Concept 14.1: Genes specify proteins via. CH 14: Gene to Protein The Flow of Genetic Information. transcription and translation

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1 H 14: ene to Protein The Flow of enetic Information The information of genes is in the sequences of nucleotides in DN DN leads to specific traits by dictating the synthesis of proteins oncept 14.1: enes specify proteins via transcription and translation How was the fundamental relationship between genes and proteins discovered? ene expression, the process by which DN directs protein synthesis, includes two stages: transcription and translation vidence from the Study of Metabolic Defects Figure 14.2 rchibald arrod (1902)suggested that genes dictate phenotypes through enzymes that catalyze specific chemical reactions He thought symptoms of an inherited disease (alkaptonuria) reflect an inability to synthesize a certain enzyme Note: The term gene wasn t actually coined until 1909 eorge Beadle and dward Tatum (1940s) disabled genes in bread mold one by one and looked for phenotypic changes Used haploid bread mold because it would be easier to detect recessive mutations They studied mutations that altered the ability of the fungus to grow on minimal medium Neurospora cells 2 ells subjected to X-rays. 1 Individual Neurospora 3 cells placed on complete growth medium. ach surviving cell forms a colony of genetically identical cells. rowth No growth rowth 4 5 ontrol: Wild-type cells in minimal medium Surviving cells tested for inability to grow on minimal medium. Mutant cells placed in a series of vials, each containing minimal medium plus one additional nutrient. The researchers started a collection of Neurospora mutant strains, catalogued by their defects For example, one set of mutants all required arginine for growth Different classes of these mutants were blocked at a different step in the biochemical pathway for arginine biosynthesis Precursor ene nzyme ene B nzyme B ene nzyme Ornithine itrulline rginine The Products of ene xpression: Developing Story Beadle and Tatum s hypothesis, basically one gene = one protein. Some proteins are not enzymes, so researchers later revised the one gene one enzyme hypothesis: one gene one protein Many proteins are composed of several s, each of which has its own gene Beadle and Tatum s hypothesis is now restated as one gene one It is common to refer to gene products as proteins rather than s 1

2 Basic Principles of Transcription and Translation RN is the bridge between DN and protein synthesis RN is chemically similar to DN, but RN has a ribose sugar and the base uracil (U) rather than thymine (T) RN is usually single-stranded etting from DN to protein requires two stages: transcription and translation Transcription is the synthesis of RN using information in DN Transcription produces messenger RN () Translation is the synthesis of a, using information in the s are the sites of translation Figure 14.4 In prokaryotes, translation of can begin before transcription has finished In eukaryotes, the nuclear envelope separates transcription from translation ukaryotic RN transcripts are modified through RN processing to yield the finished ukaryotic must be transported out of the nucleus to be translated TRNSRIPTION TRNSLTION DN TRNSRIPTION RN PROSSIN TRNSLTION DN Nuclear envelope Pre- (a) Bacterial cell (b) ukaryotic cell The enetic ode primary transcript is the initial RN transcript from any gene prior to processing The central dogma is the concept that cells are governed by a cellular chain of command DN RN Protein Instructions for assembling amino acids into proteins from DN There are 20 amino acids, but there are only four nucleotide bases in DN The flow of information from gene to protein is based on a triplet code: a series of nonoverlapping, threenucleotide words The words of a gene are transcribed into complementary nonoverlapping three-nucleotide words of These words are then translated into a chain of amino acids, forming a 2

3 First base ( end of codon) Third base ( end of codon) Figure 14.5 DN template strand Protein TRNSRIPTION TRNSLTION T T T T T T U U U U U odon Trp Phe ly Ser During transcription, one of the two DN strands, called the template strand, provides a template for ordering the sequence of complementary nucleotides in an RN transcript The template strand is always the same strand for any given gene During translation, the base triplets, called codons, are read in the to direction codon codes for an amino acid (one of 20) along a mino acid racking the ode ll 64 codons were deciphered by the mid-1960s Of the 64 triplets, 61 code for amino acids; 3 triplets are stop signals to end translation The genetic code is redundant: more than one codon may specify a particular amino acid But it is not ambiguous: no codon specifies more than one amino acid odons must be read in the correct reading frame (correct groupings) in order for the correct to be produced Figure 14.6 Second base U UUU UU UU UU U Phe Tyr ys UU U U Ser U U UU U U U Leu UU U U U Trp UU U U U U His U Leu U Pro rg ln U UU U U U U sn Ser U IIe Thr U Lys rg U Met or start UU U U U U sp U Val U la ly lu U volution of the enetic ode Figure 14.UN02 The genetic code is nearly universal, shared by the simplest bacteria and the most complex animals enes can be transcribed and translated after being transplanted from one species to another TRNSRIPTION RN PROSSIN DN Pre- TRNSLTION (a) Tobacco plant expressing a firefly gene (b) Pig expressing a jellyfish gene 3

4 Molecular omponents of Transcription Transcription is the first stage of gene expression RN synthesis is catalyzed by RN polymerase, joins together the RN nucleotides RN polymerases assemble polynucleotides in the to direction RN polymerases can start a chain without a primer The DN sequence where RN polymerase attaches is called the promoter; in bacteria, the sequence signaling the end of transcription is called the terminator The stretch of DN that is transcribed is called a transcription unit Synthesis of an RN Transcript The three stages of transcription Initiation longation Termination Figure Initiation 2 longation 3 Termination Promoter Transcription unit Start point RN polymerase Unwound DN Rewound DN RN transcript RN Template strand of DN transcript ompleted RN transcript Direction of transcription ( downstream ) RN Polymerase Binding and Initiation of Transcription Promoters usually extend several dozen nucleotide pairs upstream of the start point Transcription factors mediate the binding of RN polymerase and the initiation of transcription The completed assembly of transcription factors and RN polymerase II bound to a promoter is called a transcription initiation complex promoter called a TT box is crucial in forming the initiation complex in eukaryotes Figure 14.9 DN T T T T T T T TT box RN polymerase II Promoter Transcription factors Start point Nontemplate strand Template strand Transcription factors eukaryotic promoter Several transcription factors bind to DN. Transcription initiation complex forms. longation of the RN Strand s RN polymerase moves along the DN, it untwists the double helix, 10 to 20 bases at a time Transcription progresses at a rate of 40 nucleotides per second in eukaryotes gene can be transcribed simultaneously by several RN polymerases RN transcript Transcription initiation complex 4

5 Figure Newly made RN RN polymerase T U Nontemplate strand of DN end T T T Direction of transcription RN nucleotides Template strand of DN Termination of Transcription The mechanisms of termination are different in bacteria and eukaryotes In bacteria, the polymerase stops transcription at the end of the terminator and the can be translated without further modification In eukaryotes, RN polymerase II transcribes the polyadenylation (U) signal sequence; the RN transcript is released nucleotides past this sequence ukaryotic cells modify RN after transcription nzymes in the eukaryotic nucleus modify pre- (RN processing) before the genetic messages go to the cytoplasm TRNSRIPTION RN PROSSIN TRNSLTION DN Pre- lteration of nds ach end of a pre- molecule is modified The end receives a modified nucleotide cap The end gets a poly- tail These modifications share several functions Facilitating the export of to the cytoplasm Protecting from hydrolytic enzymes Helping ribosomes attach to the end Poly tail length varies , s Figure Split enes and RN Splicing modified guanine nucleotide added to the end P Protein-coding segment adenine nucleotides added to the end Polyadenylation signal P P U Start ap UTR UTR Poly- tail codon codon Most eukaryotic s have long noncoding stretches of nucleotides that lie between coding regions The noncoding regions are called intervening sequences, or introns The other regions are called exons and are usually translated into amino acid sequences RN splicing removes introns and joins exons, creating an molecule with a continuous coding sequence 5

6 Figure Pre- Intron ap Intron Introns cut out and exons spliced together Poly- tail Many genes can give rise to two or more different s, depending on which segments are used as exons This process is called alternative RN splicing RN splicing is carried out by spliceosomes ap UTR oding segment Poly- tail UTR Spliceosomes consist of proteins and small RNs Ribozymes can cut on own U Figure Spliceosome Small RNs Ribozymes Pre- xon 1 xon 2 Ribozymes are RN molecules that function as enzymes RN splicing can occur without proteins, or even additional RN molecules Intron The introns can catalyze their own splicing Spliceosome components xon 1 xon 2 ut-out intron oncept 14.4: Translation is the RN-directed synthesis of a : a closer look Figure 14.UN04 enetic information flows from to protein through the process of translation cell translates an message into protein with the help of transfer RN (trn) trns transfer amino acids to the growing in a ribosome TRNSRIPTION TRNSLTION DN 6

7 Figure The Structure and Function of Transfer RN U odons U U U mino acids trn with amino acid attached ly trn nticodon ach trn can translate a particular codon into a given amino acid The trn contains an amino acid at one end and at the other end has a nucleotide triplet that can basepair with the complementary codon on trn molecule consists of a single RN strand that is only about 80 nucleotides long trn molecules can base-pair with themselves One end of a trn contains the anticodon that base-pairs with an codon Figure mino acid attachment site U U U U U * U * U U * * * U * * U * * * U nticodon U Hydrogen bonds (a) Two-dimensional structure * * Hydrogen bonds nticodon (b) Three-dimensional structure mino acid attachment site nticodon (c) Symbol used in this book ccurate translation requires two steps First: a correct match between a trn and an amino acid, done by the enzyme aminoacyl-trn synthetase Second: a correct match between the trn anticodon and an codon Flexible pairing at the third base of a codon is called wobble and allows some trns to bind to more than one codon Figure mino acid and trn enter active site. Tyr-tRN Tyrosine (Tyr) (amino acid) Tyrosyl-tRN synthetase s s facilitate specific coupling of trn anticodons with codons during protein synthesis U TP The large and small ribosomal are made of proteins and ribosomal RNs (rrns) omplementary trn anticodon 3 minoacyl trn released. MP 2 P i 2 Using TP, synthetase catalyzes covalent bonding. In bacterial and eukaryotic ribosomes the large and small s join to form a ribosome only when attached to an molecule 7

8 Figure rowing trn molecules P xit tunnel Large Figure 14.17a rowing trn molecules xit tunnel P site (Peptidyl-tRN binding site) site (xit site) binding site P Small (a) omputer model of functioning ribosome xit tunnel Large Small (b) Schematic model showing binding sites site (minoacyltrn binding site) mino end rowing odons Next amino acid to be added to chain trn (c) Schematic model with and trn P Large Small (a) omputer model of functioning ribosome Figure 14.17b Figure 14.17c P site (Peptidyl-tRN binding site) site (xit site) binding site xit tunnel P Large Small (b) Schematic model showing binding sites site (minoacyltrn binding site) mino end rowing odons Next amino acid to be added to chain trn (c) Schematic model with and trn Building a ribosome has three binding sites for trn The site holds the trn that carries the next amino acid to be added to the chain The P site holds the trn that carries the growing chain The site is the exit site, where discharged trns leave the ribosome The three stages of translation Initiation longation Termination ll three stages require protein factors that aid in the translation process 8

9 ssociation and Initiation of Translation The initiation stage of translation brings together, a trn with the first amino acid, and the two ribosomal s small ribosomal binds with and a special initiator trn Then the small moves along the until it reaches the start codon (U) Figure Initiator trn U U TP P i DP P site Large ribosomal Start codon Small binding site ribosomal Translation initiation complex 1 Small ribosomal binds to. 2 Large ribosomal completes the initiation complex. longation of the hain The start codon establishes the reading frame for the The addition of the large ribosomal is last and completes the formation of the translation initiation complex Proteins called initiation factors bring all these components together During elongation, amino acids are added one by one to the previous amino acid dded at -terminus of the growing chain ach addition involves proteins called elongation factors and occurs in three steps: codon recognition, peptide bond formation, and translocation Translation proceeds along the in a to direction Figure mino end of 1 odon recognition Figure mino end of 1 odon recognition P site site TP P site site TP DP P i DP P i P P 2 Peptide bond formation P 9

10 Figure mino end of 1 odon recognition Termination of Translation ready for next aminoacyl trn P site site TP Termination occurs when a stop codon in the reaches the site of the ribosome DP P i The site accepts a protein called a release factor The release factor causes the addition of a water molecule instead of an amino acid P 3 Translocation DP P i TP P 2 Peptide bond formation This reaction releases the, and the translation assembly then comes apart P Figure Figure Release factor Release factor Free 1 codon (U, U, or U) reaches a stop codon on. 1 codon (U, U, or U) reaches a stop codon on. 2 Release factor promotes hydrolysis. Figure ompleting and Targeting the Functional Protein 1 Release factor codon (U, U, or U) reaches a stop codon on. Free 2 TP 2 DP P i 2 Release factor 3 promotes hydrolysis. Ribosomal s and other components dissociate. chains are modified after translation or targeted to specific sites in the cell During synthesis, a chain spontaneously coils and folds into its three-dimensional shape Proteins may also require post-translational modifications before doing their jobs 10

11 Targeting s to Specific Locations Two populations of ribosomes are are in cells: free ribosomes (in the cytosol) bound ribosomes (attached to the R) Free ribosomes mostly synthesize proteins that function in the cytosol Bound ribosomes make proteins of the endomembrane system and proteins that are secreted from the cell synthesis always begins in the cytosol Synthesis finishes in the cytosol unless the signals the ribosome to attach to the R s destined for the R or for secretion are marked by a signal peptide signal-recognition particle (SRP) binds to the signal peptide The SRP brings the signal peptide and its ribosome to the R Figure synthesis begins. SRP YTOSOL Signal peptide R LUMM SRP binds to signal peptide. SRP binds to receptor protein. SRP receptor protein Translocation complex SRP detaches and synthesis resumes. Signalcleaving enzyme cuts off signal peptide. Signal peptide removed ompleted folds into final conformation. R membrane Protein Making Multiple s in Bacteria and ukaryotes Multiple ribosomes translate an at the same time Once a ribosome is far enough past the start codon, another ribosome can attach to the Strings of ribosomes called polyribosomes (or polysomes) can be seen with an electron microscope Figure rowing s ompleted Incoming ribosomal s Start of ( end) nd of ( end) (a) Several ribosomes simultaneously translating one molecule s multiple s can be transcribed from the same gene In bacteria, the transcription and translation can take place simultaneously In eukaryotes, the nuclear envelope separates transcription and translation (b) large polyribosome in a bacterial cell (TM) 0.1 m 11

12 Figure RN polymerase Figure TRNSRIPTION DN RN polymerase DN Polyribosome Direction of transcription 0.25 m DN RN transcript RN PROSSIN YTOPLSM RN polymerase xon RN transcript (pre-) Intron minoacyl-trn synthetase NULUS mino acid MINO ID trn TIVTION Polyribosome (amino end) P Ribosomal s minoacyl (charged) trn TRNSLTION U U U U U nticodon ( end) odon oncept 14.5: Mutations of one or a few nucleotides can affect protein structure and function Mutations are changes in the genetic material of a cell or virus Point mutations are chemical changes in just one or a few nucleotide pairs of a gene The change of a single nucleotide in a DN template strand can lead to the production of an abnormal protein Figure Wild-type hemoglobin Wild-type hemoglobin DN T Normal hemoglobin lu Sickle-cell hemoglobin Mutant hemoglobin DN T U Sickle-cell hemoglobin Val Types of Small-Scale Mutations Point mutations within a gene can be divided into two general categories Nucleotide-pair substitutions One or more nucleotide-pair insertions or deletions Figure DN template strand Protein mino end (a) Nucleotide-pair substitution Wild type T T T T T T T T T T U U U U U Met Lys Phe ly arboxyl end (b) Nucleotide-pair insertion or deletion instead of xtra T T T T T T T T T T T T T T T T T T T T T T U instead of xtra U U U U U U U U U U U U U U Met Lys Phe ly Met Silent (no effect on amino acid sequence) Frameshift causing immediate nonsense (1 nucleotide-pair insertion) T instead of missing T T T T T T T T T T T T T T T T T T T T instead of U missing U U U U U U U U U Met Lys Phe Ser Met Lys Leu la Missense Frameshift causing extensive missense (1 nucleotide-pair deletion) instead of T T T missing T T T T T T T T T T T T T T T T T T U instead of missing U U U U U U U U U U U U Met Met Phe ly Nonsense No frameshift, but one amino acid missing (3 nucleotide-pair deletion) 12

13 Substitutions nucleotide-pair substitution replaces one nucleotide and its partner with another pair of nucleotides Silent mutations have no effect on the amino acid produced by a codon because of redundancy in the genetic code Figure 14.26a Wild type DN template strand T T T T T T T T T T U U U U U Protein Met Lys Phe ly mino end arboxyl end Nucleotide-pair substitution: silent instead of T T T T T T T T T T T U instead of U U U U U U Met Lys Phe ly Figure 14.26b Missense mutations still code for an amino acid, but not the correct amino acid Substitution mutations are usually missense mutations Nonsense mutations change an amino acid codon into a stop codon, nearly always leading to a nonfunctional protein Wild type DN template strand T T T T T T T T T T U U U U U Protein Met Lys Phe ly mino end arboxyl end Nucleotide-pair substitution: missense T instead of T T T T T T T T T T T instead of U U U U U Met Lys Phe Ser Figure 14.26c Wild type DN template strand T T T T T T T T T T U U U U U Protein Met Lys Phe ly mino end arboxyl end Nucleotide-pair substitution: nonsense instead of T T T T T T T T T T T U instead of U U U U U U Met Insertions and Deletions Insertions and deletions are additions or losses of nucleotide pairs in a gene These mutations have a disastrous effect on the resulting protein more often than substitutions do Insertion or deletion of nucleotides may alter the reading frame of the genetic message, producing a frameshift mutation 13

14 Figure 14.26d Figure 14.26e Wild type DN template strand T T T T T T T T T T U U U U U Protein Met Lys Phe ly mino end arboxyl end Nucleotide-pair insertion: frameshift causing immediate nonsense xtra T T T T T T T T T T T xtra U U U U U U U Met Wild type DN template strand T T T T T T T T T T U U U U U Protein Met Lys Phe ly mino end arboxyl end Nucleotide-pair deletion: frameshift causing extensive missense missing T T T T T T T T T U missing U U U U Met Lys Leu la Figure 14.26f Wild type DN template strand T T T T T T T T T T U U U U U Protein Met Lys Phe ly mino end arboxyl end 3 nucleotide-pair deletion: no frameshift, but one amino acid missing T T missing T T T T T T T T missing U U U U U Met Phe ly Mutagens Spontaneous mutations can occur during DN replication, recombination, or repair Mutagens are physical or chemical agents that can cause mutations Most cancer-causing chemicals (carcinogens) are mutagenic, and the converse is also true What Is a ene? Revisiting the Question The definition of a gene has evolved through the history of genetics We have considered a gene as discrete unit of inheritance region of specific nucleotide sequence in a chromosome DN sequence that codes for a specific chain gene can be defined as a region of DN that can be expressed to produce a final functional product, either a or an RN molecule 14