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Protein Synthesis (Sections 5.1 to 5.4) Inherited instructions stored in DNA direct the production of proteins A Brief History Gregor Mendel = factors are responsible for inherited traits How genes control metabolism? 1. One gene – one enzyme Archibald Garrod (1909) = hypothesis states “one gene Cells synthesize and degrade organic compounds via metabolic pathways, with each sequential step catalyzed by a specific enzyme (figure 2, p. 234) Beadle and Tatem experiment (3o years later) were able to demonstrate this experimentally i. Neurospora (bread mold) - can survive with minimal nutrients, however some mutants were not able to survive why? ii. Looked at metabolic pathway for the synthesis of arginine- they distinguished mutants by which substance needed to be added to the medium in order for the mold to grow Precursor enzyme A ornithine enzyme B citrulline enzyme C arginine enzyme D arginine Succinate iii. Conclusion: each mutant lacked a different functional enzyme, thus blocked at different parts of the metabolic pathway (see fig. 2, p. 235) 2. One gene – one polypeptide Today, we understand that genes code for proteins, and not all proteins are enzymes. Also, some proteins consist of more that one polypeptide, each peptide coded by a different gene (Vernon Ingram) see p. 235 The Central Dogma The central dogma of molecular biology (Francis Crick,1958) The central dogma of molecular biology deals with the detailed residue-by-residue transfer of sequential information. It states that information cannot be transferred back from protein to either protein or nucleic acid. DNA RNA protein DNA cannot leave the nucleus (keeps it from being damaged) An Overview of Protein Synthesis RNA links the genetic code stored in DNA to the production of Proteins – RNA copies the code in DNA (transcription) and translates the message into a polypeptide chain DNA structure vs RNA structure: o o o o DNA DNA DNA DNA double-stranded, RNA single-stranded has thymine, RNA has uracil has Deoxyribose sugar, RNA has ribose sugar is the genetic code, RNA interprets the code The Flow of genetic information: The linear sequence of nucleotides in DNA ultimately determines the linear sequence of amino acid in a protein. DNA transcription RNA RNA processing RNA translation polypeptide Protein Synthesis occurs in Two Stages: 1. Transcription = the synthesis of messenger RNA (mRNA) using DNA as a template Resulting mRNA strand carries this transcript (small strand) to the protein-synthesizing machinery 2. Translation = synthesis of a polypeptide, which occurs under the direction of mRNA Linear sequence of bases in mRNA is translated into the linear sequence of amino acids Translation occurs at protein-synthesizing machinery, which consists of ribosomes, ribosomal RNA (rRNA), and proteins that facilitate the addition of amino acids to form polypeptide NOTE: ****Prokaryotes and eukaryotes differ in how protein synthesis is organized within their cells. In Prokaryotes – happens in rapid successions (no nucleus) In Eukaryotes – mRNA is modified prior to leaving the nucleus Ribonucleic Acid - RNA There are three major classes of RNA: o Messenger RNA (mRNA) – hold code o Transfer RNA (tRNA) – help in carrying amino acids to ribosomal complex o Ribosomal RNA (rRNA) – structural component of ribosomal complext Transcription – In General Flow of information from gene to protein is based on a triplet code (p. 240) o Recall, there are only 4 different types of nucleotide, but there are 20 different amino acids. How can we code for all these amino aicds? o RNA is read 3 nucleotide bases at a time = a codon Ex. AUG = methionine – start codon / UUA, UAG, UGA = stop o The ability to extract the intended message depends on how the code is read o The reading frame dictate that this is done 3 bases at a time with no overlap ex. THE RED DOG Genes are first transcribed into mRNA: o For each gene, only one of the two DNA strands (template strand) is transcribed o The strand that is not transcribed is called the parent strand o Which strand serves as the template stand varies for each gene mRNA stand is complimentary to DNA template strand o Recall, uracil (U) in RNA is used in place of thymine (T) ie. U pairs with A Translation – In General Linear sequence of codons in mRNA is translated into linear sequence of amino acids in polypeptide o Each mRNA codon specifies which amino acid is incorporated next into growing polypeptide o Thus, the number of nucleotides is 3 times > a.a. Cracking the Genetic Code The first codon was deciphered in 1961 by Nirenberg of the NIH 61 of 64 triplets code for amino acids AUG is the start signal for translation and codes for methionine UAA, UAG, UGA code only for signal termination = stop codon There can be two or more codons for each amino acid (ex. UUU, UUC both code for phenylalanine) Codons only code for one amino acid (ex. UUU only codes for phenylalanine) The correct ordering (called the reading frame) and grouping of nucleotides is important in the molecular language of cells. A change in the reading frame leads to a change in the peptide sequence! mutation Proteins Synthesis – The Specifics! A. Transcription = synthesis of mRNA transcription of mRNA is catalyzed by RNA polymerase (separates DNA and builds mRNA in 5’ to 3’ direction) specific DNA nucleotide sequences mark where transcription of a gene begins (initiation) and ends (termination). The nucleotide sequence that is transcribed into mRNA by RNA polymerase is called a transcription unit. 1. Binding of RNA polymerase = RNA poly. Binds to specific DNA region called the promoter region marks initiation In eukaryotes, the promoter region is approx. 100 nucleotides long and consists of: i. Promoter region ii. Binding site – where DNA binding proteins attach DNA binding proteins (aka transcription factors) = bind to TATA box just upstream to initiation site TATA Box = short repeating nucleotide sequence (~25) upstream to promoter region (initiation site) RNA polymerase recognizes transcription factors and binds to promoter region. 2. Elongation of RNA Strand = RNA polymerase continues to unwind DNA and add RNA nucleotides in 5’ to 3’ direction mRNA strand grows about 30-60 nucleotides/second several molecules of RNA polymerase can simultaneously transcribe the same gene! 3. Termination of Transcription = transcription continues until RNA polymerase transcribes a terminator sequence in eukaryotes the most common terminator sequence is AAUAA 4. Modification of RNA Transcript – Eukaryotes only! Primary transcript = general term for initial RNA strand transcribed from DNA Pre-mRNA = primary transcript that will be processed to functional mRNA Transcript can be processed in two ways: a. alteration of both the 3’ and 5’ ends - 5’ CAP = guanosine triphosphate is added (protects the mRNA strand from degradation, helps in initiation of translation - 3’ END = poly A tail (protects mRNA from degredation, facilitates attachment to ribosomal complex, assists in export of nucleus) b. removal of intervening sequences - coding regions (exons) are interrupted by non coding regions (introns) - during processing introns are removed by splicosomes, and exons are joined (RNA splicing) are removed before mRNA leaves the nucleus - small nuclear ribonucleoproteins (SnRNPs) play a key role in RNA splicing Why have introns? Intron DNA sequences may control gene activity The splicing process may help regulate the export of mRNA Introns may allow a single genet to direct the synthesis of different proteins (i.e if the same RNA transcript is processed differently) B. Translation = synthesis of peptide, coordinated by mRNA We Need: mRNA tRNA amino acids polypeptide ribosomes protein Ribosomes: are made up of two subunits (60% rRNA and 40% protein) that hold the mRNA in place in order for translation to occur. Each ribosome has three biding sites: P site = holds tRNA carrying growing peptide A site = holds tRNA carrying next amino acid E site = where tRNA exits from Transfer RNA, tRNA: RNA strand (~80 bp) transcribed from DNA in nucleus 3D shape held together by H-bonds can be used repeatedly during translation proteins are synthesized according to the sequence of codons, tRNA helps in translation of RNA code to a.a sequence. How? tRNA aligns the appropriate amino acid by: - transfers amino acid from cytoplasm to ribosomal complex - each tRNA strand has an amino acid attachment site at one end and a anticodon at the opposite end. - Anticodon is complimentary to the RNA codon for that amino acid - The attaching of the a.a. to its tRNA is catalyzed by a specific aminoacyl-tRNA synthetase (required ATP) each amino acid has its own synthetase Building a polypeptide – Occurs in Thee Stages 1. Initiation of Translation Small (40) subunit of ribosome binds to mRNA strand (5’ cap of mRNA helps binding) Initiator Met-tRNA (start anticodon) finds AUG (start codon) downstream on mRNA and binds to it, large subunit then binds to initiator tRNA forming initiation complex (small ribosome unit, mRNA and met-tRNA) Large ribosomal subunit (60) attached to small one; initiator tRNA fits into P-site of ribosome vacant A site is ready for next amino acid 2. Elongation Codon recognition: mRNA codon in A site of ribosome forms H-Bonds with anticodon of entering tRNA carrying the next amino acid Peptide bond formation: a peptide bond is formed between growing polypeptide in P-site and new amino acid in A-site by peptidyl transferase polypeptide is transferred to Asite Translocation (ribosome moves forward one codon): the tRNA that was in the P-site is released (E-site). The tRNA in the Asite is translocated to the P-site (taking growing peptide with it) this repeats along the length of mRNA ….. 3. Termination Occurs when a stop codon is reached (UGA,UAG, or UAA) Stop codons do not code for any amino acid Reaching stop sequence causes a release factor or bind to the A-site of the ribosome and facilitates the release of the polypeptide. Small and large ribosomal units separate Polypeptide is now ready for further processing and folding! Recall, A protein’s sequence (primary structure) determines how the peptide will coil and fold into its 3D shape Some proteins undergo post-translational modifications before they become fully functional (ie chemical modifications, or chain length modifications) Multiple Roles of RNA in the cell RNA has many other critical roles in the cell: 1. information carrier = mRNA carries genetic info form DNA to ribosomes 2. adaptor molecule = tRNA translate info from mRNA into protein SRP RNA directs the translation complex to ER 3. catalysts and structural molecule – rRNA plays structural and enzymatic role in ribosomes; snRNA catalyzses RNA splicing 4. viral genomes – some viruses use RNA as genetic material Control Mechanisms (Section 5,5) Not all of our 42,000 genes are needed at all times, so transcription factors turn genes on and off as required Note: some housekeeping genes are always being transcribed since cells need these at all times. In eukaryotes, there are 4 levels of gene control (expression): i) transcriptional, ii) posttranscriptional, iii) translational, and iv) iv) posttranslational. See table 1.p 255 know them! Operons Operons = are clusters of genes under the control of one promoter and one operator ex lac operon, trp operon Lac operon o Form of control only used in bacteria o In bacteria such as E.coli found in the digestive system of mamals, β-galactosidase is the enzyme responsible for breaking down lactose (enzyme is not always required) o β-galactosidase is part of an operon o lacl protein binds to operon blocking transcription o in the presence of lactose a repressor protein (lacl protein) normally bound to operon leaves and binds to lactose transcription of the lac operon no longer blocked (no enzymes are made) see fig. 2 p. 256 o Why? When there is no milk, no enzyme is needed The trp Operon Works in the opposite manner to the lac operon tryptophan is an amino acid used by bacteria to produce proteins, when available in its environment bacteria stop producing tryptophan and absorb it from its environment operon acitivity is inhibited when the concentration of tryptophan in the environment increases tryptophan binds to operator region of operon see fig. 3, p. 257