General Biology. Genes specify proteins via transcription and translation Evidence from the Study of Metabolic Defects

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1 ourse No: BN003 redits: 3.00 eneral Biology 10. enetics: From enes to Proteins Overview: The Flow of enetic Information The information content of DN is in the form of specific sequences of nucleotides along the DN strands The DN inherited by an organism leads to specific traits by dictating the synthesis of proteins The process by which DN directs protein synthesis, gene expression includes two stages, called transcription and translation The ribosome is part of the cellular machinery for translation, polypeptide synthesis Prof. Dr. Klaus Heese enes specify proteins via transcription and translation vidence from the Study of Metabolic Defects In 1909, British physician rchibald arrod was the first to suggest that genes dictate phenotypes through enzymes that catalyze specific chemical reactions in the cell Nutritional in Neurospora: Scientific Inquiry Beadle and Tatum causes bread mold to mutate with X- rays creating mutants that could not survive on minimal medium sing genetic crosses, they determined that their mutants fell into three classes, each mutated in a different gene XPRIMNT RSLTS Working with the mold Neurospora crassa, eorge Beadle and dward Tatum had isolated mutants requiring arginine in their growth medium and had shown genetically that these mutants fell into three classes, each defective in a different gene. From other considerations, they suspected that the metabolic pathway of arginine biosynthesis included the precursors ornithine and citrulline. Their most famous experiment, shown here, tested both their one gene one enzyme hypothesis and their postulated arginine pathway. In this experiment, they grew their three classes of mutants under the four different conditions shown in the Results section below. The wild-type strain required only the minimal medium for growth. The three classes of mutants had different growth requirements Minimal medium (MM) (control) MM + Ornithine Wild type lass I lass II lass III MM + itrulline MM + rginine (control) 1

2 ONLSION ene ene B ene From the growth patterns of the mutants, Beadle and Tatum deduced that each mutant was unable to carry out one step in the pathway for synthesizing arginine, presumably because it lacked the necessary enzyme. Because each of their mutants was mutated in a single gene, they concluded that each mutated gene must normally dictate the production of one enzyme. Their results supported the one gene one enzyme hypothesis and also confirmed the arginine pathway. (Notice that a mutant can grow only if supplied with a compound made after the defective step.) Wild type nzyme nzyme B nzyme lass I (mutation in gene ) lass II (mutation in gene B) lass III (mutation in gene ) Precursor Precursor Precursor Precursor Ornithine Ornithine Ornithine Ornithine B B B itrulline itrulline itrulline itrulline rginine rginine rginine rginine Beadle and Tatum developed the one gene one enzyme hypothesis - which states that the function of a gene is to dictate the production of a specific enzyme The Products of ene xpression: Developing Story s researchers learned more about proteins - they made minor revision to the one gene one enzyme hypothesis genes code for polypeptide chains or for RN molecules Basic Principles of Transcription and Translation Transcription is the synthesis of RN under the direction of DN - producing messenger RN () Translation is the actual synthesis of a polypeptide, which occurs under the direction of it occurs on ribosomes In prokaryotes transcription and translation occur together In eukaryotes RN transcripts are modified before becoming true Nuclear envelope TRNSRIPTION TRNSLTION DN TRNSRIPTION RN PROSSIN DN Pre- ukaryotic cell. The nucleus provides a separate compartment for transcription. The original RN transcript, called pre-, is processed in various ways before leaving the nucleus as. Prokaryotic cell. In a cell lacking a nucleus, produced by transcription is immediately translated without additional processing. TRNSLTION

3 ells are governed by a cellular chain of commands DN --> RN --> protein The enetic ode enetic information is encoded as a sequence of non-overlapping base triplets, or codons odons: Triplets of Bases How many bases correspond to an amino acid? During transcription the gene determines the sequence of bases along the length of an molecule DN molecule DN strand (template) TRNSRIPTION TRNSLTION Protein ene 1 mino acid ene ene 3 T odon Trp Phe ly Ser racking the ode codon in messenger RN is either translated into an amino acid or serves as a translational stop signal odons must be read in the correct reading frame for the specified polypeptide to be produced First base (end) The genetic code is nearly universal shared by organisms from the simplest bacteria to the most complex animals Second base lle Met or start Phe Leu Leu Val Tyr ys Ser Trp Pro Thr la His ln sn Lys sp lu 64 options rg Ser rg ly Third base (end) In laboratory experiments genes can be transcribed and translated after being transplanted from one species to another Transcription is the DN-directed synthesis of RN: a closer look Molecular omponents of Transcription RN synthesis is catalyzed by RN polymerase, which pries the DN strands apart and hooks together the RN nucleotides follows the same base-pairing rules as DN, except that in RN, uracil substitutes for thymine Synthesis of an RN Transcript The stages of transcription are: Initiation, longation, Termination longation Newly made RN RN polymerase Non-template strand of DN T end T T T RN nucleotides Direction of transcription ( downstream ) Template strand of DN Promoter RN polymerase nwound DN Rewound RN RN transcript Transcription unit Start point DN Template strand of RN DN longation. The polymerase moves transcript downstream, unwinding the DN and elongating the RN transcript --> 3 In the wake of ompleted RN transcript 1 Initiation. fter RN polymerase binds to the promoter, the DN strands unwind, and the polymerase initiates RN synthesis at the start point on the template strand. transcription, the DN strands re-form a double helix. 3 Termination. ventually, the RN transcript is released, and the polymerase detaches from the DN. 3

4 TRNSRIPTION RN PROSSIN TRNSLTION RN Polymerase Binding and Initiation of Transcription Promoters signal the initiation of RN synthesis Transcription factors help eukaryotic RN polymerase recognize promoter sequences 1 DN longation of the RN Strand s RN polymerase moves along the DN, it continues to untwist the double helix, exposing about 10 to 0 DN bases at a time for pairing with RN nucleotides Termination of Transcription The mechanisms of termination are different in prokaryotes and eukaryotes Transcription factors RN polymerase II Pre- T T T T T T T Promoter TT box Start point Template DN strand ukaryotic promoters Transcription initiation complex 3 Several transcription factors dditional transcription factors Transcription factors RN transcript ukaryotic cells modify RN after transcription nzymes in the eukaryotic nucleus modify pre- in specific ways before the genetic messages are dispatched to the cytoplasm - 7-H 3- lteration of nds ach end of a pre- molecule is modified in a particular way the end receives a modified nucleotide cap the end gets a poly- tail TRNSRIPTION RN PROSSIN TRNSLTION DN Pre- modified guanine nucleotide added to the end P P P ap TR Protein-coding segment Start codon codon 50 to 50 adenine nucleotides added to the end Polyadenylation signal TR Poly- tail Split enes and RN Splicing RN splicing removes introns and joins exons TRNSRIPTION RN PROSSIN TRNSLTION DN Pre- xon Intron Pre- ap ap Is carried out by spliceosomes in some cases Ribozymes Ribozymes are catalytic RN molecules that function as enzymes and can splice RN xon oding segment Intron xon Poly- tail Introns cut out and exons spliced together Poly- tail TR TR 1 3 RN transcript (pre-) xon 1 Intron xon Protein snrn Other proteins snrnps Spliceosome Spliceosome components xon 1 xon ut-out intron The Functional and volutionary Importance of Introns The presence of introns allows for alternative RN splicing Proteins often have a modular architecture consisting of discrete structural and functional regions called domains In many cases different exons code for the different domains in a protein ene DN Domain xon 1 Intron xon Intron xon 3 Transcription RN processing Translation Domain 3 Domain 1 4

5 Translation: the basic concept Translation is the RNdirected synthesis of a polypeptide: a closer look Molecular omponents of Translation cell translates an message into protein with the help of transfer RN (trn) Molecules of trn are not all identical each carries a specific amino acid on one end each has an anticodon on the other end TRNSRIPTION TRNSLTION DN mino acids trn with amino acid attached ly trn nticodon odons (a) The Structure and Function of Transfer RN trn molecule consists of a single RN strand that is only about 80 nucleotides long is roughly L-shaped Two-dimensional structure. The four base-paired regions and three loops are characteristic of all trns, as is the base sequence of the amino acid attachment site at the end. The anticodon triplet is unique to each trn type. (The asterisks mark bases that have been chemically modified, a characteristic of trn.) nticodon (b) Three-dimensional structure mino acid attachment site Hydrogen bonds nticodon (c) Symbol used in this book mino acid attachment site nticodon Hydrogen bonds specific enzyme called an aminoacyl-trn synthetase joins each amino acid to the correct trn Phosphates 3 ppropriate trn covalently Bonds to amino cid, displacing MP. mino acid minoacyl-trn synthetase (enzyme) P P P denosine TP P denosine Pyrophosphate P Pi Pi Pi trn P denosine MP 4 ctivated amino acid is released by the enzyme. minoacyl trn (an activated amino acid ) 1 ctive site binds the amino acid and TP. TP loses two P groups and joins amino acid as MP. s facilitate the specific coupling of trn anticodons with codons during protein synthesis The ribosomal s are constructed of proteins and RN molecules named ribosomal RN or rrn TRNSRIPTION TRNSLTION trn molecules DN rowing polypeptide P xit tunnel Large Small omputer model of functioning ribosome. This is a model of a bacterial ribosome, showing its overall shape. The eukaryotic ribosome is roughly similar. ribosomal is an aggregate of ribosomal RN molecules and proteins. 5

6 TRNSRIPTION TRNSLTION The ribosome has three binding sites for trn: the P site, the site and the site P site (Peptidyl-tRN binding site) site (xit site) binding site P site (minoacyltrn binding site) Large Small Schematic model showing binding sites. ribosome has an binding site and three trn binding sites, known as the, P, and sites. This schematic ribosome will appear in later diagrams. mino end rowing polypeptide odons Next amino acid to be added to polypeptide chain trn Schematic model with and trn. trn fits into a binding site when its anticodon base-pairs with an codon. The P site holds the trn attached to the growing polypeptide. The site holds the trn carrying the next amino acid to be added to the polypeptide chain. Discharged trn leaves via the site. TM showing R and ribosomes ytosol ndoplasmic reticulum (R) Free ribosomes Bound ribosomes 80S 5S, 5.8S, 8S rrn 0.5 µm 18S rrn 60S Large 40S Small Diagram of a ribosome Building a We can divide translation into three stages: Initiation, longation and Termination ssociation and Initiation of Translation The initiation stage of translation brings together, trn bearing the first amino acid of the polypeptide, and two s of a ribosome Large 1 Initiator trn binding site Small ribosomal TP DP P site Start codon small ribosomal binds to a molecule of. In a prokaryotic cell, the binding site on this recognizes a specific nucleotide sequence on the just upstream of the start codon. n initiator trn, with the anticodon, base-pairs with the start codon,. This trn carries the amino acid methionine (Met). Translation initiation complex ribosomal The arrival of a large ribosomal completes the initiation complex. Proteins called initiation factors (not shown) are required to bring all the translation components together. TP provides the energy for the assembly. The initiator trn is in the P site; the site is available to the trn bearing the next amino acid. longation of the hain In the elongation stage of translation amino acids are added one by one to the preceding amino acid DN ready for next aminoacyl trn 3 Translocation. The ribosome translocates the trn in the site to the P site. The empty trn in the P site is moved to the site, where it is released. The moves along with its bound trns, bringing the next codon to be translated into the site. P mino end of polypeptide DP TP P site site P TP DP 1 odon recognition. The anticodon of an incoming aminoacyl trn base-pairs with the complementary codon in the site. Hydrolysis of TP increases the accuracy and efficiency of this step. P Peptide bond formation. n rrn molecule of the large catalyzes the formation of a peptide bond between the new amino acid in the site and the carboxyl end of the growing polypeptide in the P site. This step attaches the polypeptide to the trn in the site. 6

7 Termination of Translation The final stage of translation is termination when the ribosome reaches a stop codon in the Release factor Free polypeptide codon (,, or ) 1 When a ribosome reaches a stop The release factor hydrolyzes 3 The two ribosomal s codon on, the site of the the bond between the trn in and the other components of ribosome accepts a protein called the P site and the last amino the assembly dissociate. a release factor instead of trn. acid of the polypeptide chain. The polypeptide is thus freed from the ribosome. Polyribosomes number of ribosomes can translate a single molecule simultaneously - forming a polyribosome Incoming ribosomal s Start of ( end) rowing polypeptides nd of ( end) (a) n molecule is generally translated simultaneously by several ribosomes in clusters called polyribosomes. s ompleted polypeptide (b) This micrograph shows a large polyribosome in a prokaryotic cell (TM). 0.1 µm ompleting and Targeting the Functional Protein chains undergo modifications after the translation process Protein Folding and Post-Translational Modifications fter translation proteins may be modified (e.g. glycosylation) in ways that affect their three-dimensional shape (so called chaperone and co-chaperone proteins involved in folding (e.g. HSPs and FKBPs)). Targeting s to Specific Locations Folding, Post-Translational modifications and Targeting Proteins destined for the endomembrane system or for secretion: a) must be transported into the R; b) have signal peptides to which a signal-recognition particle (SRP) binds, enabling the translation ribosome to bind to the R The signal mechanism for targeting proteins to the R 1 n SRP binds 3 The SRP binds to a 4 The SRP leaves, and 5 The signalcleaving the completed 6 The rest of synthesis begins to the signal receptor protein in the R the polypeptide resumes on a free peptide, halting membrane. This receptor growing, meanwhile enzyme polypeptide leaves ribosome in synthesis is part of a protein complex translocating across the cuts off the the ribosome and the cytosol. momentarily. (a translocation complex) membrane. (The signal signal peptide. folds into its final that has a membrane pore peptide stays attached conformation. and a signal-cleaving enzyme. to the membrane.) Two populations of ribosomes are evident in cells - free and bound ribosomes (rough R). Free ribosomes in the cytosol initiate the synthesis of all proteins Signal peptide Signalrecognition particle (SRP) SRP receptor YTOSOL protein Signal peptide removed R membrane Protein RLMN Translocation complex 7

8 RN plays multiple roles in the cell: a review RN - can hydrogen-bond to other nucleic acid molecules - can assume a specific three-dimensional shape - has functional groups that allow it to act as a catalyst ncrn = non-coding RN Types of RN in a ukaryotic ell omparing gene expression in prokaryotes and eukaryotes reveals key differences Prokaryotic cells lack a nuclear envelope allowing translation to begin while transcription is still in progress RN polymerase RN polymerase Polyribosome (amino end) DN Polyribosome Direction of transcription ( end) 0.5 µm In a eukaryotic cell the nuclear envelope separates transcription from translation; extensive RN processing occurs in the nucleus DN Types of Point Mutations Point mutations within a gene can be divided into two general categories base-pair substitutions base-pair insertions or deletions base-pair substitution is the replacement of one nucleotide and its partner with another pair of nucleotides can cause missense or nonsense Wild type Protein Met Lys Phe ly mino end Base-pair substitution No effect on amino acid sequence Missense Nonsense arboxyl end Met Lys Phe ly instead of Met Lys Phe Ser Met instead of instead of Point mutations can affect protein structure and function Mutations are changes in the genetic material of a cell Point mutations are changes in just one base pair of a gene The change of a single nucleotide in the DN s template strand leads to the production of an abnormal protein Wild-type hemoglobin DN Mutant hemoglobin DN T T T Normal hemoglobin lu Sickle-cell hemoglobin Val In the DN, the mutant template strand has an where the wild-type template has a T. The mutant has a instead of an in one codon. The mutant (sickle-cell) hemoglobin has a valine (Val) instead of a glutamic acid (lu). 8

9 Insertions and Deletions Insertions and deletions are additions or losses of nucleotide pairs in a gene and may produce frameshift mutations (change of natural ORF (open reading frame)) Protein Wild type mino end Met Lys Phe ly Base-pair insertion or deletion Frameshift causing immediate nonsense xtra arboxyl end Mutagens Spontaneous mutations can occur during DN replication, recombination, or repair Mutagens are physical or chemical agents that can cause mutations Met Frameshift causing extensive missense Missing Met Lys Leu la Insertion or deletion of 3 nucleotides: no frameshift but extra or missing amino acid Missing Met Phe ly What is a gene? revisiting the question Overview of four basic molecular genetic processes gene is a region of DN whose final product is either a polypeptide or an RN molecule summary of transcription and translation in a eukaryotic cell TRNSRIPTION DN 1 RN is transcribed from a DN template. 3 5 RN RN transcript polymerase RN PROSSIN xon In eukaryotes, the RN transcript RN transcript (pre) is spliced and (pre-) Intron modified to produce, which moves minoacyl-trn from the nucleus to the synthetase cytoplasm. NLS mino FORMTION OF acid INITITION OMPLX MINO ID TIVTION YTOPLSM trn 3 fter leaving the 4 ach amino acid nucleus, attaches attaches to its proper trn to the ribosome. with the help of a specific enzyme and TP. rowing polypeptide ctivated amino acid Ribosomal s TRNSLTION 5 succession of trns add their amino acids to the polypeptide chain nticodon as the is moved through the ribosome one codon at a time. odon (When completed, the polypeptide is released from the ribosome.) 9