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CAMPBELL BIOLOGY TENTH EDITION Reece • Urry • Cain • Wasserman • Minorsky • Jackson 17 Gene Expression: From Gene to Protein Lecture Presentation by Nicole Tunbridge and Kathleen Fitzpatrick © 2014 Pearson Education, Inc. Overview: The Flow of Genetic Information • The information in DNA is in the form of specific sequences of nucleotides • The DNA inherited by an organism leads to specific traits by dictating the synthesis of proteins • Proteins are the links between genotype and phenotype • Gene expression, the process by which DNA directs protein synthesis, includes two stages: transcription and translation © 2014 Pearson Education, Inc. How does a single faulty gene result in the dramatic appearance of an albino deer or racoon ? Gene Expression: From Gene to Protein Figure 17.1 Evidence from the Study of Metabolic Defects • In 1902, British physician Archibald Garrod first suggested that genes dictate phenotypes through enzymes that catalyze specific chemical reactions • He thought symptoms of an inherited disease reflect an inability to synthesize a certain enzyme • Linking genes to enzymes required understanding that cells synthesize and degrade molecules in a series of steps, a metabolic pathway © 2014 Pearson Education, Inc. Genes specify proteins via transcription and translation • How was the fundamental relationship between genes and proteins discovered? • The study of metabolic defects provided evidence that genes specify proteins. • Scientific experiments suggested that the symptoms of an inherited disease reflect a person’s inability to synthesize a particular enzyme. – Such diseases are referred to as “inborn errors of metabolism.” • Several studies provided strong evidence for the one gene–one enzyme hypothesis. • It was found that not all proteins are enzymes. • This tweaked the hypothesis to one gene–one protein. © 2014 Pearson Education, Inc. Nutritional Mutants in Neurospora: Scientific Inquiry • George Beadle and Edward Tatum exposed bread mold to X-rays, creating mutants that were unable to survive on minimal medium as a result of inability to synthesize certain molecules • Using crosses, they identified three classes of arginine-deficient mutants, each lacking a different enzyme necessary for synthesizing arginine • They developed a one gene–one enzyme hypothesis, which states that each gene dictates production of a specific enzyme © 2014 Pearson Education, Inc. Figure 17.2 Precursor Classes of Neurospora crassa Results Table Enzyme A Wild type Class I mutants Class II mutants Class III mutants Can grow with or without any supplements Can grow on ornithine, citrulline, or arginine Can grow only on citrulline or arginine Require arginine to grow Class II mutants (mutation in gene B) Class III mutants (mutation in gene C) Minimal medium (MM) (control) Ornithine Enzyme B Citrulline Enzyme C MM + ornithine Condition Arginine Growth: Wild-type cells growing and dividing No growth: Mutant cells cannot grow and divide Control: Minimal medium MM + citrulline MM + arginine (control) Summary of results Gene (codes for enzyme) Wild type Precursor Gene A Enzyme A Ornithine Gene B Enzyme B Citrulline Gene C Enzyme C Arginine Class I mutants (mutation in gene A) Precursor Enzyme A Ornithine Enzyme B Citrulline Enzyme C Arginine Precursor Enzyme A Ornithine Enzyme B Citrulline Enzyme C Arginine Precursor Enzyme A Ornithine Enzyme B Citrulline Enzyme C Arginine The Products of Gene Expression: A Developing Story • Some proteins aren’t enzymes, so researchers later revised the hypothesis: one gene–one protein • Many proteins are composed of several polypeptides, each of which has its own gene • Therefore, Beadle and Tatum’s hypothesis is now restated as the one gene–one polypeptide hypothesis • Note that it is common to refer to gene products as proteins rather than polypeptides © 2014 Pearson Education, Inc. The language of DNA-gene produce proteins DNA and RNA, are polymers made from four different nucleotides- A, T/U, G and C arranged in specific orders. Genes are made by thousands of nucleotides long and each gene has a specific sequence of nucleotide bases. Genes provide the instructions for making specific proteins. The bridge between DNA and protein synthesis is the RNA. Proteins are made from specific orders and types of twenty amino acids. Two processes ,Transcription and translation are the two main processes linking gene to protein. Transcription DNA → mRNA Translation → Protein Basic Principles of Transcription and Translation • RNA is the intermediate between genes and the proteins for which they code • Transcription is the synthesis of RNA under the direction of DNA. Transcription produces messenger RNA (mRNA) • Translation is the synthesis of a polypeptide, which occurs under the direction of mRNA • Ribosomes are the sites of translation • Proteins are made from 2 or more polypeptide chains and each polypeptide is specified by its own gene. © 2014 Pearson Education, Inc. Transcription is synthesis of mRNA from DNA • To get from DNA, written in one chemical language (nucleotides), to protein, written in another (amino acids), requires two major stages: transcription and translation. • During transcription, a DNA strand provides a template for the synthesis of a complementary RNA strand. The information is transcribed, or copied from DNA to mRNA. – Just as a DNA strand provides a template for the synthesis of each new complementary strand during DNA replication, it provides a template for assembling a sequence of RNA nucleotides. © 2014 Pearson Education, Inc. Translation is the synthesis of a polypeptide • mRNA carries the genetic message from the DNA to the protein-synthesizing machinery of the cell. • Translation is the synthesis of a polypeptide (via mRNA). – During translation there is a change in language: The cell must translate the nucleotide sequence of mRNA molecules into amino acid sequences of proteins. • Ribosomes are the sites of translation Transcription and translation occur in prokaryotes • A bacterial cell does not have a nucleus, so the mRNA produced by transcription is immediately translated into protein by the ribosomes. – Ribosomes attach to the leading end of an mRNA molecule while transcription is still in progress. © 2014 Pearson Education, Inc. Figure 17.3 Transcription and translation occur in eukaryotes Figure 17.3 • The nucleus of a eukaryotic cell provides a separate compartment for transcription, yielding premRNA, which is modified in the nucleus making mRNA through RNA processing to yield finished mRNA. • mRNA is then exported out of the nucleus translation occurs at ribosomes in the cytoplasm, producing polypeptides. © 2014 Pearson Education, Inc. Fig. 17.3 p329 • A primary transcript is the initial RNA transcript from any gene • The central dogma is the concept that cells are governed by a cellular chain of command: DNA © 2014 Pearson Education, Inc. RNA Protein The Genetic Code • How do the nucleotide bases of DNA provide the instructions for making proteins that contain specific amino acids? • There are 20 amino acids, but there are only four nucleotide bases in DNA: C,G,A, and T • How many nucleotide bases = an amino acid? • How do nucleotide bases result in the production of specific amino acids? © 2014 Pearson Education, Inc. Genetic codon is a three nucleotide sequence of DNA→RNA that produces an amino acid • The flow of information from gene to protein is based on a triplet code: a series of nonoverlapping , 3 consecutive nucleotide bases. • The genetic instructions for a polypeptide chain are written in the DNA as a series of non-overlapping 3 nucleotide “words”. • These triplets are the smallest units of uniform length that can code for all the amino acids. • Example: AGT at a particular position on a DNA strand results in the transcription of AGU mRNA. An AGU sequence in mRNA results in the placement of the amino acid serine at the correct position of the polypeptide that is produced at the ribosome. © 2014 Pearson Education, Inc. The Triplet Code Figure 17.4 • During transcription, one of the two DNA strands called the template strand provides a template for ordering the sequence of nucleotides for transcription of mRNA. The base-pairing rules for DNA replication also govern transcription (but U is used instead of T) of RNA. • During translation, the mRNA is read as a sequence of base triplets (codons). • Each codon specifies a particular amino acid to be added in the growing polypeptide chain at the ribosome. © 2014 Pearson Education, Inc. Codons are read in the 5′3′ direction along the mRNA • During translation, mRNA base triplets, called the codons read in the 5′3′ direction along the mRNA molecule is translated into a sequence of amino acids making up the polypeptide chain. • Each codon specifies which one of the 20 amino acids will be incorporated at the corresponding position along a polypeptide. • Because codons are base triplets, the number of nucleotides making up a genetic message must be three times the number of amino acids making up the protein product. • 300 nucleotides makes 100 triplet codon which translate into 100 amino acids long protein. © 2014 Pearson Education, Inc. Cracking the Code • There are 64 codons – Of the 64 triplets, 61 code for amino acids – 3 triplets are UAA, UGA and UAG“stop” signals to end translation. – AUG =methionine or “start” signal • The genetic code is redundant but no codon specifies more than one amino acid © 2014 Pearson Education, Inc. Figure 17.5 Reading Frame • Processing the message from the genetic code requires specifying the correct starting point. • The starting point establishes the reading frame; subsequent codons are read in groups of three nucleotides. • The cell’s protein-synthesizing machinery reads the message as a series of nonoverlapping three-letter words (codons) each of which is translated into a specific amino acid during protein synthesis.. © 2014 Pearson Education, Inc. The genetic code must have evolved very early in the history of life • The genetic code is nearly universal, shared by organisms from the simplest bacteria to the most complex plants and animals. • Genes can be transcribed and translated after being transplanted from one species to another. • The evolutionary significance of the near universality of the genetic code is clear: A language shared by all living things arose very early in the history of life—early enough to be present in the common ancestors of all modern organisms. © 2014 Pearson Education, Inc. Genes can be transcribed and translated after being transplanted from one species to another (a) Tobacco plant expressing a firefly gene Figure 17-6 (b) Pig expressing a jellyfish gene Transcription is the DNA-directed synthesis of RNA • Transcription is the first stage of gene expression • Messenger RNA is the carrier of information from DNA to the cell’s protein-synthesizing machinery (ribosomes). It is transcribed from the template strand of a gene. • RNA synthesis is catalyzed by RNA polymerase, which seprate the DNA strands apart and hooks together the RNA nucleotides. • RNA synthesis follows the same base-pairing rules as DNA, except uracil substitutes for thymine. • Like DNA polymerases, RNA polymerases can assemble a polynucleotide only in a 5′3′ direction. • Unlike DNA polymerases, RNA polymerases are able to start a chain from scratch; they don’t need a primer. © 2014 Pearson Education, Inc. RNA polymerase attaches and initiates transcription at the promoter • Specific sequences of nucleotides along the DNA mark where gene transcription begins and ends. • RNA polymerase attaches and initiates transcription at the promoter. • The sequence that signals the end of transcription is called the terminator. • Molecular biologists refer to the direction of transcription as “downstream” and the other direction as “upstream.” • The terms downstream and upstream are also used to describe the positions of nucleotide sequences within the DNA or RNA. • The promoter sequence in DNA is upstream from the terminator. • The stretch of DNA that is transcribed into an RNA molecule is called a transcription unit. © 2014 Pearson Education, Inc. Transcription can be separated into three stages: initiation, elongation, and termination Figure 17.7 • The presence of a promoter sequence determines which strand of the DNA helix is the template. – Within the promoter is the starting point for the transcription of a gene. A DNA sequence called a TATA box is located near the start site. – The promoter also includes a binding site for RNA polymerase several dozen nucleotides from the start point. • In eukaryotes, proteins called transcription factors regulate the binding of RNA polymerase and the initiation of transcription. • Only after certain transcription factors are attached to the promoter does RNA polymerase II bind to it and start transcription. • The completed assembly of transcription factors and RNA polymerase II bound to a promoter is called a transcription initiation complex. © 2014 Pearson Education, Inc. Transcription Initiation at the Promoter • The DNA strand will contain a TATA box (containing a sequence of thymine/adenine nucleotides) very close to the transcription start point. • One transcription factor binds specifically to the TATA box, then other transcription factors bind together at this area to form a complex. • After all of the required transcription factors have bound to the DNA RNA polymerase II can bind to the DNA strand, forming the transcription initiation complex. • The DNA double helix unwinds and RNA synthesis begins at the start point. Figure 17.8 Initiation Promoter Transcription unit 3′ 5′ 5′ 3′ 1 Initiation Start point RNA polymerase 3′ 5′ 5′ 3′ Figure 17.7 Unwound DNA Template strand of DNA RNA transcript After RNA polymerase II binds to the promoter: The DNA strands unwind. RNA polymerase II initiates RNA synthesis at the start point along the template DNA strand. Elongation Promoter Transcription unit 3′ 5′ 5′ 3′ 1 Initiation Figure 17.7 Start point RNA polymerase 3′ 5′ 5′ 3′ Unwound DNA 2 Elongation 5′ 3′ Template strand of DNA RNA transcript Rewound DNA 3′ 5′ RNA transcript 3′ 5′ Direction of transcription (“downstream”) The RNA polymerase II moves downstream both untwist the DNA 10 to 20 bases at a time and synthesizing the RNA transcript in the 5’→3’ direction. Nucleotides are added to the 3′ end of the growing RNA molecule Several RNA polymerase molecules can work together to transcribe a gene. The DNA strands simultaneously reform a double helix just after RNA polymerase has made the transcript. Transcription progresses at a rate of 40 nucleotides per second in eukaryotes. Figure 17.9 Nontemplate strand of DNA RNA nucleotides RNA polymerase A 3′ T C C A A 5′ 3′ end A U C C U A 3′ 5′ T 5′ A G G T T Direction of transcription Template strand of DNA Newly made RNA Termination Promoter Transcription unit 3′ 5′ 5′ 3′ 1 Initiation Start point RNA polymerase 3′ 5′ 5′ 3′ Unwound DNA 2 Elongation 5′ 3′ Template strand of DNA RNA transcript Rewound DNA 5′ RNA transcript 3 Termination 3′ 5′ 3′ Direction of transcription (“downstream”) 3′ 5′ 5′ 3′ 5′ Completed RNA transcript Figure 17.7 3′ Termination • The mechanisms of termination are different in bacteria and eukaryotes. • In bacteria, the polymerase stops transcription at the end of the terminator and the mRNA can be translated without further modification • In eukaryotes, the pre-mRNA is cleaved from the growing RNA chain while RNA polymerase II continues to transcribe the DNA. • The RNA polymerase transcribes a DNA sequence called the polyadenylation signal sequence, (AAUAAA) in the pre-mRNA. • At a point about 10 to 35 nucleotides past this sequence, the pre-mRNA is cut from the enzyme. • The completed single-stranded RNA transcript is released and the RNA polymerase will detach from the DNA strand. Eukaryotic cells modify RNA after transcription • RNA processing-Enzymes in the eukaryotic nucleus modify pre-mRNA before it is released into the cytoplasm • During RNA processing, both ends of the mRNA transcript are usually altered • Some interior parts of the molecule are cut out, and the other parts spliced together © 2014 Pearson Education, Inc. Alteration of mRNA Ends occurs in the Nucleus Figure 17.10 • Each end of a pre-mRNA molecule is modified in a particular way: – The 5′ end receives a modified nucleotide 5′ cap consisting of guanine – The 3′ end gets a poly-A tail consisting of 50-250 adenine nucleotides • These modifications share several functions: – They facilitate the export of mRNA into the cytoplasm – They protect mRNA from hydrolytic enzymes – They help ribosomes (in the cytoplasm) attach to the 5′ end © 2014 Pearson Education, Inc. Split Genes and RNA Splicing Figure 17.11 • Most eukaryotic genes and their RNA transcripts have long noncoding stretches of nucleotides that lie between coding regions • These noncoding regions are called introns • The other regions are called exons-they are eventually translated into amino acid sequences • RNA splicing removes introns and joins exons, creating an mRNA molecule with a continuous coding sequence The signal for RNA splicing is a short nucleotide sequence at each end of an intron • Particles called small nuclear ribonucleoproteins (snRNPs) recognize the splice sites. • snRNPs are located in the cell nucleus and are composed of RNA and protein molecules. • Each snRNP has several protein molecules and a small nuclear RNA molecule (snRNA). • Several different snRNPs join with a variety of proteins to form a larger spliceosome. • The spliceosome interacts with specific sites along an intron, releasing the intron and joining together the two exons that surrounded the intron. © 2014 Pearson Education, Inc. Figure 17-12 RNA transcript (pre-mRNA) 5′ Exon 1 Intron Protein snRNA Exon 2 Other proteins snRNPs Spliceosome 5′ Spliceosome components 5′ mRNA Exon 1 Exon 2 Cut-out intron Ribozymes • Ribozymes are catalytic RNA molecules that function as enzymes and can splice RNA • The discovery of ribozymes rendered obsolete the belief that all biological catalysts were proteins • Three properties of RNA enable it to function as an enzyme – It can form a three-dimensional structure because of its ability to base pair with itself – Some bases in RNA contain functional groups – RNA may hydrogen-bond with other nucleic acid molecules © 2014 Pearson Education, Inc. Why is RNA splicing important? The Functional and Evolutionary Importance of Introns • • • Split genes can encode for more than one polypeptide, depending on which segments are treated as exons during RNA splicing. Alternative RNA splicing gives rise to two or more different polypeptides, depending on which segments are treated as exons. Because of alternative splicing, the number of different protein products an organism can produce is much greater than its number of genes. – The presence of introns increases the probability of crossing over between genes. – Introns increase the opportunity for recombination between two alleles of a gene. – Recombination raises the probability that a crossover will switch one version of an exon for another version found on the homologous chromosome. – There may also be occasional mixing and matching of exons between completely different genes. – Either way, exon shuffling can lead to the production of new proteins. – Proteins often have a modular architecture consisting of discrete regions called domains. In many cases, different exons code for the different domains in a protein © 2014 Pearson Education, Inc. Figure 17.13 Gene DNA Exon 1 Intron Exon 2 Intron Exon 3 Transcription RNA processing Translation Domain 3 Domain 2 Domain 1 Polypeptide Translation is the RNA-directed synthesis of a protein • Genetic information flows from mRNA to protein through the process of translation. • In the process of translation, a cell interprets a series of codons along an mRNA molecule and builds a protein. • The interpreter is transfer RNA (tRNA), which transfers amino acids from the cytosol to a ribosome. • The ribosome adds each amino acid carried by tRNA to the growing end of the polypeptide chain. • During translation, each type of tRNA links an mRNA codon with the appropriate amino acid. © 2014 Pearson Education, Inc. tRNA • The tRNA (transfer RNA), molecule is a translator because it can read a the mRNA codon and translate it into a protein. • Each tRNA arriving at the ribosome carries a specific amino acid at one end and has a specific nucleotide triplet, an anticodon at the other. • The anticodon base-pairs with a complementary codon on mRNA. • Example: If the codon on mRNA is UUU, a tRNA with an AAA anticodon and carrying phenylalanine amino acid will bind to it. • Codon by codon, tRNAs deposit amino acids in the prescribed order, and the ribosome joins them into a polypeptide chain. © 2014 Pearson Education, Inc. Translation • As a molecule of mRNA moves through a ribosome, codons are translated into amino acids one at a time. • tRNA have a specific anticodon of 3 nucleotide complementary sequence to the mRNA on one end and the corresponding amino acid at the other end. • When H bonding between the anticodon and mRNA occurs, tRNA adds the amino acid to the growing polypeptide chain. Figure 17.14 tRNA- Structure and Function • • • • • tRNA is transcribed in the nucleus from a DNA template and must enter the cytoplasm, where the ribosomes are located. Each tRNA is used repeatedly, picking up its designated amino acid in the cytosol, depositing the amino acid at the ribosome, and returning to the cytosol to pick up another copy of that amino acid. A tRNA molecule consists of a single RNA strand that is only about 80 nucleotides long that folds back on itself to form a three-dimensional structure. Flattened into one plane to reveal its base pairing, a tRNA molecule looks like a cloverleaf. tRNA includes a loop containing the anticodon and an attachment site at the 3′ end for an amino acid. Because of hydrogen bonds, tRNA actually twists and folds into a three-dimensional molecule tRNA is roughly L-shaped Figure 17.15 • Accurate translation requires two steps: – First: a correct match between a tRNA and an amino acid, done by the enzyme aminoacyltRNA synthetase – Second: a correct match between the tRNA anticodon and an mRNA codon • Flexible pairing at the third base of a codon is called wobble and allows some tRNAs to bind to more than one codon © 2014 Pearson Education, Inc. Amino acids joined to the mRNA • Each amino acid is joined to the correct tRNA by aminoacyl-tRNA synthetase. • The 20 different synthetases match the 20 different amino acids. • Each has active sites for only a specific tRNA–amino acid combination. • The synthetase catalyzes a covalent bond between them in a process driven by ATP hydrolysis. • The result is an aminoacyl-tRNA or charged amino acid. • The second recognition process involves a correct match between the tRNA anticodon and an mRNA codon. © 2014 Pearson Education, Inc. 1 Amino acid and tRNA enter active site. Tyr-tRNA A U A Complementary tRNA anticodon Figure 17.16-1 Tyrosine (Tyr) (amino acid) Tyrosyl-tRNA synthetase 1 Amino acid and tRNA enter active site. Tyrosine (Tyr) (amino acid) Tyrosyl-tRNA synthetase Tyr-tRNA A U A Complementary tRNA anticodon Figure 17.16-2 Aminoacyl-tRNA synthetase ATP AMP + 2 P i 2 Using ATP, synthetase catalyzes covalent bonding. tRNA Amino acid Computer model 1 Amino acid and tRNA enter active site. Figure 17.16-3 Tyrosine (Tyr) (amino acid) Tyrosyl-tRNA synthetase Tyr-tRNA A U A Complementary tRNA anticodon 3 Aminoacyl tRNA released. Aminoacyl-tRNA synthetase ATP AMP + 2 P i 2 Using ATP, synthetase catalyzes covalent bonding. tRNA Amino acid Computer model The Ribosome is the site of translation • Ribosomes facilitate specific coupling of tRNA anticodons with mRNA codons in protein synthesis. • Each ribosome is made up of a large and a small subunit. • The subunits are composed of proteins and ribosomal RNA (rRNA). • After rRNA genes are transcribed to rRNA in the nucleus, the rRNA and proteins are assembled to form the subunits with proteins from the cytoplasm. • The large and small subunits join to form a functional ribosome only when they attach to an mRNA molecule. • Bacterial and eukaryotic ribosomes are somewhat similar but have significant differences: some antibiotic drugs specifically target bacterial ribosomes without harming eukaryotic ribosomes © 2014 Pearson Education, Inc. Ribosomes • • • • • • • Each ribosome has a binding site for mRNA and three binding sites for tRNA molecules. The P site (peptidyl-tRNA site) holds the tRNA carrying the growing polypeptide chain. The A site (aminoacyl-tRNA site) holds the tRNA with the next amino acid to be added to the chain. Discharged tRNAs leave the ribosome at the E (exit) site. 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. The ribosome then catalyzes the formation of the peptide bond (via rRNA). As the polypeptide becomes longer, it passes through an exit tunnel in the ribosome’s large unit and is released to the cytosol. © 2014 Pearson Education, Inc. Figure 17.17 Building a Polypeptide • The three stages of translation: – Initiation – Elongation – Termination • All three stages require protein “factors” that aid in the translation process © 2014 Pearson Education, Inc. Initiation of the Polypeptide Chain • Initiation brings together mRNA, a tRNA, the first amino acid, and the two ribosomal subunits. • First, the small ribosomal subunit binds with mRNA and a special initiator tRNA, which carries methionine. • The small subunit binds to the 5′ cap of the mRNA and then moves, or scans, downstream along the mRNA until it reaches the start codon AUG, which signals the start of translation. • This establishes the reading frame for the mRNA. • The initiator tRNA, already associated with the complex, then hydrogen-bonds with the start codon. • Initiation factors (proteins) and GTP are required for complex formation. © 2014 Pearson Education, Inc. Figure 17.18 Large ribosomal subunit 3′ U A C 5′ 5′ A U G 3′ P site Pi Initiator tRNA + GTP GDP E mRNA 5′ Start codon mRNA binding site © 2014 Pearson Education, Inc. 3′ Small ribosomal subunit 5′ A 3′ Translation initiation complex Elongation of the Polypeptide Chain • During the elongation stage, amino acids are added one by one to the preceding amino acid at the C-terminus of the growing chain • Each addition involves proteins called elongation factors and occurs in three steps: codon recognition, peptide bond formation, and translocation • Translation proceeds along the mRNA in a 5′ to 3′ direction © 2014 Pearson Education, Inc. Figure 17.19-1 Amino end of polypeptide E 5′ 3′ mRNA P A site site 1 Codon recognition GTP GDP + P i STEP-1 Codon Recognition-The anticodon of an incoming aminoacyl tRNA (containing an amino acid at one end) base pairs with the complementary mRNA codon in the A site. GTP is needed for this step of elongation. E P A Figure 17.19-2 Amino end of polypeptide E 5′ 3′ mRNA P A site site STEP-2 Peptide Bond Formation- During peptide bond formation, an rRNA molecule catalyzes the formation of a peptide bond between the polypeptide in the P site and the new amino acid in the A site. This step separates the tRNA at the P site from the growing polypeptide chain and transfers the chain, now one amino acid longer, to the tRNA at the A site. 1 Codon recognition GTP GDP + P i E P A 2 Peptide bond formation E P A STEP-3 Translocation - ribosome moves the tRNA with the attached polypeptide from the A site to the P site. ● Because the anticodon remains bonded to the mRNA codon, the mRNA moves along with it. ● The next codon is now available at the A site. ● The tRNA that had been in the P site is moved to the E site and then leaves the ribosome. ●Translocation is fueled by the hydrolysis of GTP. ●Effectively, translocation ensures that the mRNA is “read” 5′3′ codon by codon. © 2014 Pearson Education, Inc. Figure 17.19-3 Amino end of polypeptide E Ribosome ready for next aminoacyl tRNA 3′ mRNA P A site site 5′ 1 Codon recognition GTP GDP + P i E E P A P A GDP + P i 3 Translocation 2 Peptide bond formation GTP E These three steps of elongation continue to add amino acids codon by codon until the polypeptide chain is completed. P A Figure 17.20 Termination Release factor Free polypeptide 5′ 3′ 3′ 5′ 5′ Stop codon (UAG, UAA, or UGA) 3′ GTP 2 2 GDP + 2 P i 2 Release factor promotes hydrolysis. 3 Ribosomal subunits and other components dissociate. Termination occurs when one of the three stop codons (UAG, UAA, or UGA). When a ribosome reaches a stop codon along the mRNA strand, the A site accepts a “release factor”- a protein shaped like a tRNA. The release factor hydrolyzes the bond between the tRNA in the P site and the last amino acid of the polypeptide chain by the addition of a water molecule . The polypeptide is then released from the chain. The two ribosomal machinery subunit disassembles-this requires GTP. This reaction releases the polypeptide, and the translation assembly then comes apart 1 Ribosome reaches a stop codon on mRNA. Completing and Targeting the Functional Protein • Often translation is not sufficient to make a functional protein • Polypeptide chains are modified after translation or targeted to specific sites in the cell Protein folding and post-translational modifications • During and after synthesis, a polypeptide coils and folds to its three-dimensional shape spontaneously. • The primary structure, the order of amino acids, determines the secondary and tertiary structure. • Chaperone proteins may aid correct folding. • In addition, proteins may require post-translational modifications before doing their particular job. – These modifications may require additions such as sugars, lipids, or phosphate groups to amino acids. – Enzymes may remove some amino acids from the leading (amino) end of the polypeptide chain. – In some cases, a single polypeptide chain may be enzymatically cleaved into two or more pieces. – In other cases, two or more polypeptides may join to form a protein with quaternary structure. © 2014 Pearson Education, Inc. Targeting polypeptides to specific locations in the cell • Two populations of ribosomes are evident in cells: free ribsomes and bound ribosomes • Free ribosomes are suspended in the cytosol and synthesize proteins that reside in the cytosol. • Bound ribosomes are attached to the cytosolic side of the endoplasmic reticulum (ER) or to the nuclear envelope. • Bound ribosomes synthesize proteins of the endomembrane system as well as proteins secreted from the cell. • Although bound and free ribosomes are identical in structure, their location depends on the type of protein they are synthesizing. • Polypeptide synthesis always begins in the cytosol. – The polypeptide that is being synthesized will cause the ribosome to attach to the ER if it contains a signal peptide sequence. Polypeptides of membrane proteins and proteins that are to be secreted contain signal peptide sequences. – Signal peptides are around 20 amino acids long and are located near the leading end of the polypeptide chain. • The signal peptide is recognized by a protein-RNA complex called a signal-recognition complex. © 2014 Pearson Education, Inc. SRP’s • A signal recognition particle (SRP) binds to the signal peptide and attaches it and its ribosome to a receptor protein in the ER membrane. • The SRP, which consists of a protein–RNA complex, functions as an adapter to bring the ribosome to a receptor protein built into the ER membrane. • The receptor is part of a multiprotein translocation complex. • Protein synthesis resumes, with the growing polypeptide snaking across the membrane into the ER lumen via a protein pore. • An enzyme usually cleaves the signal polypeptide. • Secretory proteins are released into solution within the ER lumen, but membrane proteins remain partially embedded in the ER membrane. © 2014 Pearson Education, Inc. Figure 17.21 2 1 3 Polypeptide SRP SRP synthesis binds to binds to begins. signal receptor peptide. protein. 4 5 SRP detaches and polypeptide synthesis resumes. Signalcleaving enzyme cuts off signal peptide. 6 Completed polypeptide folds into final conformation. Ribosome mRNA Signal peptide Signal peptide removed SRP CYTOSOL SRP receptor protein ER LUMEN Translocation complex ER membrane Protein Targeting Proteins to the ER 1. Polypeptide synthesis begins on a free ribosome in the cytosol. 2. An SRP binds to the signal peptide and temporarily stops polypeptide synthesis. 3. The SRP binds to a receptor protein in the ER membrane. 4. The SRP leaves and polypeptide synthesis resumes and translocation across the ER membrane occurs. 5. The signal-cleaving enzyme cuts off the signal peptide. 6. The rest of the completed polypeptide leaves the ribosome and folds. Making Multiple Polypeptides in Bacteria and Eukaryote- Polyribosomes • Typically a single mRNA is used to make many copies of a polypeptide simultaneously. • Multiple ribosomes, polyribosomes or polysomes, may trail along the same mRNA. • Polyribosomes can be found in prokaryotic and eukaryotic cells. Figure 17.23 • A bacterial cell ensures a streamlined process by coupling transcription and translation • In this case the newly made protein can quickly diffuse to its site of function • In eukaryotes, the nuclear envelop separates the processes of transcription and translation • RNA undergoes processes before leaving the nucleus Figure 17.24 DNA TRANSCRIPTION 3′ 5′ RNA polymerase RNA transcript Exon RNA PROCESSING RNA transcript (pre-mRNA) Intron Aminoacyl-tRNA synthetase NUCLEUS Amino acid CYTOPLASM AMINO ACID ACTIVATION tRNA mRNA 3′ E P A Aminoacyl (charged) tRNA Ribosomal subunits TRANSLATION E A A A A U G G U U U A U G Codon Ribosome Anticodon Point mutations (in DNA) can affect protein structure and function • Mutations result in changes in genetic material of a cell or virus. • Mutations can be either large-scale, in which long segments of DNA are affected (for example, translocations, duplications, and inversions), or point mutations- changes in just one base pair of a gene. • If a point mutation occurs in a gamete or in a cell that produces gametes, it may be transmitted to future generations. • If the mutation has an adverse effect on the phenotype of an organism, the mutant condition is referred to as a genetic disorder or hereditary disease- sickle-cell disease . • A change in a single nucleotide in the DNA’s template strand can lead to an abnormal protein. Types of Point Mutations • Point mutations within a gene can be divided into two general categories – Base-pair substitutions – Base-pair insertions or deletions © 2014 Pearson Education, Inc. Substitutions • A point mutation that results in the replacement of a pair of complementary nucleotides with another nucleotide pair is called a base-pair substitution. • Some base-pair substitutions have little or no impact on protein function. • In silent mutations, altered nucleotides still code for the same amino acids because of redundancy in the genetic code. • Missense mutations change an amino acid but have little effect on protein function but not necessarily the right amino acid. • Nonsense mutations change an amino acid codon into a stop codon, causing premature termination of translation nearly always leading to a nonfunctional protein. • In some cases, the mutation switches one amino acid for another with similar properties. © 2014 Pearson Education, Inc. Figure 17.25 Sickle cell Disease is caused by a point mutation Wild-type β-globin Sickle-cell β-globin Wild-type β-globin DNA 3′ 5′ C T C G A G Mutant β-globin DNA 5′ 3′ 3′ 5′ mRNA 5′ C A C G T G 5′ 3′ G U G 3′ mRNA G A G Normal hemoglobin Glu 3′ 5′ Sickle-cell hemoglobin Val For example, sickle-cell disease is caused by a mutation of a single base pair in the gene that codes for one of the polypeptides of hemoglobin. Insertions and Deletions • Insertions and deletions are additions or losses of nucleotide pairs in a gene. • Insertions and deletions have a disastrous effect on the resulting protein more often than substitutions do. • Unless insertion or deletion mutations occur in multiples of 3, they cause a frameshift mutation. • All the nucleotides downstream of the deletion or insertion will be improperly grouped into codons. • The result will be extensive missense, ending sooner or later in nonsense—premature termination. • Mutations can occur during DNA replication, DNA repair, or DNA recombination © 2014 Pearson Education, Inc. Figure 17.26 Wild type Types of Point Mutations DNA template strand 3′ T A C T T C A A A C C G A T T 5′ 5′ A T G A A G T T T G G C T A A 3′ mRNA5′ A U G A A G U U U G G C U A A 3′ Protein Met Lys Phe Gly Stop Carboxyl end Amino end (b) Nucleotide-pair insertion or deletion (a) Nucleotide-pair substitution Extra A A instead of G 3′ T A C T T C A A A C C A A T T 5′ 5′ A T G A A G T T T G G T T A A 3′ 3′ T A C A T T C A A A C C G A T T 5′ 5′ A T G T A A G T T T G G C T A A 3′ Extra U U instead of C 5′ A U G A A G U U U G G U U A A 3′ Met Lys Phe Gly Stop Silent (no effect on amino acid sequence) 5′ A U G U A A G U U U G G C U A A 3′ Met Stop Frameshift causing immediate nonsense (1 nucleotide-pair insertion) T instead of C A missing 3′ T A C T T C A A A T C G A T T 5′ 5′ A T G A A G T T T A G C T A A 3′ 3′ T A C T T C A A C C G A T T 5′T ′ 5′ A T G A A G T T G G C T A A 3A A instead of G U missing 5′ A U G A A G U U U A G C U A A 3′ Met Lys Phe Ser Stop Missense 5′ A U G A A G U U G G C U A A Met Lys Leu 3′ Ala Frameshift causing extensive missense (1 nucleotide-pair deletion) A instead of T 3′ T A C A T C A A A C C G A T T 5′ 5′ A T G T A G T T T G G C T A A 3′ U instead of A 5′ A U G U A G U U U G G C U A A 3′ Met Nonsense Stop T T C missing 3′ T A C A A A C C G A T T 5′ 5′ A T G T T T G G C T A A 3′ A A G missing ′ A A 5′ A U G U U U G G C U A A 3U Met Phe Gly Stop No frameshift, but one amino acid missing (3 nucleotide-pair deletion) Mutagens • Mutagens are chemical or physical agents that interact with DNA to cause mutations. • Physical agents include high-energy radiation like X-rays and ultraviolet light. – Ultraviolet light can cause disruptive thymine dimers in DNA. • Chemical mutagens cause mutations in different ways. – Some chemicals may be substituted into DNA, but they pair incorrectly during DNA replication. – Other mutagens interfere with DNA replication by inserting into DNA and distorting the double helix. – Still others cause chemical changes in nucleotide bases that change their pairing properties. © 2014 Pearson Education, Inc. While gene expression differs among the domains of life, the concept of a gene is universal • Archaea are prokaryotes, but share many features of gene expression with eukaryotes • Bacteria and eukarya differ in their RNA polymerases, termination of transcription and ribosomes; archaea tend to resemble eukarya in these respects • Bacteria can simultaneously transcribe and translate the same gene • In eukarya, transcription and translation are separated by the nuclear envelope • In archaea, transcription and translation are likely coupled © 2014 Pearson Education, Inc. What Is a Gene? Revisiting the Question • The idea of the gene itself is a unifying concept of life • We have considered a gene as: – A discrete unit of inheritance – A region of specific nucleotide sequence in a chromosome – A DNA sequence that codes for a specific polypeptide chain • In summary, a gene can be defined as a region of DNA that can be expressed to produce a final functional product, either a polypeptide or an RNA molecule © 2014 Pearson Education, Inc. Figure 17.UN09 Type of RNA Functions Messenger RNA (mRNA) Transfer RNA (tRNA) Plays catalytic (ribozyme) roles and structural roles in ribosomes Primary transcript Small RNAs in the spliceosome