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

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1 ourse No: BN2003 redits: 3.00 eneral Biology 10. enetics: From enes to roteins 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 rof. 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 XRIMNT 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 ) recursor recursor recursor recursor 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 roducts 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 rinciples of Transcription and Translation Transcription is the synthesis of RN under the direction of DN - producing messenger RN (mrn) Translation is the actual synthesis of a polypeptide, which occurs under the direction of mrn it occurs on ribosomes In prokaryotes transcription and translation occur together In eukaryotes RN transcripts are modified before becoming true mrn Nuclear envelope TRNSRITION TRNSLTION DN mrn TRNSRITION RN ROSSIN mrn DN re-mrn ukaryotic cell. The nucleus provides a separate compartment for transcription. The original RN transcript, called pre-mrn, is processed in various ways before leaving the nucleus as mrn. rokaryotic cell. In a cell lacking a nucleus, mrn produced by transcription is immediately translated without additional processing. TRNSLTION 2

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 mrn molecule DN molecule DN strand (template) TRNSRITION mrn TRNSLTION rotein ene 1 mino acid ene 2 ene 3 T odon Trp he 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 The genetic code is nearly universal shared by organisms from the simplest bacteria to the most complex animals First mrn base (end) Second mrn base lle Met or start he Leu Leu Val Tyr ys Ser Trp ro Thr la His ln sn Lys sp lu 64 options rg Ser rg ly Third mrn 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 RN polymerase T Newly made RN Non-template strand of DN RN nucleotides T end T T T T Direction of transcription ( downstream ) T Template strand of DN romoter RN polymerase RN RN transcript Transcription unit DN Start point Template strand of nwound RN DN longation. The polymerase moves DN transcript downstream, unwinding the 2 DN and elongating the RN transcript --> 3 In the wake of transcription, the DN strands re-form a Rewound double helix. 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. 3 Termination. ventually, the RN transcript is released, and the polymerase detaches from the DN. 3

4 RN olymerase Binding and Initiation of Transcription romoters signal the initiation of RN synthesis Transcription factors help eukaryotic RN polymerase recognize promoter sequences TRNSRITION DN 1 longation of the RN Strand s RN polymerase moves along the DN, it continues to untwist the double helix, exposing about 10 to 20 DN bases at a time for pairing with RN nucleotides Termination of Transcription The mechanisms of termination are different in prokaryotes and eukaryotes RN ROSSIN TRNSLTION mrn re-mrn T T Transcription factors RN polymerase II T T T T T romoter TT box Start point Template DN strand ukaryotic promoters Transcription initiation complex 3 2 Several transcription factors dditional transcription factors Transcription factors RN transcript ukaryotic cells modify RN after transcription nzymes in the eukaryotic nucleus modify pre-mrn in specific ways before the genetic messages are dispatched to the cytoplasm - 7-H 3 - lteration of mrn nds ach end of a pre-mrn molecule is modified in a particular way the end receives a modified nucleotide cap the end gets a poly- tail TRNSRITION RN ROSSIN TRNSLTION mrn DN re-mrn modified guanine nucleotide added to the end ap TR rotein-coding segment Start codon codon 50 to 250 adenine nucleotides added to the end olyadenylation signal TR oly- tail Split enes and RN Splicing RN splicing removes introns and joins exons TRNSRITION RN ROSSIN TRNSLTION mrn DN re-mrn xon Intron re-mrn ap mrn 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 oly- tail Introns cut out and exons spliced together oly- tail TR TR RN transcript (pre-mrn) xon 1 Intron xon 2 rotein snrn Other proteins snrns Spliceosome Spliceosome components mrn xon 1 xon 2 ut-out intron The Functional and volutionary Importance of Introns The presence of introns allows for alternative RN splicing roteins 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 2 xon 1 Intron xon 2 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 mrn 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 TRNSRITION TRNSLTION DN mrn mrn Trp he 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 mino acid attachment site 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.) mino acid nticodon (b) Three-dimensional structure attachment site Hydrogen bonds nticodon (c) Symbol used in this book nticodon Hydrogen bonds specific enzyme called an aminoacyl-trn synthetase joins each amino acid to the correct trn mino acid T yrophosphate 3 ppropriate trn covalently Bonds to amino cid, displacing M. denosine i hosphates 4 ctivated amino acid is released by the enzyme. i i trn denosine M minoacyl trn (an activated amino acid ) minoacyl-trn synthetase (enzyme) denosine 1 ctive site binds the amino acid and T. 2 T loses two groups and joins amino acid as M. s facilitate the specific coupling of trn anticodons with mrn codons during protein synthesis The ribosomal s are constructed of proteins and RN molecules named ribosomal RN or rrn TRNSRITION TRNSLTION trn molecules DN mrn rowing polypeptide mrn 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 The ribosome has three binding sites for trn: the site, the site and the site site (eptidyl-trn binding site) site (xit site) mrn binding site site (minoacyltrn binding site) Large Small Schematic model showing binding sites. ribosome has an mrn binding site and three trn binding sites, known as the,, and sites. This schematic ribosome will appear in later diagrams. mrn mino end rowing polypeptide odons Next amino acid to be added to polypeptide chain trn Schematic model with mrn and trn. trn fits into a binding site when its anticodon base-pairs with an mrn codon. The 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, 28S 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 mrn, trn bearing the first amino acid of the polypeptide, and two s of a ribosome Large Initiator trn mrn mrn binding site Met Small ribosomal T D site Start codon 1 small ribosomal binds to a molecule of mrn. In a prokaryotic cell, the mrn binding site on this recognizes a specific nucleotide sequence on the mrn 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). Met Translation initiation complex ribosomal 2 The arrival of a large ribosomal completes the initiation complex. roteins called initiation factors (not shown) are required to bring all the translation components together. T provides the energy for the assembly. The initiator trn is in the 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 TRNSRITION TRNSLTION DN mrn ready for next aminoacyl trn 3 Translocation. The ribosome translocates the trn in the site to the site. The empty trn in the site is moved to the site, where it is released. The mrn moves along with its bound trns, bringing the next codon to be translated into the site. mino end of polypeptide mrn D T site site 2 T 2 D 1 odon recognition. The anticodon of an incoming aminoacyl trn base-pairs with the complementary mrn codon in the site. Hydrolysis of T increases the accuracy and efficiency of this step. 2 eptide 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 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 mrn Release factor Free polypeptide codon (,, or ) 1 When a ribosome reaches a stop 2 The release factor hydrolyzes 3 The two ribosomal s codon on mrn, the site of the the bond between the trn in and the other components of ribosome accepts a protein called the 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. olyribosomes number of ribosomes can translate a single mrn molecule simultaneously - forming a polyribosome Incoming ribosomal s Start of mrn ( end) rowing polypeptides olyribosome nd of mrn ( end) (a) n mrn molecule is generally translated simultaneously by several ribosomes in clusters called polyribosomes. s mrn ompleted polypeptide 0.1 µm (b) This micrograph shows a large polyribosome in a prokaryotic cell (TM). ompleting and Targeting the Functional rotein chains undergo modifications after the translation process rotein Folding and ost-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. HSs and FKBs)). Targeting s to Specific Locations Folding, ost-translational modifications and Targeting roteins 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 (SR) binds, enabling the translation ribosome to bind to the R The signal mechanism for targeting proteins to the R 1 2 n SR binds 3 The SR binds to a 4 The SR leaves, and 5 The signalcleaving 6 The rest of synthesis begins on a free ribosome in the cytosol. to the signal peptide, halting synthesis momentarily. receptor protein in the R membrane. This receptor is part of a protein complex (a translocation complex) that has a membrane pore and a signal-cleaving enzyme. the polypeptide resumes growing, meanwhile translocating across the membrane. (The signal peptide stays attached to the membrane.) enzyme cuts off the signal peptide. the completed polypeptide leaves the ribosome and folds into its final conformation. 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 Signalrecognition particle (SR) YTOSOL mrn Signal peptide SR receptor protein Signal peptide removed R membrane rotein 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 rokaryotic cells lack a nuclear envelope allowing translation to begin while transcription is still in progress RN polymerase RN polymerase olyribosome (amino end) DN olyribosome Direction of transcription mrn ( end) mrn 0.25 µm In a eukaryotic cell the nuclear envelope separates transcription from translation; extensive RN processing occurs in the nucleus DN Types of oint Mutations oint 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 mrn rotein Met Lys he ly mino end Base-pair substitution arboxyl end No effect on amino acid sequence instead of Missense Met Lys he ly Met Lys he Ser Nonsense instead of Met instead of oint mutations can affect protein structure and function Mutations are changes in the genetic material of a cell oint 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 mrn T T T Normal hemoglobin mrn lu Sickle-cell hemoglobin Val In the DN, the mutant template strand has an where the wild-type template has a T. The mutant mrn 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)) mrn rotein Wild type Met Lys he ly mino end Base-pair insertion or deletion Frameshift causing immediate nonsense arboxyl end Met Frameshift causing extensive missense xtra Met Lys Leu la Missing Mutagens Spontaneous mutations can occur during DN replication, recombination, or repair Mutagens are physical or chemical agents that can cause mutations Insertion or deletion of 3 nucleotides: no frameshift but extra or missing amino acid Missing Met he 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 TRNSRITION DN 1 RN is transcribed from a DN template. 3 5 RN RN transcript polymerase RN ROSSIN xon 2 In eukaryotes, the RN transcript RN transcript (premrn) is spliced and (pre-mrn) Intron modified to produce mrn, which moves minoacyl-trn from the nucleus to the synthetase cytoplasm. NLS mino FORMTION OF acid INITITION OMLX MINO ID TIVTION trn YTOLSM 3 fter leaving the 4 ach amino acid nucleus, mrn attaches attaches to its proper trn to the ribosome. with the help of a specific enzyme and T. mrn rowing polypeptide ctivated amino acid ap oly- ap oly- Ribosomal s TRNSLTION odon 5 succession of trns add their amino acids to nticodon the polypeptide chain as the mrn is moved through the ribosome one codon at a time. (When completed, the polypeptide is released from the ribosome.) oly- 9