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TRANSCRIPT
Chapter 17: From Gene to Protein
17.1: Genes specify proteins via transcription and translation
Evidence from the Study of Metabolic Defects
· Gene expression = process by which DNA directs synthesis of proteins
· Garrod = first to suggest that genes dictate phenotypes through enzymes that catalyze specific chemical reactions in the cell
· Suggested symptoms of a disease reflect inability to make an enzyme
· Recognized that Mendel’s principles of heredity apply to humans
· Nutritional Mutants in Neurospora: Scientific Inquiry
· Beadle & Tatum worked with bread mold, bombarding it with X-rays to show genetic changes
· Identified that mutants couldn’t survive on minimal medium because they couldn’t synthesize certain molecules from medium
· needed to grow on complete growth medium that supplemented needed nutrients
· Distributed samples from mutant into different vials containing minimal medium plus an extra nutrient
· Supplement that allowed growth indicated metabolic defect
· Srd & Horowitz pinned down each mutant’s defect realizing that mutants in each class required a different set of compounds along the pathway which has three steps
· Suggests that each class was blocked at a different step
· Saw that since each mutant was defective in a single gene, it supported one gene one enzyme hypothesis that the function of a gene dictates the production of a specific enzyme
· The Products of Gene Expression: A Developing Story
· Not all proteins are enzymes one gene one protein
· But many proteins are constructed are constructed from two or more polypeptide chains, each chain of which is specified by its own gene
· One gene one polypeptide hypothesis
· Not totally accurate, many eukaryotic genes can each code for a set of closely related polypeptides via alternative splicing or code for RNA
Basic Principles of Transcription and Translation
· A gene doesn’t build a protein directly- nucleic acid RNA bridges DNA and protein synthesis
· Transcription = the synthesis of RNA using info in DNA
· 2 nucleic acids written n different forms of the same language, and info is transcribed/rewritten from DNA to RNA, where DNA serves as a template for assembling a complementary sequence of RNA
· Resulting RNA molecules = messenger RNA/mRNA because it carries a genetic message from DNA to protein synthesizing machinery
· Translation = synthesis of a polypeptide using info from mRNA
· Change in language: cell must translate nucleotide sequence of mRNA into amino acid sequence of a polypeptide
· Ribosomes = translation sites
· Transcription and translation occur in all organisms
· Basic mechanics for bacteria and eukaryotes are similar but difference in flow of genetic info
· Because bacteria don’t have nuclei their DNA is not separated by nuclear membranes from ribosomes
· Lack of compartmentalization allows translation of mRNA to begin while transcription is still in progress
· In a eukaryotic cell the nuclear envelope separates transcription from translation
· Transcription occurs in the nucleus resulting in pre-mRNA
· Initial RNA transcript = primary transcript
· RNA transcripts are modified to produce final mRNA
· mRNA is transported to cytoplasm where translation occurs
· Central dogma dubbed by Francis crick: DNA RNA Protein
The Genetic Code
· Codons: Triplets of Nucleotides
· Problem: getting from four nucleotide bases to 20 amino acids
· Triplets of nucleotide bases are the smallest units of uniform length that can code for all amino acids 43=64 possible code words = more than enough to specify all amino acids
· Triplet code = genetic instructions for a polypeptide chain written in the DNA as a series of non-overlapping, three nucleotide words
· Transcribed into a complementary series of non-overlapping, three nucleotide words of mRNA
· Gene determines the sequence of nucleotide bases along mRNA
· For each gene only one of the two DNA strands is transcribed = template strand provides the patter for mRNA
· mRNA molecules is complementary rather than identical to its DNA template because RNA nucleotides are assembled on the template according to base-pairing rules (with U instead of T and ribose instead of deoxyribose)
· RNA molecule is synthesized in an antiparallel direction to the template DNA strand
· Codons are written in the 5’3’ direction
· Complementary DNA strand has the same code as the RNA strand (except with T instead of U) = coding strand
· Because codons are nucleotide triplets the number of nucleotides making up a genetic message must be three times the number of amino acids in the protein product
· Cracking the Code
· Cracked the genetic code of life when experiments disclosed the amino acid translations of each RNA codons
· First codon deciphered by Marshall Nirenberg by synthesizing an artificial mRNA by linking identical RNA nucleotides with uracil as the base: UUU
· Added to a test-tube with amino acids, ribosomes, and other components required translated UUU into polypeptide with units of phenylalanine amino acid
· Used same techniques for AAA, CCC, GGG
· More elaborate techniques were required to code mixed triplets, all 64 codons were deciphered
· Three codons don’t code- they code for stop signals marking the end
· AUG both codes for methionine and functions as a start signal
· There is a redundancy in the genetic code but no ambiguity
· Multiple codons can code for the same amino acid but never specify any other amino acid
· Often codons that are synonyms vary only in third nucleotide
· Need the correct reading frame to extract the intended message
· Wrong chain will be made if nucleotides are read from left to right (5’3’)
· Evolution of the Genetic Code
· The genetic code is nearly universal shared by most organisms
· Codons code for the same amino acids in every organism
· Some exceptions where codons differ from standard ones in unicellular eukaryotes
· Evolutionary significance of code’s near universality = language must have been operating very early in the history of life, early enough so be present in the common ancestor of all present day organisms
17.2: Transcription is the DNA-directed synthesis of RNA
Molecular Components of Transcription
· RNA polymerase pries the two strands of DNA apart and joins together RNA nucleotides complementary to the DNA template strand, elongating the RNA polynucleotide
· Assembles in the 5’3’ direction
· Unlike DNA polymerase it can start the chain from scratch w/o primer
· Specific sequences of nucleotides along DNA mark where transcription of a gene begins and ends
· RNA polymerase attaches and initiates transcription at the promoter DNA sequence
· Terminator = sequence that signals end of transcription in bacteria
· Promoter is upstream of the terminator
· Stretch of DNA between promoter and terminator that is transcribed into RNA = transcription unit
· Bacteria have one type of RNA polymerase that synthesizes not only mRNA but other RNA as well like ribosomal RNA
· Eukaryotes have at least three types of RNA polymerase in their nuclei
· RNA polymerase II used for mRNA synthesis
· Others transcribe RNA molecules that aren’t translated into protein
Synthesis of an RNA Transcript
· RNA Polymerase Binding and Initiation of Transcription
· Promoter includes start point=nucleotide where RNA synthesis begins
· Extends several dozen nucleotide pairs upstream from start
· RNA polymerase binds in a precise location and orientation on the promoter determining where transcription starts and which of the two strands of the helix is used as the template
· Transcription factors = collection of proteins that mediate the binding of RNA polymerase and the initiation of transcription in eukaryotes
· Only after they are attached to promoter does RNA polymerase II bind transcription initiation complex
· TATA box = DNA sequence that helps it form
· Protein=protein interactions in controlling eukaryotic transcription
· Elongation of the RNA Strand
· As RNA polymerase moves along DNA it continues to untwist double helix, exposing 10-20 nucleotides at a time for pairing with RNA
· Adds nucleotides to the 3’ end of the growing RNA molecule
· The new RNA molecule peels away from its template and the DNA double helix reforms
· A single gene can be transcribed simultaneously by sever molecules of RNA polymerase following each other
· Growing strand of RNA trails off from each polymerase
· Congregation of many polymerase molecules simultaneously transcribing a single gene increases about of mRNA transcribed from it
· Termination of Transcription
· In bacteria: transcription proceeds through DNA terminator sequence
· Transcribed terminator functions as termination signal causing polymerase to detach from DNA and release transcript
· Requires no further modification
· In eukaryotes: RNA polymerase II transcribes DNA polyadenylation signal sequence which codes for signal in pre-mRNA (AAUAAA)
· At a point 10-35 nucleotides downstream from the signal proteins associated with RNA transcript cut it free from the polymerase releasing the pre-mRNA
17.3: Eukaryotic cells modify RNA after transcription
Eukaryotic cells modify RNA after transcription
· RNA processing = when enzymes in the eukaryotic nucleus modify pre-mRNA before its dispatched to the cytoplasm
· Alteration of mRNA Ends
· At the 5’ end receives a 5’ cap, a modified form of guanine added after the first 20-40 nucleotides
· At 3’ end enzyme adds 50-250 adenine nucleotides forming poly-A tail
· Cap and tail functions:
· Facilitate the export of mRNA form the nucleus
· Protect mRNA from degradation by hydrolytic enzymes
· Help ribosomes attach to the 5’ end in the cytoplasm
· Split Genes and RNA Splicing
· RNA splicing = removal of portions of RNA = cut and paste job
· Average transcription unit is 27,000 nucleotides but it only takes 1,200 nucleotides to code for the average protein
· Most genes and RNA transcripts have long noncoding stretches which are interspersed between coding segments
· Introns = noncoding segments/intervening sequences
· Exons = other regions that are expressed/translated
· Exit the nucleus
· RNA polymerase translates introns and exons but the introns are cut out and the exons joined together
· Short nucleotide sequence at each end of an intron gives the signal for RNA splicing
· Small nuclear ribonucleoproteins/snRNPs, located in the nucleus recognize splice sites
· snRNA = RNA in a snRNP
· Several snRNPs join other proteins to form spliceosome
· Interacts with sites along an intron, releasing the intron which is degraded and joining the exons
· Catalyze processes as well as participate in it
· Ribozymes = RNA molecules that function as enzymes
· In some organisms the intron RNA functions as a ribozyme and catalyzes its own excision = self-splicing
· RNA is single-stranded so a region can base-pair with a complementary region elsewhere on the same molecule giving it a 3-D structure
· Some of the bases in RNA contain functional groups that participate in catalysis
· The ability of RNA to hydrogen-bond with other nucleic acids adds specificity to catalytic activity complementary base paring between RNA of splicesome and RNA of primary RNA precisely locates the region to catalyze splicing
· The Functional and Evolutionary Importance of Introns: Evolution
· Specific functions have not been identified for most introns but some contain sequences that regulate gene expression effecting products
· A single gene can encode more than one kind of polypeptide
· many genes are known to give rise to 2+ different polypeptides depending on which segments are treated as exons = alternative RNA splicing
· Ex: sex differentiation in fruit flies
· Proteins have a modular architecture with discrete structural and functional regions = domains
· Different exons code for different domains of a protein
· Ex: include an active site
· Introns may facilitate evolution of new and potentially beneficial proteins through exon shuffling
· Introns increase the probability of crossing over between the exons of alleles of a gene by providing more terrain for crossovers without interrupting code sequences
· new combinations of exons and proteins with altered structure and function
· Occasional mixing and matching of exons between different genes
17.4: Translation is the RNA-directed synthesis of a polypeptide
Molecular Components of Translation
· During translation a cell reads a genetic message and builds a polypeptide
· Translator = transfer RNA/tRNA used to transfer amino acids from the cytoplasmic pool of amino acids to a growing polypeptide in a ribosome
· A cell keeps its cytoplasm stocked will all 20 amino acids either by synthesizing them from other compounds or by taking them up from the surrounding solution
· The Structure and Function of Transfer RNA
· tRNA are not all identical, each type of tRNA molecule translates a particular mRNA codon into an amino acid
· tRNA arrives at a ribosome bearing a specific amino acid at one end
· At the other end it has an anticodon which base pairs with a complementary codon on mRNA
· As an mRNA molecule is moved through a ribosome the amino acid will be added to the polypeptide chain whenever the codon is presented for translation
· Codon by codon the genetic message is translated as tRNAs deposit amino acids and the ribosome joins the amino acids in the chain
· tRNA molecule is a translator between a codon to an amino acid
· tRNA is transcribed from DNA templates
· Each tRNA is used repeatedly
· tRNA consists of a single RNA strand that is ~80 nucleotides long
· Has complementary stretches of nucleotide bases that can hydrogen bond to each other the strand can fold back upon itself and form a molecule with a 3D structure
· Amino acid attaches at the 3’ end
· Translation requires two instances of molecular recognition:
· tRNA binds to an mRNA codon specifying a particular amino acid must carry that specific amino acid to the ribosome Aminoacyl-tRNA synthases carry out matching
· Active site of each type only fits a specific combination of amino acid and tRNA
· 20 different synthases, one for each amino acid
· Catalyzes the covalent attachment of the amino acid to its tRNA driven by hydrolysis of ATP aminoacyl tRNA (charged tRNA)
· Pairing of tRNA anticodon with the appropriate mRNA codon
· Some tRNAs bind to more than one codon because base pairing between the third nucleotide base of a codon and the corresponding base of the tRNA codon are relaxed compared to those at the other codon positions
· Wobble = flexible base pairing at third codon position
· Ribosomes facilitate the coupling of tRNA anticodons with mRNA codons
· Ribosomes are made up of a large and small subunit made of ribosomal RNAs (rRNAs) proteins
· In eukaryotes subunits are made in the nucleolus
· Ribosomal RNA genes are transcribe and RNA is processed and assembled with proteins imported from cytoplasm
· Large and small subunits join to form a functional ribosome when they attach to an mRNA molecule
· Most abundant type of cellular RNA
· Eukaryotic ribosomes are large and differ in molecular composition from bacterial ribosomes
· Some drugs can inactivate bacterial ribosomes without inactivating eukaryotic ribosomes
· Ribosome structure reflects its function of bringing together mRNA and tRNA with amino acids:
· P site (peptidyl-tRNA binding site) holds the tRNA carrying the growing polypeptide chain
· A site (aminoacyl-tRNA binding site) holds the tRNA carrying the next amino acid to be added to the chain
· E site (exit site) is where discharged tRNAs leave
· 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
· Catalyzes formation of the peptide bond
· rRNA, not protein, is primarily responsible for both structure and function of the ribosme
· Proteins support the shape changes of the rRNA molecules as they carry out catalysis during translation
Building a Polypeptide
· Divide translation into three stages: initiation, elongation, and termination
· Energy is required, provided by hydrolysis of GTP (similar to ATP)
· Ribosome Association and Initiation of Translation
· Initiation stage of translation brings together mRNA, tRNA with the first amino acid of the polypeptide and the two subunits of a ribosome
· First a small ribosomal subunit binds to mRNA and initiator tRNA which carries methionine
· Complementary base pairing between site and rRNA involved
· In eukaryotes the small subunit (with initiator tRNA bound) binds to the 5’ cap of mRNA and then scans downstream along mRNA until it reaches the start codon
· Initiator tRNA hydrogen-bonds to AUG start codon, signaling start of translation and establishing the codon reading frame
· Next the large ribosomal subunit attaches completing the translation initiation complex
· At the end the initiator tRNA sits in the P site of the ribosome and the vacant A site is ready for the next tRNA
· Initiation factors are required to bring all the pieces together
· Cell expends energy through hydrolysis of GTP to form initiation complex
· Polypeptide is always synthesized in one direction, from the N-terminus end (methionine) toward C-terminus (the final amino acid)
· Elongation of the Polypeptide Chain
· Elongation stage adds amino acids one by one to the previous amino acid at the C-terminus end of the growing chain
· Each addition involves several elongation factor proteins
· mRNA is moved through ribosome in one direction, 5’3’ end
· Move relative to each other, codon by codon
· Occurs in three step cycle (energy used in steps 1 and 3), see picture
1. Codon recognition: anticodon of incoming tRNA base pairs with complementary mRNA codon in the A site
a. Hydrolysis of GTP increases accuracy and efficiency
2. Peptide bond formation: rRNA molecule of the large ribosomal subunit catalyzes the formation of a peptide bond between amino group of the new amino acid at the A site and the carboxyl end of the growing chain in the P site
a. Removes polypeptide form tRNA in the P site and attaches it to the amino acid on the tRNA in the A site
3. Translocation: ribosome translocates tRNA in A site to the P site
a. Empty tRNA in the P site is moved to the E site where it’s released
b. mRNA moves along with its bound tRNAs, bringing the next codon to be translated into the A site
· Termination of Translation
· Termination stage occurs when a stop codon in the mRNA reaches the A site of the ribosome
· Release factor = protein shaped like tRNA binds directly to the stop codon in the A site, causing the addition of a water molecule instead of an amino acid to the polypeptide chain
· breaks (hydrolyzes) the bond between the completed polypeptide and the tRNA in the P site, releasing the polypeptide through the E site
· Translation assembly comes apart
· Polyribosomes:
· A single ribosome can make a polypeptide in less than a minute
· Multiple ribosomes translate an mRNA at the same time- a single mRNA is used to make many copies of a polypeptide simultaneously
· Once a ribosome is far enough past the start codon a second ribosome can attach to the mRNA, resulting in a number of ribosomes trailing along the mRNA = polyribosomes/polysomes
· Allow a cell to make many copies of polypeptide very quickly
Completing and Targeting the Functional Protein
· Protein Folding and Post-Translational Modifications
· Translation isn’t enough to make a functional protein
· During synthesis the polypeptide chain begins to coil and fold spontaneously due to its amino acid sequence (primary structure)
· Chaperonin protein helps the polypeptide fold correctly
· Post-translational modifications may be required before the protein can begin doing its job
· Amino acids may be chemically modified by attachment of sugars, lipids, phosphate groups, etc.
· Enzymes may remove one or more amino acids from les the leading end or enzymatically cleave the chain
· Two or more polypeptides are synthesized separately and may come together becoming a protein of quaternary structure
· Targeting Polypeptides to Specific Locations
· Two types of ribosomes are found in the cell: free and bound
· Free are suspended in cytosol
· Synthesize proteins that stay in the cytosol
· Bound are attached to the endoplasmic reticulum or the nuclear envelope
· Make proteins of the endomembrane system and proteins secreted from the cell
· Polypeptide synthesis always begins in the cytosol as a free ribosome starts to translate mRNA
· Continues to completion unless the growing polypeptide cues the ribosome to attach to the ER
· Polypeptides destined for the endomembrane system or secretion are marked by a signal peptide which targets the protein to the ER
· Sequence of 20 amino acids near the end
· Recognized as it emerges from the ribosome by signal-recognition particle (SRP) protein-RNA complex
· Functions as an escort bringing the ribosome to a receptor protein on the ER membrane
· Part of a multiprotein translocation complex where synthesis continues, the growing polypeptide snaking across the membrane into ER lumen via protein pore
· Removed by an enzyme and either is released or remains embedded
· Other types are used to target polypeptides to mitochondria, chloroplasts, interior of the nucleus, and other organelles not part of the endomembrane system
· Translation is completed in the cytosol before the polypeptide is imported into the organelle
· Bacteria use to target proteins to membrane for secretion
17.5: Mutations of one or a few nucleotides can affect protein structure and function
Types of Small-Scale Mutations
· Mutations = changes in genetic info of a cell, responsible for diversity of genes in organisms (source of new genes)
· Point mutations = changes in a single nucleotide pair of a gene
· If it occurs in a gamete that gives rise to gametes it can be transmitted to offspring and future generations
· Ex: hemoglobin: single point mutation that encodes polypeptide of hemoglobin leading to production of abnormal protein
· Nucleotide-pair substitution = replacement of one nucleotide and its partner with another pair of nucleotides
· Some have no phenotypic effect because of redundancy of the genetic code = silent mutation
· Missense mutations do change the amino acid can or can’t have a phenotypic effect
· Nonsense mutation = missense mutation that changes a codon to a stop codon terminating translation prematurely
· nonfunctional proteins
· Insertions and deletions are additions or losses of nucleotide pairs in a gene
· Have a disastrous effect because they alter the reading frame = frameshift mutation all nucleotides that are downstream of the deletion/insertion will be improperly grouped into codons
· nonfunctional proteins
Mutagens = physical and chemical agents that interact with DNA causing mutations
· Incorrect base will be used as a template in the next round of replication = spontaneous mutations
· Change is passed on to the next generation of cells
· Hermann Muller discovered that X-rays caused genetic changes in fruit flies
· Pose hazards to genetic material of people
· Chemical mutagens fall into many categories
· Nucleotide analogs = chemicals similar to normal DNA nucleotides but pair incorrectly during replication
· Others interfere with correct DNA replication by inserting themselves into the DNA, distorting the double helix
· Others cause chemical changes in bases changing pairing properties
· Tests of mutagenic activity of chemicals used to identify carcinogens
17.6: While gene expression differs among the domains of life, the concept of a gene is universal
Comparing Gene Expression in Bacteria, Archaea, and Eukarya
· Bacterial and eukaryotic RNA polymerases differ (archaeal RNA polymerase resembles eukaryotic ones)
· Archaea and eukaryotes use transcription factors, unlike accessory proteins in bacteria
· Transcription is terminated differently between eukaryote and bacteria (Achaea similar to eukaryotes probs)
· Archaeal ribosomes are the same size as bacterial ribosomes but sensitivities to chemical inhibitors are more similar to eukaryotic ribosomes
· Initiation of translation is different in bacteria and eukaryotes (archaea is more like bacteria)
· Most important differences between bacteria and eukaryotes arise from bacteria’s lack of compartmental organization
· Streamlined operation in a bacterial cell- without a nucleus it can simultaneously transcribe and translate the same gene
· Newly made protein quickly diffuses to site of function
· Similar in archaeal cells which lack a nuclear envelope
· Eukaryotic cell’s nuclear envelope segregates transcription from translation providing a compartment for RNA processing
· Includes additional steps to coordinate elaborate activities
What Is a Gene? Revisiting the Question
· Functional definition of a gene as a DNA sequence that codes for a specific polypeptide chain
· Most eukaryotic genes contain noncoding segments so large portions of these genes have no corresponding segments in polypeptides
· Promoters and regulatory regions considered part of the gene
· DNA that transcribes rRNA, tRNA, and other RNAs that aren’t translated are also part of the gene
· A gene is a region of DNA that can be expressed to produce a final functional product that is either a polypeptide or an RNA molecule