A.P. Biology CHAPTER 17: FROM GENE TO PROTEIN The DNA inherited by an organism leads to specific traits by dictating the synthesis of proteins. This process is called gene expression and includes two stages: transcription and translation. Proteins are the link between genotype and phenotype GENES SPECIFY PROTEINS VIA TRANSCRIPTION AND TRANSLATION HISTORY: 1909: Archibald Garrod first suggested that genes dictate phenotypes through enzymes 1930’s: George Beadle and Edward Tatum formulated the one gene-one enzyme hypothesis based on their data on bread mold (Neurospora crassa) in different growth mediums (See Figure 17.2, page 310). Today: With continued research, we now have revised Beadle & Tatum’s conclusion and have created the one gene-one polypeptide hypothesis. BASIC PRINCIPLES OF TRANSCRIPTION AND TRANSLATION: DNA→Transcription→RNA→Translation→Protein Ribonucleic Acid (RNA): a nucleic acid that uses information from DNA to synthesize proteins. RNA and DNA compared: 1. RNA is single stranded and DNA has two strands 2. RNA has a ribose sugar and DNA has deoxyribose 3. RNA has a base of Uracil (U) and DNA has the base Thymine **There is no T in RNA, so A=U and C=G** TRANSCRIPTION: The synthesis of RNA under the direction of DNA A gene’s unique nucleotide sequence is transcribed from the DNA template to a complementary nucleotide sequence in messenger RNA (mRNA). The resulting mRNA carries this transcript of protein-building instructions to the cell’s protein- synthesizing machinery. TRANSLATION: The synthesis of a polypeptide, which occurs under the direction of mRNA During translation, the linear sequence of bases in mRNA is translated into the linear sequence of amino acids in a polypeptide Translation occurs on ribosomes (in the cytoplasm of the cell). Ribosomes are complex particles composed of ribosomal RNA (rRNA) and protein that facilitate the orderly linking of amino acids into polypeptide chains. PROKARYOTIC/EUKARYOTIC DIFFERENCES IN GENE TRANSFER Because bacteria lack nuclei, their DNA is not segregated from ribosomes and other protein- synthesizing equipment (See Figure 17.3, page 312). THE GENETIC CODE The template strand of DNA provides the template for ordering the sequence of nucleotides in an RNA transcript. The RNA instructions are written in a series of three-nucleotide sequences called a triplet code. Each mRNA triplet code (codon) “codes” for a specific amino acid. There are 20 different amino acids and 64 codons (See Figure 17.5, page 314). The amino acids are linked together to form a protein (See Fig. 17.4, page 313) This process of protein synthesis occurs in the 5’ to 3’ direction along the mRNA. TIDBITS: All 64 codons were deciphered by the mid 1960’s. The reading frame is the order in which the codons should be translated (in bases of three) The genetic code is nearly universal, shared by organisms from the simplest bacteria to the most complex animals. TRANSCRIPTION IS THE DNA-DIRECTED SYNTHESIS OF RNA Transcription involves three main steps: Initiation, Elongation, and Termination STEPS OF TRANSCRIPTION: Figure 17.7, page 315 1. RNA Polymerase (an enzyme that adds and links complementary RNA nucleotides) binds to a gene’s promoter (a sequence of DNA that acts like a start signal, it typically extends several dozen nucleotide pairs from the start point). 2. In Eukaryotes, a collection of proteins, called transcription factors assist the binding of RNA polymerase and the initiation of transcription. The transcription factors and a DNA sequence called TATA box initiate transcription (See Figure 17.8, page 316). 3. RNA polymerase unwinds the double helix, exposing DNA nucleotides. 4. RNA polymerase adds nucleotides to the 3’ end and links complementary RNA nucleotides together. The stretch of DNA that is transcribed into an RNA molecule is called a transcription unit. 5. RNA polymerase transcribes until it reaches a DNA sequence known as a terminator…Details become “murky”, but transcription is terminated when the polymerase eventually falls off the DNA. EUKARYOTIC CELLS MODIFY RNA AFTER TRANSCRIPTION: RNA transcripts in eukaryotes are modified, or processed, before leaving the nucleus to yield Functional mRNA. Eukaryotic RNA transcripts can be processed in two ways: a) covalent alteration of both the 3’ & 5’ ends and b) removal of intervening sequences. Alteration of mRNA ends: Fig. 17.9, pg. 317 Primary transcript: General term for initial RNA transcribed from DNA Pre-mRNA: Primary transcript that will be processed to functional mRNA The 5’ end is capped off with a modified form of guanine, which forms a 5’cap. At the 3’ end, an enzyme adds 50 to 250 adenine nucleotides, forming a poly-A tail. The 5’ cap and poly-A tail facilitate the export of mRNA from the nucleus, protect the mRNA from degradation by enzymes, and help ribosomes attach to the 5’ end of the mRNA. Split genes and RNA splicing. Fig. 17.10, pg. 318 Introns: Noncoding sequences in DNA that intervene between coding sequences (exons). They are initially transcribed, but not translated, because they are excised from the transcript before mature RNA 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 pre- mRNA; produces mature mRNA that will move into the cytoplasm from the nucleus. This is a “cut and paste” job. Pre-mRNA splicing is carried out by small nuclear ribonucleoproteins (snRNPs). Several snRNPs join with additional proteins to form a splicosome. The spliceosome interacts with certain sites along an intron, releasing the intron and joining together the two exons that flanked the intron (See Figure 17.11, page 319). Ribozymes are RNA molecules that function as enzymes. In some organisms, RNA splicing occurs when the ribozyme catalyzes its own excision. Introns may play regulatory roles in the cell. Depending on which segments of are treated as exons during RNA processing, some genes can encode more than one type of polypeptide (alternative RNA splicing). Also, introns increase the probability of potentially beneficial crossing over between the exons of alleles (creating more potentially useful proteins) TRANSLATION IS THE RNA-DIRECTED SYNTHESIS OF A POLYPEPTIDE (Fig. 17.13, pg. 320) 1. mRNA leaves the nucleus and enters the cytoplasm. There, each type of tRNA associates a distinct mRNA codon with one of the 20 amino acids used to make proteins. The tRNA has an amino acid on one end and an anticodon (nucleotide triplet in tRNA that base pairs with a complementary nucleotide codon in mRNA). (Fig. 17. 14, pg 321 for tRNA structure) 2. Initiation: The tRNA reads the start code of mRNA (AUG) and orients itself in a region of a ribosome called the P site 3. Elongation: Amino Acids are added one by one to the first amino acid. The A site of the ribosome is ready to receive the next tRNA. The tRNA then binds to the codon by hydrogen bonds. 4. The P and A sites are holding tRNA molecules (each with its own amino acid). The amino acids are linked together by a peptide bond. 5. Translocation: The tRNA in the P site moves to the E site, the tRNA in the A site moves over (translocates), and a new tRNA molecule binds to the codon in the A site (See page 322 & 324). 6. Termination) The amino acids continue to join together until a stop codon is reached (UAG, UAA, or UGA). Fig 17.19, pg 325. A protein is thus created. Once the protein is made, it is often times not ready to be functional. The protein has to go through post-translational modifications. Free and Bound ribosomes will direct the protein in the right direction. A signal recognition particle (SRP) is a protein-RNA complex that functions as an adapter that brings the ribosome to a receptor protein in the ER membrane. RNA PLAYS MULTIPLE ROLES IN THE CELL: See Table 17.1, page 327 POINT MUTATIONS CAN AFFECT PROTEIN STRUCTURE AND FUNCTION: Mutations: Heritable changes in the genetic material of a call (or virus). Point mutations: A mutation limited to about one or a few base pairs in a single gene Types of point mutations: 1. Substitutions (Base-pair substitution) The replacement of one base pair with another; occurs when a nucleotide and its partner in the complementary DNA strand are replaced with another pair of nucleotides according to base-pairing rules (Figure 17.24, page 329). Missense mutation: Base-pair substitution that alters an amino acid codon (sense codon) to a new codon that codes for a different amino acid. These alterations make sense, but not necessarily “the right sense” Nonsense mutation: Base-pair substitution that changes an amino acid codon to a chain termination codon. Leads to nonfunctional proteins. 2. Insertions and Deletions: Usually have a greater negative effect on proteins then substitutions. Whenever the number of nucleotides inserted or deleted is not or a multiple of 3, it will result in a frameshift mutation. (See Figure 17.25, page 330) Framesift 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. 3. Mutagenesis: The creation of mutations. May be caused naturally or because of exposure to mutagens. Mutagen: Physical or chemical agents that interact with genetic material to cause mutations a. Radiation is the most common physical mutation b. The Ames test is one of he most widely used tests for measuring the mutagenic strength of various chemicals, such as carcinogens. See Overall Summary of Protein Synthesis: Page 331.