The Connection Between Genes and Proteins

Chapter 17 Study Guide – Science Olympiad By Julia Beamesderfer (2005)

The Connection Between Genes and Proteins I. Evidence for genes specifying proteins was first hypothesized by British physician Archibald Garrod, who believed that genes dictate phenotypes through enzymes that catalyze specific chemical processes within the cell II. Decades later, George Beadle and Edward Tatum tested this hypothesis with mutants of a bread mold called Neurospora crassa. A. Auxotrophs- The mutants of the bread mold could not survive on the minimal medium (modest food requirements) that the wild types of the mold could, because they could not synthesize certain necessary ingredients from the minimal medium B. To pinpoint their metabolic defect, Beadle and Tatum put the mutants into vials of the minimal medium plus single additional nutrients to pinpoint the ones that would aid the mutants in surviving. C. They discovered that the mutants needed the amino acid arginine to survive, and that there were three precursors of arginine needed to produce it D. From this three classes of mutants were discovered- those who only needed arginine, those who needed its 1st precursor, or those who needed its 2nd precursor E. From this it was concluded that each mutant type lacked a different enzyme III. One Gene—One Polypeptide Hypothesis A. Since not all proteins are enzymes, and some proteins were made of multiple polypeptide chains, the one gene-one enzyme hypothesis was altered to the above name IV. Quick Facts Regarding Transcription and Translation A. RNA- chemically similar to DNA except it consists of a ribose sugar (instead of a deoxyribose sugar) and the possible nitrogenous base of uracil U (rather than thymine T) B. Transcription- synthesis of RNA under direction of DNA C. Messenger RNA (mRNA)- a faithful transcript of the gene’s protein-building instructions which carries genetic message from DNA to protein-synthesizing machinery D. Translation- actual synthesis of polypeptide occurring under direction of mRNA. Also, cell must translate base sequence of mRNA into amino acid sequence of a polypeptide E. RNA Processing- the process which takes the initial RNA transcript (pre-mRNA or primary transcript) and yields the finished mRNA F. Triplet Code- since there are 20 amino acids and only 4 nitrogenous bases, it is necessary for an amino acid to be coded by a triplet of nucleotide bases- yielding 64 possible codes. The genetic instructions for a polypeptide chain are written in the DNA as a series of three- nucleotide words. G. Template Strand- the DNA strand that is transcribed during RNA transcription H. Codons- mRNA base triplets EX- UGG is codon for amino acid tryptophan (Trp) V. The Genetic Code A. In 1961 Marshall Nirenberg deciphered the first codon (UUU) by synthesizing an artificial mRNA by linking uracil nucleotides. When he added the strand to amino acids, ribosomes, and other components, he got a polypeptide containing a string of a single amino acid, phenylaline. B. Start Signal (AUG)- it was also discovered that AUG could serve as methionine (Met) as well as functioning as initiation codon, to start translating mRNA with methionine. C. Stop Signals (UAA, UAG, and UGA)- these three codons are not designated as amino acids, but rather they mark the end of translation acting as termination codons. D. Codon Redundancy- in many cases, codons that are synonyms for a certain amino acid differ only in the third base of the triplet. E. Because the genetic code is nearly universal, the RNA codon CCG, ex, is translated to amino acid proline in all studied organisms, though there are a few exceptions Transcription- the DNA-directed synthesis of RNA I. Initiation A. Promoter- the region of DNA where RNA polymerase attaches and initiates transcription 1. RNA Polymerase- enzyme that pries DNA strands apart and hooks together RNA nucleotides with base pairing along DNA template 2. The promoter determines where transcription starts and which DNA helix strand is used as template

1 Chapter 17 Study Guide – Science Olympiad By Julia Beamesderfer (2005)

3. TATA Box- A crucial promoter DNA sequence used to form initiation complex B. Transcription Factors- in eukaryotes, these proteins bind to the promoter and causes RNA polymerase to bind to it C. Transcription Initiation Complex- completed assembly of transcription factors and RNA polymerase bound to the promoter II. Elongation of RNA Strand A. RNA polymerase continues to move and untwist DNA, adding nucleotides to the 3’ end of the growing RNA molecule B. In its wake, DNA re-twists and RNA molecule peels away from its template C. Several molecules of RNA polymerase can transcribe a single gene by following each other, causing the growing strands to trail from each polymerase, helping to make protein in large amounts III. Termination A. Terminator- sequence that calls for termination of transcription 1. Transcribed terminator (RNA sequence) acts as actual termination signal 2. In eukaryotes, shortly after the polymerase has passed the termination signal, AAUAAA, it cuts free from enzyme. RNA Processing- the modifying of pre-mRNA by enzymes in a eukaryotic nucleus I. Alteration of mRNA Ends A. 5’ End 1. 5’ Cap- during RNA processing the 5’ end of the pre-mRNA molecule is capped off with a modified form of a guanine G nucleotide 2. Function a. First, this helps protect the mRNA from degradation by hydrolygic enzymes b. Second, after mRNA reaches cytoplasm, 5’ cap functions as part of an “attach here” sign for ribosomes B. 3’ End 1. Poly (A) Tail- this is added by an enzyme to the 3’ end of the pre-mRNA. It consists of 30 to 200 adenine nucleotides 2. Function a. Inhibits degradation of the RNA b. Helps ribosomes attach to it c. Facilitates the export of mRNA from the nucleus II. RNA Splicing- the removal of a large portion of the pre-mRNA molecule A. Introns- noncoding segments of nucleic acid that lie between coding regions 1. Importance a. Maybe introns play regulatory roles in the cell b. Domains- discrete structural and functional components (EX- active site, area where protein attaches to membrane, etc.) that are thought to be coded by a split genes’ exons c. Since exons are separated by intron DNA, frequency of recombination within a split gene can be higher than for a gene lacking introns, since they increase opportunity for crossing over between two alleles of a gene B. Exons- the other regions that are eventually expressed, or translated into amino acid sequences C. Process 1. snRNPs- small nuclear ribonucleoproteins that recognize short nucleotide sequences at the ends of introns, signaling RNA splicing a. Composed of RNA (called small nuclear RNA, or snRNA) and protein molecules 2. Spliceosomes- consist of different snRNPs joined with additional proteins. Spliceosomes interact with splice sites and cuts ends of introns to release them and then join the two exons D. Ribozymes- Sometimes primary transcripts are spliced by intron RNA itself (Ex- tetrahymena protozoan use self-splicing 1. Proves that not all biological catalysts are proteins

The Synthesis of Proteins I. Transfer RNA (tRNA)- molecule which transfers amino acids from the cytoplasm to ribosome. A. Cytoplasm is stocked with all 20 amino acids, gained by synthesizing them from other compounds or taking them up from surrounding solution B. tRNA molecules are not identical; each type of tRNA molecule links a particular mRNA codon with a certain amino acid C. When tRNA arrives at ribosome, one end bears the specific amino acid and the other has a nucleotide triplet called an anticodon, which binds to complementary codon on mRNA. D. Structure and Function 1. Made in nucleus and travels to cytoplasm where translation occurs 2. Consists of a single, short RNA strand (looking like a clover) with the anticodon on one end and the 3’ end that is an attachment site for the amino acid. E. Wobble- there are only 45 different tRNA molecules needed (instead of 61) because some can recognize two or more different codons (due to the fact that the third base of the codon is not strict. 1. When tRNAs have inosine I (a modified base) in the wobble position of a codon, it can bond with U,C, or A 2. Explains why synonymous codons for a given amino acid can often differ in 3rd base F. Aminoacyl-tRNA Synthetase- enzyme that joins amino acid to correct RNA. 1. There are 20 of these enzymes in a cell, one for each amino acid 2. It’s active site fits only certain combination of amino acid and tRNA 3. It attaches amino acid to tRNA, driven by hydrolysis of ATP II. Ribosomal RNA (rRNA)- the type of RNA which, along with proteins, constructs the ribosomal subunits A. Ribosomes consist of large subunit and small subunit 1. Each subunit is made in nucleus and sent to cytoplasm, where they join to form ribosome when they attach to mRNA B. Each ribosome has 3 binding sites 1. P Site (peptidyl-tRNA site)- holds the tRNA carrying the growing polypeptide chain 2. A Site (aminoacyl-tRNA site)- holds the tRNA carrying the next amino acid to be added to the chain 3. E Site (exit site)- site where discharged tRNAs leave the ribosome III. Translation- Building a Polypeptide A. Initiation- when mRNA, tRNA with amino acid, and two ribosome subunits are joined 1. Small ribosomal subunit attaches to leader segment (5’ end) of mRNA. 2. Then initiation codon, AUG, signals start of translation (initiator tRNA carrying amino acid Met attaches to the initiation codon) 3. Large ribosomal subunit is added, completing translation initiation complex. 4. Initiation Factors- required to bring all components together, by use of GTP molecule 5. Results- initiator tRNA sits in the P site of the ribosome, and vacant A site is ready for the next aminoacyl tRNA. B. Elongation- amino acids are added one by one to the first amino acid, involving elongation factors 1. Codon Recognition- mRNA codon in the A site of ribosome bonds with incoming tRNA molecule (which carries appropriate amino acid) a. Requires energy from GTP 2. Peptide Bond Formation- an rRNA molecule catalyzes formation of peptide bond that joins the polypeptide chain from the P site to the new amino acid in the A site 3. Translocation- 1. tRNA in the A site is translocated to the P site (bonds remain, the mRNA moves along with it and brings the next codon into the A site) 2. tRNA in the P site moves to the E site and leaves the ribosome. 3. Requires energy from GTP 4. Ribosome moves along the mRNA in direction of 5’ to 3’, and they move relative to each other C. Termination

1. Elongation continues until a stop codon (UAA, UAG, or UGA) reaches the A site of a ribosome 2. Release factor- protein that binds to stop codon in A site and causes addition of water molecule instead of amino acid to polypeptide chain, and frees polypeptide from ribosome. D. Polyribosomes- when a string of ribosomes trails along a single mRNA strand to produce polypeptides more efficiently IV. From Polypeptide to Functional Protein A. During and after synthesis, a polypeptide chain coils and folds spontaneously forming a functional protein of specific conformation B. Posttranslational Modifications- steps that may be required before a protein can begins functioning 1. Certain amino acids may be chemically modified by attaching sugars, lipids, phosphate groups, etc. 2. Enzymes may remove one or more amino acids from the leading (amino) end of the polypeptide chain 3. A single chain may be enzymatically cleaved into two or more pieces 4. Two or more polypeptides may join to become subunits of a protein with quaternary structure V. Signal Peptides- since there are free (cytosol) ribosomes and bound ribosomes (attached to ER), bound ribosomes and secretory ribosomes are marked by signal peptides, targeting them to the ER A. Signal peptides are short sequences of amino acids near the leading end of a polypeptide B. Signal-Recognition Particle (SRP)- protein-RNA complex that recognize signal peptides when they emerge from a ribosome and brings them to a receptor protein on the ER membrane C. Once attached to the ER membrane, protein synthesis continues, signal peptide is removed by an enzyme, and secretory proteins are released into the cisternal space of ER via a protein pore and a membrane protein remains embedded in ER membrane D. Other signal peptides target polypeptides to other organelles in the cell, but translation is completed before they are translocated. Prokaryotes (Bacteria) Vs. Eukaryotes I. Differences A. Contain different RNA polymerases; eukaryotes’ depend on transcription factors B. Transcription is terminated differently C. Slightly different ribosomes D. Cell organization 1. Prokaryotic cell assures a streamlined operation, simultaneously transcribing and translating the same gene 2. Eukaryotic cell’s nuclear envelope segregates transcription from translation and provides compartment for extensive RNA processing E. Eukaryotes have complicated mechanisms for targeting proteins to the appropriate cellular compartment. Mutations- changes in the genetic material of a cell or virus I. Large Scale Mutations- chromosomal rearrangements that affect long segments of DNA (previous . chapters II. Point Mutations- chemical changes in just one or a few pairs in a single gene A. Genetic Disorder- if a mutation has an adverse effect on the phenotype of a human or other animal B. Two Main Types 1. Base-Pair Substitution- the replacement of one nucleotide and its partner in the complementary DNA strand with another pair of nucleotides a. Silent Substitutions- due to the redundancy of the genetic code, a change in a base pair may transform one codon into another that has the same amino acid b. Other changes of a single nucleotide pair may switch an amino acid but have little effect on the protein if the new amino acid has similar properties

c. Some alterations of a single amino acid in a crucial area of a protein will significantly alter protein activity d. Missense Mutations- when the altered codon still codes for an amino acid and still makes sense, although it’s incorrect e. Nonsense Mutation- alterations that change an amino acid codon to a stop signal, usually leading to a nonfunctional protein 2. Insertions and Deletions- additions or losses of one or more nucleotide pairs in a gene a. These have disastrous effects because mRNA is read as a series of nucleotide triplets, and an insertion or deletion that is not a multiple of three would throw everything off b. Frameshift Mutation- when number of inserted or deleted nucleotides is not a multiple of three; almost certainly producing non-functional protein III. Mutagens- physical and chemical agents that interact with DNA to cause mutations A. In 1920 Hermann Muller discovered that X-rays caused an increased frequency in genetic changes B. Base Analogues- chemicals similar to normal DNA bases but pair incorrectly during DNA replication C. Other mutagens insert themselves in DNA and distort double helix. D. Ames Test- method to test the mutagenic activity of different chemicals, developed by Bruce Ames 1. Uses colonies of bacteria with mutations rendering them unable to synthesize amino acid histidine. 2. When suspected mutagenic chemical is added to culture, the only bacteria that will form colonies are those who have undergone a back-mutation to restore histidine-producing ability 3. Therefor, the number of colonies that grow increases measure of strength of mutagen 4. Ames test is often used to screen chemicals to identify those that cause cancer, because most cancer-causing chemicals, or carcinogens, are mutagenic.