How did this organisms nutritional requirements facilitate this research?

Beadle and Tatum used Neurospora Crassa for their experiments. They used this bread mold because it was easy to grow and had simple nutritional needs. Also, this bread mold had a single set of chromosomes so scientists could observe changes easily. 7. How were Neurospora spores treated to increase the mutation rate? Neurospora spores were treated with complete growth medium, which consisted of minimal medium supplemented with all 20 amino acids and a few other nutrients. 8. Study Figure 17.2 carefully. On the figure below, outline the technique used to identify and isolate mutant fungi. Working with Neurospora Crassa, Beadle and Tatum isolated mutants that required arginine in their growth medium. The research showed that these mutants fell into three classes, each defective in a different gene.

9. Cite two significant findings that resulted from the research of Beadle and Tatum. From the growth requirements of the mutants, Beadle and Tatum deduced that each class of 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. 10. What revision of detail (but not of basic principle) did this hypothesis undergo as more information was gained? Write this restatement and then box or highlight it. This is an important concept! The one gene- one enzyme hypothesis was later revised to become the one geneone polypeptide hypothesis.

Basic Principles of Transcription and Translation This section will introduce you to the processes and associated terminology in the form of an overview. Once you have the big picture, you will take a closer look in the next few concepts. 11. From the first paragraph in this section, find three ways in which RNA differs from DNA. DNA contains deoxyribose, while RNA contains ribose. RNA contains uracil rather than thymine in DNA. An RNA molecule usually consists of a single strand. 12. What are the monomers of DNA and RNA? Of proteins? In DNA and RNA, the monomers are the four types of nucleotides, which differ in their nitrogenous bases. The monomers of proteins are made up of 20 different and unique amino acids. 13. Define each of these processes that are essential to the formation of a protein: transcription- is the synthesis of RNA under the direction of DNA translation- is the synthesis of a polypeptide, which occurs under the direction of mRNA 14. Complete the following table to summarize each process.

15. In eukaryotes, what is the pre-mRNA called? Primary transcript. 16. Write the central dogma of molecular genetics, as proclaimed by Francis Crick, in the box below. Cells are governed by a molecular chain of command with a directional flow of genetic information: DNA RNA Protein.

17. How many nucleotide bases are there? 4 (in DNA) How many amino acids? 20 18. How many nucleotides are required to code for these 20 amino acids? 3 19. So, the language of DNA is a triplet code. How many unique triplets exist? 20 20. DNA is double-stranded, but for each protein, only one of these two strands is used to produce an mRNA transcript. What is the coding strand called? The coding strand is called a template strand. 21. Here is a short DNA template. Below it, assemble the complementary mRNA strand. 3'A C G A C C A G T A A A 5' 5'U

G C U G G U C A U U U 3'

22. How many codons are there above? 4. Label one codon. UGC. 23. Describe Nirenbergs experiment in which he identified the first codon. Nirenberg synthesized an artificial mRNA by linking identical RNA nucleotides containing uracil as their base. No matter where the message began or ended, it could contain only one codon in repetition: UUU or poly-U. He then created his own protein synthesis and isolated poly-U. Once he did that, he realized that there was many unites of the amino acid phenylalanine. Thus, Nirenberg determined that the mRNA codon UUU specified the amino acid phenylalanine. 24. What was the first codonamino acid pair to be identified? UUU or Poly-U 25. Of the 64 possible codons, how many code for amino acids? 61 26. What event is coded for by UAA, UAG and UGA? Stop 27. What is the start codon? AUG 28. Why is the genetic code said to be redundant but not ambiguous? There is redundancy in the genetic code, but no ambiguity. For example, although codons GAA and GAG both specify glutamic acid (redundancy), neither of them ever specifies any other amino acid (no ambiguity). The redundancy in the code is not altogether random. In many cases, codons that are synonyms for a particular amino acid differ only in the third base of the triplet. 29. Explain the concept of reading frame. Our ability to extract the intended message from a written language depends on

the reading the symbols in the correct groupings. 30. Now here is an important idea: DNA is DNA is DNA. By this we mean that the code is nearly universal, and because of this, jellyfish genes can be inserted into pigs, or firefly genes can make a tobacco plant glow. Enjoy a look at Figure 17.6 in your text . . . and no question to answer here! Concept 17.2 Transcription is the DNA-directed synthesis of RNA: A closer look 31. Name the enzyme that uses the DNA template strand to transcribe a new mRNA strand. RNA polymerase is the enzyme that uses the DNA template strand to transcribe a new mRNA strand. 32. You will recall from Chapter 16 that DNA polymerase III adds new nucleotides to the template DNA strand to assemble each new strand of DNA. Both enzymes can assemble a new polynucleotide only in the 5' direction. Which enzyme, DNA polymerase III or RNA polymerase, does not require a primer to begin synthesis? RNA polymerase does not require a primer to begin synthesis. 33. What is a transcription unit? Transcription unit is the stretch of DNA that is transcribed into an RNA molecule. 34. Figure 17.7 in your text will require a bit of study. Use it to label the following elements on the figure below: promoter, RNA polymerase, transcription unit, DNA template, nontemplate DNA, and RNA transcript. Then, to the right of the figure, name the three stages of transcription and briefly describe each stage. 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 reform a

double helix. 3. Termination: Eventually, the RNA transcript is released, and the polymerase detaches from the DNA.

35. Lets now take a closer look at initiation. Read the paragraph titled RNA Polymerase Binding and Initiation of Transcription carefully. List three important facts about the promoter here. a. b. c. Serves as a binding site for RNA polymerase Determines where transcription starts Determines which of the two strands of the DNA helix is used as a template

36. Use Figure 17.8 in your text to label the following elements of the figure below: TATA box, RNA polymerase II, transcription factors, template DNA strand, start point, 5' and 3', and mRNA transcript. To the right of the figure, explain the three stages of initiation that are shown. 1) A eukaryotic promoter commonly includes a TATA box, a nucleotide sequence containing TATA, about 25 nucleotides upstream from the transcriptional start point. 2) Several transcription factors, one recognizing the TATA box, must bind to the DNA before the RNA polymerase II can do so. 3) Additional transcription factors bind to DNA along with RNA polymerase II, forming the transcription initiation complex. The DNA double helix then unwinds, and RNA synthesis begins at the start point on the template strand

37. What is the TATA box? How do you think it got this name? TATA box is a DNA sequence in eukaryotic promoters crucial in forming the transcription initiation complex. It got this name because the nucleotide sequence follows a TATA pattern. 38. What comprises a transcription initiation complex? The whole complex of transcription factors and RNA polymerase II bound to the promoter is called a transcription initiation complex. 39. Now it is time to put all of the elements of transcription together. Write an essay below to describe the process by which mRNA is formed. Use these terms correctly in your essay, and underline each one: TATA box, gene, terminator,

promoter, elongation, 5 to 3', termination, initiation RNA, polymerase RNA nucleotides, template, start point, termination signal, and transcription factors. This essay is typical of what you might be asked to write on the AP Biology exam. The formation of mRNA: The synthesis of mRNA is called transcription. First, RNA polymerase binds to the promoter, the DNA stand begins to unwind and the polymerase initiates RNA synthesis at the start point on the template strand. Then, the RNA polymerase moves along the temple strand of the DNA, creating complementary sequences that form RNA transcript in a 5 3 direction. As the RNA polymerase copies the sequence, the DNA re-forms its double helix. Eventually, the RNA transcript is released, and the polymerase detaches from the DNA entirely. A eukaryotic promoter commonly includes a TATA box, a nucleotide sequence containing TATA, about 25 nucleotides, upstream from the transcriptional start point. Several transcription factors, one recognizing the TATA box, must bind to the DNA before RNA polymerase II can do so. Additional transcriptional factors bind to the DNA along with RNA polymerase, forming the transcription initiation complex. The DNA double helix then unwinds, and RNA synthesis beings at the start point on the template strand. It is also important to note that the congregation of many polymerase molecules simultaneously transcribing a single gene increase the amount of mRNA transcribed from it, which helps the cell makes the encoded protein in large amounts. Concept 17.3 Eukaryotic cells modify RNA after transcription 40. RNA processing occurs only in eukaryotic cells. The primary transcript is altered at both ends, and sections in the middle are removed. a. What happens at the 5' end? A modified guanine nucleotide added to the 5 end (5 cap) b. What happens at the 3' end? 50 to 250 adenine nucleotides added to the 3 end (poly-A tail) 41. What are three important functions of the 5' cap and poly-A tail? The modified ends may promote the export of mRNA from the nucleus, and they help protect the mRNA from degradation. When the mRNA reaches the cytoplasm, the modified ends, in conjunction with certain cytoplasmic proteins, facilitate ribosome attachment as well. 42. Distinguish between introns and exons. Perhaps it will help to remember this: Exons are expressed. Introns are non-coding regions in the nucleic acid. The other regions that are expressed are called exons.

43. On the figure below, label: pre-mRNA, 5' cap, poly-A tail, introns, and exons.

44. What are snRNPs? What two types of molecules make up a snurp? (We like the word snurp! It reminds us of little cartoon characters that wore blue hoods and were called smurfs. SnRNPs are short nucleotide sequences at each end of an intron. SnRNPs are made up of proteins and RNA molecules. 45. You will be introduced to a number of small RNAs in this course. What type is the RNA in a snRNP? The RNA in a snRNP particle is called a small nuclear RNA (snRNA). 46. Snurps band together in little snurp groups to form spliceosomes. How do spliceosomes work? Several different snRNPs join with additional proteins to form an even larger assembly called a spliceosome, which is almost as bid as a ribosome. The spliceosome interacts with certain sites along an intron releasing the intron and joining together the two exons that flanked the intron. SnRNAs catalyzes these processes, as well as participation in splicosome assembly and splice site recognition. 47. On the figure below, label the following: pre-mRNA, snRNPs, snRNA, protein, spliceosomes, intron, and other proteins.

48. Study the figure and text carefully to explain how the splice sites are recognized. First, snRNPs and other proteins form a molecular complex called a spliceosome on a pre-mRNA molecule containing exons and introns. Within the spliceosome, snRNP base pairs with nucleotides at specific sites along the intron. The spliceosome cuts the pre-mRNA, releasing the intron, and at the same time splices the exons together. The spliceosome then comes apart, releasing mRNA, which now contains exons. 49. What is a ribozyme? Ribozymes are RNA molecules that function as enzymes. 50. What commonly held idea was rendered obsolete by the discovery of ribozymes? The discovery of ribozymes rendered obsolete the idea that all biological catalysts are proteins. 51. What are three properties of RNA that allow it to function as an enzyme? First, because RNA is single-stranded, a region of a RNA molecule may base-pair with a complementary region elsewhere in the same molecule which gives the molecule a particular 3D structure. A specific structure is essential to the catalytic function of ribozymes, just as it is for enzymatic proteins. Second, like certain amino acids in an enzymatic protein, some of the bases in RNA contain functional groups that may participate in catalysis. Third, the ability of RNA to hydrogen bond with other nucleic acid molecules adds specific to its catalytic activity. 52. What is the consequence of alternative splicing of identical mRNA transcripts? One consequence of the presence of introns in genes is that a single gene can encode more than one kind of polypeptide. Many genes are known to give rise to two or more different polypeptides, depending on which segments are treated as exons during RNA processing; this is called alternative RNA splicing. Concept 17.4 Translation is the RNA-directed synthesis of a polypeptide: A closer look 53. You may need to read on in this section in order to answer this question as well as think back to earlier information about mRNA. Come back to this question later if you wish. Three types of RNA are needed for protein synthesis. Complete the chart below.

54. W h a t i s an anticodon? An anticodon is a nucleotide triplet which base pairs with a complementary codon on mRNA. 55. Transfer RNA has two attachment sites. What binds at each site? Sketch tRNA, indicate the 2 attachment sites, and note where complementary base pairing and hydrogen bonding occur to give tRNA its shape.

56. How many different aminoacyl-tRNA synthetases are there? 20 57. Scientists expected to find one aminoacyl-tRNA synthetase per codon, but far fewer have been discovered. How does wobble explain this? The flexible base pairing at this codon position is called wobble. Wobble explains why the synonymous codons for a given amino acid can differ in their third base, but usually not in their other bases. 58. Use the figure below to explain the process of a specific amino acid being joined to a tRNA. Also add these labels: aminoacyl-tRNA synthetase, ATP, amino acid, and tRNA. First, the active site binds the amino acid and ATP. ATP then loses two phosphate groups and joins amino acid as AMP. Then, appropriate tRNA covalently bonds to amino acid, displacing AMP. Finally, the enzyme releases the tRNA charged with amino acid.

59. Describe the structure of a eukaryotic ribosome. In eukaryotes, large and small subunits join to form a functional ribosome only when they attach to an mRNA molecule. About two-thirds of the mass of a ribosome consists of rRNAs, four molecules. Because most cells contain thousands of ribosomes rRNA is the most abundant type of cellular RNA. 60. How does a prokaryotic ribosome differ from a eukaryotic ribosome? What is the medical significance of this difference? Eukaryotes are slightly larger and differ from prokaryotes slightly in molecular composition. The differences can be medically significant. Some antibiotic drugs can inactivate bacterial ribosomes without inhibiting the ability of eukaryotic ribosomes to make proteins. These types of drugs are used to combat bacterial infections. 61. On this figure, label the large subunit, small subunit, A, P, and E sites, mRNA binding site. To the right of the figure, explain the functions of the A, P, and E sites. E= Exit site P= Peptidyl-tRNA binding site A= Aminoacyl-tRNA binding site A ribosome has an mRNA binding site and three tRNA binding sites, known as the A, P, and E sites. 62. Much like transcription, we can divide translation into three stages. List them. Codon recognition, peptide bond formation and translocation are the three stages of translation. 63. Summarize the events of initiation. Include these components: small ribosomal subunit, large ribosomal subunit, mRNA, initiator codon, tRNA, Met, initiation complex, P site, and GTP. The figure below may help you.

A small ribosomal subunit binds to a molecule of mRNA. In a bacterial 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). 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. 64. What is always the first amino acid in the new polypeptide? AUG or merthionine (Met). 65. Now, summarize the events of elongation. Include these components: mRNA, A site, tRNA, codon, anticodon, ribozyme, P site, and E site. Again, the figure may help you.

The events of elongation can be summarized in three steps: 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. Peptide Bond Formation: An rRNA molecule of the large ribosomal 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 removes the polyupeptide from the tRNA in the P site and attaches it to the amino aicds on the tRNA in the P site and attaches it to the amino acid on the tRNA in the A site. Translocation: the ribosome translocates the tRNA in the A site to the P site. At the same time, 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 to be translated into the A site.

66. What is a release factor? By what mechanism is termination accomplished? A protein called a release factor binds directly to the stop codon in the A site. Termination requires GTP hydrolysis as well as additional protein factors.

67. What is a polyribosome? A polyribosome is a group of several ribosomes attached to, and translating, the same messenger RNA molecule. 68. What are some of the things that will result in a final-form functional protein? During its synthesis, a polypeptide chain begins to coil and fold spontaneously as a consequence of its amino acid sequence (primary sequence), forming a protein with a specific shape: a 3D molecule with secondary and tertiary structure. Thus, a gene determines primary structure, and primary structure in turn determines shape. In many cases, a chaperone protein (chaperonin) helps the polypeptide fold correctly. 69. Describe at least three types of post-translational modifications. Certain amino acids may be chemically modified by the attachment of sugars, lipids, phosphate groups, or other additions Enzymes may remove one or more amino acids form the leading (amino) end of the polypeptide chain Two or more polypeptides that are synthesized separately may come together, becoming the subunits of a protein that has quaternary structure (i.e. hemoglobin)

70. Use the following figure to explain how proteins are targeted for the ER.

Polypeptide synthesis begins on a free ribosomes in the cytosol An SRP binds to the signal peptide, halting synthesis momentarily

The SRP binds to a receptor protein in the ER membrane. This receptor is a part of a protein complex (a translocation complect0 that has a membrane pore and a signal-cleaving enzyme The SRP leaves, and polypeptide synthesis resumes, with simultaneous translocation across the membrane The signal- cleaving enzyme cuts off the signal peptide The rest of the completed polypeptide ;eaves the ribosomes and folds into its final conformation

Concept 17.5 Point mutations can affect protein structure and function 71. Define a mutation in terms of molecular genetics. A mutation is a change in the nucleotide sequence of an organisms DNA. 72. Define point mutations. A point mutation is a change in a gene at a single nucleotide pair. 73. What are frameshift mutations? A mutation occurring when the number of nucleotides inserted or deleted is not a multiple of three, resulting in the improper grouping of the subsequent nucleotides into codons. 74. Identify two mechanisms by which frameshifts may occur. - Insertion - Deletion 75. What is the difference between a nonsense and missense mutation? Missense mutations are substitutions that change one amino acid to another one. A point mutation can also change a codon for an amino acid into a stop codon is called a nonsense mutation. 76. How can a base-pair substitution result in a silent mutation? Base-pair substitution is a type of point mutation; the replacement of one nucleotide and its partner in the complementary DNA strand by another pair of nucleotides. Silent mutations are present in the genes; theyre just not expressed. 77. What are the two categories of mutagens? Physical and chemical mutagens are the two categories of mutagens.

78. Describe the action of difference types of chemical mutagens. Chemical mutagens fall into several categories. Base analogs are chemical that similar to normal DNA bases but that pair incorrectly during DNA replication. Some other chemical mutagens interfere with correct DNA replication by inserting themselves into the DNA and distorting the double helix. Still other mutagens cause chemical changes in bases that change their pairing properties. Concept 17.6 Although gene expression differs among the domains of life, the concept of a gene is universal 79. Describe two important ways in which bacterial and eukaryotic gene expression differ. Bacteria and eukaryotic RNA polymerases differ significantly from each other. Also, transcription is terminated differently in bacteria and eukaryotes. 80. What is a gene? It used to be simply stated that one gene codes for one polypeptide. That definition has now been modified. Write below the broader molecular definition in use today. A gene is a region of DNA that can be expressed to produce a final functional product that is either a polypeptide or an RNA molecule. 81. Finally, use this summary figure to put together all that you have learned in this chapter.

Testing Your Knowledge: Self-Quiz Answers Now you should be ready to test your knowledge. Place your answers here: 1. __________2.__________3. __________4. __________5. __________ 6. __________7. _________