AP Biology Ch. 17

1. AP Biology Ch. 17 From Gene to Protein

2. Inherited instructions in DNA direct protein synthesis. The study of metabolic defects provided evidence that genes specify proteins. -Archibald Garrod was the first to propose the relationship between genes and proteins (1909). He suggested that genes dictate phenotypes through enzymes that catalyze reactions. Inherited diseases (inborn errors in metabolism) reflect the person’s inability to make particular enzymes.

3. How Genes Control Metabolism • Geneticists George Beadle and Boris Ephrussi (1930s) were able to demonstrate the relationship between genes and enzymes by studying eye color in Drosophila and Beadle and Edward Tatum studied mutations of a bread mold, Neurospora. • They found that mutants of the bread mold could not survive on minimal medium because they lacked the ability to synthesize essential molecules.

4. Terms: • Auxotroph=nutritional mutants that can only be grown on minimal medium augmented with nutrients not required by the wild type. • Minimal medium=Support medium that is mixed only with molecules required for the growth of wild-type organisms. • Complete growth medium=Minimal medium supplemented with all 20 amino acids and some other nutrients.

5. Beadle and Tatum: Conclusions • Beadle and Tatum identified specific metabolic defects from mutations by transferring fragments of auxotrophic mutants growing on complete growth medium to vials containing minimal medium each supplemented with only 1 additional nutrient. • For example: if a mutant grew on minimal medium supplemented with only arginine, it could be concluded that the mutant was defective in the arginine synthesis pathway.

6. One Gene--One Polypeptide • Beadle and Tatum’s one gene-one enzyme hypothesis has been slightly modified: • while most enzymes are proteins, many proteins are not enzymes but are still gene products. • Many proteins are composed of 2 or more polypeptide chains, each chain specified by a different gene.

7. Transcription--Translation • Transcription and translation are the 2 main steps from gene to protein: • RNA links DNA’s genetic instructions for making proteins to the process of protein synthesis. It copies or transcribes the message from DNA and then translates that message into a protein. • The linear sequence of nucleotides in DNA ultimately determines the linear sequence of amino acids in a protein.

8. DNA Double stranded molecule of nucleic acid Deoxyribose is the 5-carbon sugar. Consists of 4 nitrogen bases: adenine, guanine, cytosine, and thymine. RNA Single stranded molecule of nucleic Ribose is the 5-carbon sugar Consists of 4 nitrogen bases: adenine, guanine, cytosine, and uracil DNA/RNA Comparison

9. Transcription=The synthesis of RNA using DNA as a template A gene’s unique nucleotide sequence is transcribed from DNA to a complementary nucleotide sequence in messenger RNA (mRNA). The resulting mRNA carries this transcript of protein-building instructions to the ribosomes. Translation=Synthesis of a polypeptide, which occurs under the direction of mRNA The linear sequence of bases in mRNA is translated into the lienar sequence of amino acids in a polypeptide. Translation occurs on ribosomes, complex particles composed of ribosomal RNA (rRNA) and protein. Transcription--Translation

10. Prokaryotes lack nuclei, so DNA is not segregated from ribosomes or the protein synthesizing machinery. Thus, transcription and translation occur in rapid succession. Eukaryotes have nuclear envelopes that segregate transcription in the nucleus from the translation in the cytoplasm; mRNA is modified before it moves from the nucleus to the cytoplasm where translation occurs. Prokaryotes vs Eukaryotes

11. The Genetic Code • In the genetic code, a particular triplet of nucleotides specifies a certain amino acid. • Researchers have verified that the flow of information from a gene to a protein is based on a triplet code. • Triplets of nucleotides are the smallest units of uniform length to allow translation into all 20 amino acids with plenty to spare. • There 3-nucleotide “words” are called codons.

12. Codons • Codon=A 3-nucleotide sequence in mRNA that specifies which amino acid will be added to a growing polypeptide or that signals termination; the basic unit of the genetic code. • Genes are not directly translated into amino acids, but are first transcribed as codons in mRNA. • For each gene, only one of the 2 DNA strands is transcribed (the template strand).

13. An mRNA is complementary to the DNA template from which it is transcribed. • If the triplet nucleotide sequence on the template DNA strand is CCG; GGC, the codon for glycine, will be the complementary mRNA transcript. • Remember: uracil (U) in RNA is used in place of thymine (T) of DNA. • Each mRNA codon specifies which one of 20 amino acids will be incorporated into the polypeptide.

14. Cracking the Genetic Code • 1961--Marshall Nirenberg of the National Institute of Health deciphered the first codon. • All 64 codons have since been determined. • 61 of the 64 codons code for amino acids. • The triplet AUG has a dual function--it is the start signal for translation and codes for methionine. • 3 codons do not code for amino acids, but signal termination (UAA, UAG, and UGA).

15. Redundancy but no ambiguity • Redundancy exists in the genetic code since 2 or more codons differing only in their third base can code for the same amino acid (UUU and UUC both code for phenylalanine). • Ambiguity is absent, since codons code for only 1 amino acid.

16. The Correct Ordering • The correct ordering and grouping of nucleotides is important in the molecular language of cells. • Reading Frame=The correct grouping of adjacent nucleotide triplets into codons that are in the correct sequence on mRNA. • The cell reads the message in the correct frame as a series of non-overlapping 3-letter words.

17. Common Genetic Language • The genetic code is shared nearly universally among living organisms. • EX: The RNA codon CCG is translated into proline in all organisms whose genetic codes have been examined. • Mitochondria and chloroplasts have their own DNA codes for some proteins. • Several ciliates depart from standard code; codons UAA and UAG are not stop signals but code for glutamine.

18. Transcription • Transcription of mRNA from template DNA is catalyzed by RNA polymerases, which: • Separate the 2 DNA strands and link RNA nucleotides as they base-pair along the DNA • Add nucleotides only to the 3’ end There are several types of RNA polymerase. Prokaryotes have only 1 type of RNA polymerase but Eukaryotes have 3 types, 1 for each type of RNA. RNA polymerase II catalyzes synthesis mRNA.

19. Three Steps in Transcription • Transcription Unit—Nucleotide sequence on the template strand of DNA that is transcribed into a single RNA molecule; it includes the initiation and termination sequences, as well as the nucleotides in between. • Transcription occurs in 3 steps: • Polymerase binding and initiation • Elongation • Termination

20. Promoter—region of DNA that includes the site where RNA polymerase binds and transciption begins. In Eukaryotes, the promoter is about 100 nucleotides long and consists of the initiation site and a few nucleotide sequences that help initiate transcription. Transcription factors—DNA-binding proteins that bind to specific DNA nucleotides at the promoter that help RNA polymerase recognize and bind to the promoter region RNA polymerase Binding and Initiation

21. TATA box—a short nucleotide sequence at the promoter that is rich in thymine and adenine, about 25 nucleotides from the initiation site. RNA polymerase II recognizes the complex between the bound TATA transcription factor and the DNA binding site TATA Box

22. Once transcription begins, RNA polymerase II moves along DNA and: Untwists and opens a short segment of DNA exposing about 10 nucleotides. Links incoming RNA nucleotides to the 3’ end of the elongating strand. During transcription, mRNA grows about 30-60 nucleotides per second. As it elongates, MRNA peels away from the DNA template. Elongation of the RNA Strand

23. Termination • Transcription proceeds until RNA polymerase reaches a termination site on DNA. • Terminator sequence—DNA sequence that signals RNA polymerase to stop transcription and to release the RNA molecule and DNA template • In Eukaryotes, the most common terminator sequence is AATAAA. • Prokaryotic mRNA is ready for translation immediately; eukaryotic mRNA must be processed before it leaves the nucleus and becomes functional.

24. Translation is RNA-directed synthesis of a polypeptide. • Transfer RNA (tRNA) is the interpreter between the mRNA and the amino acid sequence of the polypeptide. • tRNA aligns the appropriate amino acids to form a new polypeptide. tRNA must: • Transfer amino acids from the cytoplasm’s amino acid pool to a ribosome. • Recognize the correct codons in mRNA. Molecules of tRNA are specific for only 1 particular amino acid. Each type of tRNA associates a distinct mRNA codon with one of the 20 amino acids.

25. One end of a tRNA molecule attaches to a specific amino acid. The other end attaches to an mRNA codon by base pairing with its anticodon. Anticodon—a nucleotide triplet in tRNA that base pairs with a complementary triplet (codon) in mRNA ANTICODON

26. Anticodon details: • tRNAs decode the genetic message, codon by codon. • For ex: the mRNA codon UUU is translated as the amino acid phenylalanine. The tRNA that transfers phenylalanine to the ribosome has an anticodon of AAA. • As tRNAs deposit amino acids in the correct order, ribosomal enzymes link them into a chain.

27. All types of RNA are transcribed from DNA. tRNA must travel into the cytoplasm where translation occurs. tRNA can be used repeatedly. tRNA is a single-stranded RNA about 80 nucleotides long. There are 45 distinct types of tRNA; some recognize 2 or 3 of the mRNA codons. Wobble—the ability of 1 tRNA to recognize 2 or 3 different mRNA codons; occurs when the 3rd base of the tRNA anticodon has some play or wobble, so that it can H-bond with more than 1 kind of base in the 3rd position of the codon. Ex: The base U in the wobble position of tRNA can pair with either A or G in the codon. Structure and Function of tRNA

28. A Unique Base • Some tRNA molecules contain a modified base called inosine (I), which is in the wobble position and can base pair with U, C, or A in the 3rd position of an mRNA. • For example, a single tRNA with the anticodon CCI will recognize 3 mRNA codons: GGU, GGC, or GGA—all of which code for glycine.

29. Aminoacyl-tRNA Synthetases • Aminoacyl-tRNA synthetase—a type of enzyme that catalyzed the attachment of amino acid to its tRNA. • Each of the 20 amino acids has a specific aminoacyl-tRNA synthetase. • 2 steps in attachment of an amino acid: • Activation of the amino acid with AMP. The synthetase’s active site binds the amino acid and ATP; the ATP loses 2 phosphate groups and attaches to the amino acid as AMP. • Attachment of the amino acid to tRNA. The appropriate tRNA covalently bonds to the amino acid, displacing AMP from the enzyme’s active site. • The aminoacyl-tRNA complex releases from the enzyme and transfers its amino acid to a growing polypeptide on the ribosome.

30. Ribosomes have 2 subunits (small and large) which are separate when not involved in protein synthesis. Ribosomes are composed of about 60% ribosomal RNA (rRNA) and 40% protein. The large and small subunits of eukaryotes are constructed in the nucleolus, move through nuclear pores, and assembled in the cytoplasm only when attached to mRNA. Ribosomes

31. Prokaryotic ribosomes are smaller with different molecular composition than eukaryotic. Antibiotics tetracycline and streptomycin can be used to combat bacterial infections because they inhibit bacterial protein synthesis but not eukaryotic. In addition to a mRNA binding site, each ribosome has 2 tRNA binding sites (A and P). The P site (Peptidyle-tRNA) holds the tRNA carrying the growing polypeptide chain. The A site (Aminoacyl-tRNA) holds the tRNA carrying the next amino acid to be added. A and P Sites

32. Building A Polypeptide • Translation occurs in 3 stages: • Initiation • Elongation • Termination • All 3 stages require enzymes and other protein factors. • Initiation and elongation also require energy provided by GTP—a molecule closely related to ATP.

33. Initiation must bring together the mRNA, the first amino acid attached to its tRNA, and the 2 ribosomal subunits. In eukaryotes, the small ribosomal subunit binds first to an initiator tRNA with the anticodon UAC and the amino acid methionine. The small ribosomal subunit next binds to the 5’ end of mRNA. The bound initiator tRNA finds and base pairs with initiation or start codon on mRNA, AUG. The assembly of the initiation complex—small ribosomal subunit, initiator tRNA and mRNA—requires: Protein initiation factors that are bound to the small ribosomal subunit. One GTP molecule to stabilize the binding of initiation factors and once hydrolyzed, drives the attachment of the large ribosomal subunit. A large ribosomal subunit binds to the small one to form a functional ribosome. Iniation factors attached to the small subunit are released allowing the large subunit to bind with the small one. The initiator tRNA fits into the P site on the ribosome. The vacant A site is ready for the next aminoacyl-tRNA. Initiation

34. Elongation • Several proteins called elongation factors take part in this 3-step process which adds amino acids one by one. • Codon recognition. The mRNA codon in the A site forms hydrogen bonds with the anticodon of an entering tRNA carrying the next amino acid. An elongation factor directs tRNA into the A site. Hydrolysis of GTP provides the energy. • Peptide bond formation. An enzyme, peptidyl transferase, catalyzes the formation of a peptide bond between the polypeptide in the P site and the new animo acid in the A site. Peptidyl transferase is part of the large ribosomal subunit and consists of ribosomal proteins and rRNA. • Translocation. The tRNA in the P site releases from the ribosome, and the tRNA in the A site is translocated to the P site. GTP hydrolysis provides energy for each translocation step.

35. Termination • Termination codon (stop codon)—Base triplet (codon) on mRNA that signals the end of translation • UAA, UAG, and UGA are stop codons and do not code for an amino acid. • When a stop codon reaches the ribosome’s A site, a protein release factor binds to the codon and initiates the hydrolyzes of the bond between the completed polypeptide and the tRNA in the P site; this frees the polypeptide and the tRNA; the 2 ribosomal subunits dissociate from mRNA and separate into the small and large subunits.

36. Other Facts • Polyribosomes—a cluster of ribosomes simultaneously translating an mRNA molecule. • Prokaryotes lack nuclei, so transcription is not segregated form translation and may begin before transcription is complete. • Eukaryotes process or modify mRNA before it leaves the nucleus.

37. mRNA Modification • 5’Cap—modified guanine nucleotide (GTP) that is added to the 5’ end of mRNA shortly after transcription begins; protects mRNA from degradation and helps small ribosomal subunits recognize the attachment site on mRNA’s 5’ end. • Poly-A tail—sequence of about 200 adenine nucleotides added to the 3’ end of mRNA before it exits the nucleus

38. Gene Splicing • The original transcript, or precursor mRNA, is heterogeneous nuclear RNA (hnRNA); it is processed before leaving the nucleus. • Introns—noncoding sequences in DNA that intervene between coding sequences (exons); are initially transcribed but are not translated because they are excised before mRNA leaves the nucleus • Exons—coding sequences of a gene that are transcribed and expressed. • RNA splicing—RNA processing that removes introns and joins exons from eukaryotic hnRNA to produce mature mRNA

39. snRNPs • Small nuclear ribonucleoproteins (snRNPs)-complexes of proteins and small nuclear RNAs that are found only in the nucleus; some participate in RNA splicing. • Composed of small nuclear RNA (snRNA) with less than 300 nucleotides, and 7 or more proteins Spliceosome—a large molecular complex that catalyzes RNA splicing reactions; composed of snRNPs and other proteins. Ribozymes—RNA molecules that can catalyze reactions by breaking and forming covalent bonds; rRNA functions as an enzyme during translation

40. Mutation • Mutation—a permanent change in DNA that can involve large chromosomal regions or a single nucleotide pair. • Point mutation—a mutation limited to 1 or 2 nucleotides in a single gene • Mutagenesis—the creation of mutations • Mutagen—physical or chemical agents that interact with DNA to produce mutations • Radiation and certain chemicals including base analogues can cause mutations.

41. Types of Point Mutation • Base-pair substitution—the replacement of 1 base pair with another; occurs when a nucleotide and its partner from the complementary DNA strand are replaced with another pair of nucleotides according to base-pairing rules. • Missense mutation—base-pair substitution that alters an amino acid codon to a new codon that codes for a different amino acid. • Nonsense mutation—base-pair substitution that changes an amino acid codon to a chain termination codon, or vice versa

42. Types of Point Mutation • Insertions or Deletions • Base-pair insertion—the insertion of 1 or more nucleotide pairs into a gene • Base-pair deletion—the deletion of 1 or more nucleotide pairs from a gene • Frameshift mutation—a base-pair insertion or deletion that causes a shift in the reading frame, so that codons beyond the mutation will be the wrong grouping of triplets and will specify the wrong amino acids.