17: From Gene to Protein DNA inherited by an organism usually leads to specific traits by dictating the synthesis of proteins,

, which act as the link between genotype and phenotype Gene expression is the process by which DNA directs protein synthesis, and includes two stages: transcription and translation The one gene-one polypeptide hypothesis states that the function of a gene is to dictate the production of a specific polypeptide Transcription is the synthesis of RNA using information in the DNA; the complementary strand of RNA transcribed from DNA is called mRNA because it carries a genetic message from the DNA to the protein-synthesizing machinery of the cell Translation is the synthesis of a polypeptide using the information in mRNA. The site of translation are ribosomes, complex particles that facilitate the orderly linking of amino acids into polypeptide chains In bacteria, because their DNA is not separated by nuclear membranes from ribosomes and other protein-synthesizing equipment, translation of an mRNA can begin while its transcription is still in progress In eukaryotes, the nuclear envelope separates transcription from translation. Transcription occurs in the nucleus, and mRNA is then transported to the cytoplasm, where translation occurs. The transcription of a protein-coding eukaryotic gene results in pre-mRNA, and further processing yields the finished mRNA. The initial RNA transcript from any gene, including those specifying RNA that is not translated into protein, is called a primary transcript The flow of information from gene to protein is based on a triplet code: a series of non-overlapping, three-nucleotide codons For each gene, only one of the two DNA strands is transcribed, this strand is called the template strand. For any given gene, the same strand is used as the template every time the gene is transcribed The RNA molecule is synthesized in an antiparallel direction to the template strand of DNA The nontemplate strand of DNA is sometimes called the coding strand as its sequence is identical to the RNA produced Codons are read by translation machinery in the 5 -> 3 direction Because more than one codon specifies an amino acid, the code is redundant; because the gene sequence is read three base pairs at a time, the code is unpunctuated; one triplet specifies only one amino acid so the code is unambiguous AUG codes for methionine and acts as the start signal, or initiation codon. It signals the protein-synthesizing machinery to begin translating the mRNA at that location (polypeptide chains also begin with methionine when synthesized but enzymes may subsequently remove this starter amino acid from the chain) UAA, UGA, UAG are the three stop codons; they do not designate any amino acid (you are an/ ugly goat and/ you are gay)

 The genetic code is nearly universal, shared by the simplest bacteria to the most complex animals. Genes can be transcribed and translated after being transplanted from one species to another. Exceptions to the universality of the code include protistans (single-celled eukaryotes) and mitochondria mRNA is transcribed from the template strand of a gene; an enzyme called RNA polymerase pries the two strands of DNA apart and joins together RNA nucleotides complementary to the template. It can only assemble the RNA in the 5 -> 3 direction. Unlike DNA, RNA is able to start a chain from scratch; they do not need a primer Specific sequences of nucleotides along the DNA mark where transcription of a gene begins and ends. The promoter is where RNA polymerase attaches and initiates transcription; the terminator is the sequence that signals the end of transcription The stretch of DNA that is transcribed into an RNA molecule is called a transcription unit Bacteria only have a single type of RNA polymerase while eukaryotes have at least three types in their nuclei. The one used for mRNA synthesis is called RNA polymerase II; the other RNA polymerases transcribe RNA molecules that are not translated into protein The three stages of transcription: 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 The promoter of a gene includes within it the transcription start point, which is the nucleotide where RNA synthesis actually begins In bacteria, the RNA polymerase specifically recognizes and binds to the promoter. In eukaryotes, a collection of proteins called transcription factors attach to the promoter, only then does RNA polymerase II bind to it. The whole complex is called a transcription initiator complex. A crucial promoter DNA sequence called a TATA box is also present The RNA polymerase exposes about 10-20 DNA nucleotides at a time for pairing, the enzyme adds nucleotides to the 3 end of the growing RNA. The new RNA molecule peels away from the template and the double helix of the DNA reforms. Transcription progresses at about 40 nucleotides a second in eukaryotes and multiple RNA polymerases can transcribe a single gene at a given time In bacteria, the polymerase stops transcription at the end of the terminator sequence. In eukaryotes, RNA polymerase II transcribes a polyadenylation sequence (AAUAAA) and about 10-35 nucleotides downstream, proteins cut it free from the polymerase and the pre-RNA is released During RNA processing in eukaryotic cells, both ends of the primary transcript are altered and some interior sections of the RNA are cut out and the remaining parts spliced together

 The 5 end is given a 5 cap (modified form of guanine nucleotide) and the 3 end is given a poly-A tail (composed of 50-250 adenine nucleotides). These additions facilitate the export of the mRNA from he nucleus and protect the mRNA from degradation by hydrolytic enzymes, they also help ribosomes attach to the 5 end of the mRNA. There are also untranslated regions (UTRs) at both ends that are not translated into protein but help ribosome bonding In RNA splicing, introns (noncoding segments) are removed and exons (expressed sequences) are spliced together by small nuclear ribonucleoproteins (snRNPs). snRNPs recognize splice sites, are located in the nucleus, and are composed of small nuclear RNA (snRNA) and protein molecules. Several different snRNPs join with additional proteins to form a spliceosome, which releases introns (that then degrade) and join the exons. snRNA catalyzes these processes, as well as participates in spliceosome assembly and splice site recognition Three properties of RNA enable it to function as an enzyme (ribozyme): 1) it can form 3-d structures because of its ability to base pair with itself 2) some bases in RNA contain functional groups 3) RNA may hydrogen bond with other nucleic acid molecules Some introns contain sequences that regulate gene expression; some genes can encode more than one kind of polypeptide, depending on which segments are treated as exons during RNA splicing, such variations are called alternative RNA splicing. Because of alternative splicing, the number of different proteins an organism can produce is much greater than its number of genes Domains are structural and functional regions of a protein, usually different exons code for different domains of a protein. In exon shuffling, crossing over between the exons of alleles of a gene is facilitated by introns, which can lead to new proteins with novel combinations of functions, which may sometimes be beneficial As a molecule of mRNA is moved through a ribosome, codons are translated into amino acids by tRNA molecules, each type with a specific anticodon at one end and a corresponding amino acid at the other end. A tRNA adds its amino acid cargo to a growing polypeptide chain when the anticodon hydrogen-bonds to a complementary codon on the mRNA tRNA is transcribed from DNA templates in the nucleus and then travels to the cytoplasm; it is reused repeatedly A tRNA molecule consists of a single RNA strand about 80 nucleotides long. In the 2d structure, there are four base-paired regions and three loops and the amino acid attachment site is at the 3 end. The 3d structure is like an L shape, with the anticodon loop at one end and the amino acid attachment site at the other Accurate translation requires two steps: 1) a correct match between a tRNA and an amino acid created by the appropriate aminoacyl-tRNA synthetase (20 types that catalyze the covalent attachment of the amino acid to its tRNA in a process driven by the hydrolysis of ATP) 2) a correct match between the tRNA anticodon and an mRNA codon; flexible pairing at the third base of a codon is called wobble and allows some tRNAs to bind to more than one codon (only 45 tRNAs for 61 codons)

 A ribosome consists of a large subunit and a small subunit, each made up of proteins and one or more rRNAs. In eukaryotes, the subunits are made in the nucleolus. In both bacteria and eukaryotes, large and small subunits join to form a functional ribosome only when they attach to an mRNA molecule In addition to a binding site for mRNA, each ribosome has three binding sites for tRNA. The P site holds the tRNA carrying the growing polypeptide chain, the A site holds the tRNA carrying the next amino acid to be added to the chain, and discharged tRNAs leave through the E site The ribosome holds the tRNA and mRNA in close proximity and positions the new amino acid for addition to the carboxyl end of the growing polypeptide. It then catalyzes the formation of the peptide bond. As the polypeptide becomes longer, it passes through an exit tunnel in the large subunit and is later released through it as well The three stages of translation are initiation, elongation, and termination Initiation: 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. In eukaryotes, with the initiator tRNA already bound, the small subunit binds to the 5 cap and moves down the mRNA until it reaches the start codon. The initiator tRNA, with the anticodon UAC, base pairs with the start codon (AUG). The arrival of a large ribosomal subunit completes the translation initiation complex. Proteins called initiation factors are required to bring all of the translation components together. Hydrolysis of GTP provides the energy for the assembly. The initiator tRNA is in the P site; the A site is available for the next tRNA Elongation: amino acids are added one by one at the C-terminus of the growing chain and involves the participation of several proteins called elongation factors. First there is codon recognition; hydrolysis of GTP increases the accuracy and efficiency of this step. Then there is peptide bond formation, in which an rRNA molecule of the large subunit catalyzes the formation of a peptide bond between the amino group of the new amino acid in the A site and the carboxyl end of the growing polypeptide in the P site. Then there is translocation, where the ribosomes moves the tRNA in the A site to the P site and the empty tRNA in the P site is moved to the E site and released. Termination: when a ribosome reaches a stop codon on the mRNA, the A site of the ribosome accepts a release factor protein which promotes hydrolysis of the bond between the tRNA in the P site and the last amino acid of the polypeptide by bonding water instead of an amino acid. The polypeptide is freed and the two subunits and other components dissociate; GTP hydrolysis is also necessary Polyribosomes are the chains of ribosomes that translate the same mRNA at the same time, allowing a cell to make many copies of a polypeptide quickly Polypeptide chains are frequently modified after translation. A chaperone protein (chaperonin) often helps fold a polypeptide correctly. Certain amino acids are chemically modified by the attachment of sugars, lipids, phosphate groups, or other additions. Enzymes may also remove one or more amino acids from the chain, and some chains are enzymatically cleaved into pieces

 Synthesis of a polypeptide begins on a free ribosome in the cytosol; a signalrecognition particle (protein-RNA complex) will recognize a signal peptide and then bind to a receptor protein in the ER membrane. This receptor is part of a protein complex that has a membrane pore and a signal-cleaving enzyme. The signal recognition particle leaves and polypeptide synthesis resumes, with simultaneous translocation across the membrane. The signal cleaving enzyme then cuts off the signal peptide and the rest of the completed polypeptide leaves the ribosomes and folds into its final conformation Small-scale mutations include point mutations, changes in one DNA nucleotide pair, which may lead to production of nonfunctional proteins. Nucleotide-pair substitutions (the replacement of one nucleotide and its partner with another pair of nucleotides) can cause silent mutations (no observable effect), missense mutations (change one amino acid to another), or nonsense mutations (changes a codon for an amino acid into a stop codon). Nucleotide pair insertions or deletions may produce frameshift mutations, which occurs whenever the number of nucleotides inserted or deleted is not a multiple of three. All the nucleotides downstream will be improperly grouped into codons Spontaneous mutations can occur during DNA replication, recombination, or repair. Chemical and physical mutagens cause DNA damage that can alter genes Archaea are prokaryotes, but share many features of gene expression with eukaryotes. Bacteria and eukarya differ in their RNA polymerases, termination of transcription in ribosomes; archaea tend to resemble eukarya in these respects. Bacteria can simultaneously transcribe and translate the same gene; archaea probably can too. In eukarya, transcription and translation are separated by the nuclear envelope A gene is a discrete unit of inheritance, a region of DNA in a chromosome, and a DNA sequence that codes for a specific polypeptide chain A gene is a region of DNA that can be expressed to produce a final functional product, either a polypeptide or an RNA molecule