The Blueprint of Life, from DNA to Protein PDF

The Blueprint of Life, 7 from DNA to Protein KEY TERMS Codon A series of three nucleotides...

The Blueprint of Life, 7 from DNA to Protein KEY TERMS Codon A series of three nucleotides that code for a specific amino acid. DNA Polymerase Enzyme that synthesizes DNA, using an existing Ribosomal RNA (rRNA) Type of RNA molecule present in ribosomes. Ribosome Structure that facilitates the joining of amino acids during strand as a template to make a new translation; composed of ribosomal complementary strand. RNA (rRNA) and protein. DNA Replication Duplication of a RNA Polymerase Enzyme that DNA molecule. synthesizes RNA using one strand of Gene The functional unit of the DNA as a template. genome; it encodes a product, most Transcription The process by often a protein. which the information encoded in Genome Complete set of genetic DNA is copied into RNA. information in a cell or a virus. Transfer RNA (tRNA) Type of RNA Messenger RNA (mRNA) Type molecule involved in interpreting the of RNA molecule translated during genetic code; each tRNA molecule protein synthesis. carries a specific amino acid. Promoter Nucleotide sequence to Translation The process by which the which RNA polymerase binds to information carried by mRNA is used start transcription. to synthesize the encoded protein. Model of DNA double helix. A Glimpse of History all proteins are enzymes. In 1958, Beadle and Tatum were awarded a In 1866, the Czech-Austrian monk Gregor Mendel determined that Nobel Prize, largely for these pioneering studies that ushered in the traits are inherited as physical units, now called genes. The precise era of modern biology. polypeptide, p. 37 function of genes, however, was not revealed until 1941, when George Beadle and Edward Tatum published a scientific paper reporting that onsider for a moment the incredible diversity of life- genes direct the production of enzymes. Beadle and Tatum set out to discover how genes control meta- bolic reactions by studying common bread molds that have very simple nutritional requirements. These molds, Neurospora species, C forms in our world—from the remarkable variety of microorganisms, to the plants and animals consist- ing of many different specialized cells. Every characteristic can grow on media containing only sucrose, inorganic salts, and of each of these cells, from its shape to its function, is dic- the vitamin biotin. Beadle and Tatum reasoned that if they created tated by information within its deoxyribonucleic acid (DNA). mutant strains that required additional nutrients, they could use them DNA is the “blueprint,” providing instructions for building an to gain insights into the relationship between genes and enzymes. For organism’s components. example, a mutant that requires a certain amino acid probably has a DNA itself is a simple structure—a linear molecule com- defect in an enzyme required to synthesize that amino acid. posed of only four different nucleotides, each containing a par- To generate mutant strains, Beadle and Tatum treated the mold ticular nucleobase (also called a nitrogenous base or simply a cultures with X rays and then grew the resulting cells on a nutrient- base): adenine (A), thymine (T), cytosine (C), or guanine (G). rich medium that supported the growth of both the original strain and A set of three nucleotides encodes a specific amino acid; in any mutants. Next, they screened thousands of the progeny to find the turn, a string of amino acids makes up a protein, the structure nutrient-requiring mutants. To identify the metabolic defect of each and function of which is dictated by the order of the amino one, they grew them separately in various types of media containing acid subunits. Some proteins serve as structural components of different nutrients. Eventually, Beadle and Tatum established that the metabolic a cell. Others, such as enzymes, direct cellular activities includ- defect was inherited as a single gene, which ultimately led to their con- ing biosynthesis and energy conversion. Together, all these clusion that a single gene determines the production of one enzyme. proteins control the cell’s structure and activities, dictating the That assumption has been modified somewhat, because we now know overall characteristics of that cell. deoxyribonucleic acid (DNA), p. 39 that some enzymes are made up of more than one polypeptide, and not nucleotide, p. 38 nucleobase, p. 38 amino acid, p. 34 enzymes, p. 141 176  Focus Figure Gene Expression DNA Transcription Copies the information in DNA DNA Replication into RNA. Duplicates the DNA molecule so its encoded information can be passed on to the next generation. RNA Translation Interprets the information carried by RNA to synthesize the encoded protein. FIGURE 7.1 Overview of Replication, Transcription, and Translation Protein ? Which is more stable in a cell—DNA or RNA? Although at first it might seem unlikely that the vast array All cells must accomplish two general tasks in order to mul- of life-forms could be encoded by a molecule consisting of only tiply. First, the double-stranded DNA must be duplicated before four different units (the nucleotides), think about how much cell division so that its encoded information can be passed on information can be transmitted by binary code, the language to the next generation (figure 7.1). This is the process of DNA of all computers. Using only a simple series of ones and zeros, replication. Second, the information encoded by the DNA must binary can code for each letter of the alphabet. String enough be decoded so that the cell can synthesize the necessary gene prod- of these together in the right sequence and the letters become ucts. This process, gene expression, involves two related events: words. With longer and longer strings, the words can become transcription and translation. Transcription is the process by complete sentences, chapters, books, or even whole libraries. which the information encoded in DNA is copied into a slightly This chapter will focus on the processes bacteria use to repli- different molecule—RNA. In translation, the information car- cate their DNA and convert the encoded information into proteins. ried by the RNA is interpreted and used to synthesize the encoded The mechanisms used by eukaryotic cells have many similarities, protein. The flow of information from DNA → RNA → protein is but are considerably more complicated, and will only be dis- often referred to as the central dogma of molecular biology. cussed briefly. The processes in archaea are sometimes similar to those of bacteria, but often resemble those of eukaryotic cells. Characteristics of DNA DNA is usually a double-stranded, helical structure (figure 7.2). 7.1 Overview Each strand is composed of a chain of deoxyribonucleotide subunits, more commonly called nucleotides. Each nucleotide Learning Outcomes contains a 5-carbon sugar (deoxyribose), a phosphate group, 1. Compare and contrast the characteristics of DNA and RNA. and one of four different nucleobases (A, T, G, or C). They 2. Explain why gene regulation is important to a cell. are joined together by a covalent bond between the 59PO4 (5 prime phosphate) of one nucleotide and the 39OH (3 prime The complete set of genetic information of a cell is referred to hydroxyl) of the next. The designations 59 and 39 refer to the as its genome. Technically, this includes plasmids as well as numbered carbon atoms of the pentose sugar of the nucleo- the chromosome; however, the term “genome” is often used tide (see figure 2.11). Joining the nucleotides this way creates interchangeably with chromosome. The genome of all cells is a series of alternating sugar and phosphate units, called the composed of DNA, but some viruses have an RNA genome. sugar-phosphate backbone. Because of the chemical structure The functional unit of the genome is a gene. A gene encodes a of nucleotides and how they are joined to each other, a single product (called the gene product), most commonly a protein. strand of DNA will always have a 59PO4 at one end and a 39OH The study and analysis of the nucleotide sequence of DNA is at the other. These ends are often referred to as the 59 end called genomics. chromosome, p. 74 plasmid, p. 75 (5 prime end) and the 39 end (3 prime end). nucleotides, p. 38 177 178 Chapter 7 The Blueprint of Life, from DNA to Protein 5' phosphate 5' end 3' end 3' hydroxyl HO Base pairs Sugar P O A T P Sugar O Sugar P O G C P Sugar O Sugar DNA P O C G P Sugar O Sugar P O Nucleotide T A P Sugar O Sugar P O C G P Sugar O Hydrogen bonds HO 3' hydroxyl 3' end 5' end 5' phosphate FIGURE 7.2 The Structure of DNA The two strands in the double helix are complementary. Three hydrogen bonds form between a G-C base pair and two between an A-T base pair. The strands are antiparallel; one is oriented in the 59 to 39 direction, and its complement is oriented in the 39 to 59 direction. ? If a 100 base-pair double-stranded DNA fragment has 40 cytosines, how many adenines does it contain? The two strands of DNA are complementary and are Characteristics of RNA held together by hydrogen bonds between the nucleobases. RNA, like DNA, is composed of a chain of nucleotides. It is Wherever an adenine (A) is in one strand, a thymine (T) is in similar to DNA in many ways, with a few important excep- the other; these opposing A-T bases are held together by two tions. One difference is that the sugar in the nucleotides of hydrogen bonds. Similarly, wherever a guanine (G) is in one RNA is ribose, not deoxyribose; it has an oxygen molecule strand, a cytosine (C) is in the other. These G-C bases are held that deoxyribose lacks. Another distinction is that RNA con- together by three hydrogen bonds, a slightly stronger attrac- tains the nucleobase uracil in place of the thymine found in tion than that of an A-T pair. The characteristic bonding of A DNA. Also, RNA is usually a single-stranded linear molecule to T and G to C is called base-pairing and is a fundamental much shorter than DNA. ribonucleic acid (RNA), p. 40 characteristic of DNA. Because of the rules of base-pairing, RNA is synthesized using a region of one of the two one strand can always be used as a template for the synthesis strands of DNA as a template. In making the RNA molecule, of the opposing strand. or transcript, the base-pairing rules apply except that uracil, Although the two strands of DNA in the double helix are rather than thymine, pairs with adenine. The interaction of complementary, they are also antiparallel. That is, they are DNA and RNA is only temporary, however, and the transcript oriented in opposite directions. One strand is oriented in the quickly separates from the template. 59 to 39 direction and its complement is oriented in the 39 to Three different functional types of RNA are required 59 direction. for gene expression, and these are transcribed from different The duplex structure of double-stranded DNA is gener- sets of genes (figure 7.3). Most genes encode proteins and ally quite stable because of the numerous hydrogen bonds are transcribed into messenger RNA (mRNA). The informa- that occur along its length. Short fragments of DNA have cor- tion encrypted in mRNA is decoded according to the genetic respondingly fewer hydrogen bonds, so they are easily sepa- code, which correlates each set of three nucleotides to a par- rated into single strands. Separating the two strands of DNA ticular amino acid. Some genes are never translated into pro- is called melting, or denaturing. hydrogen bonds, p. 25 teins; instead the RNAs themselves are the final products.  Part I Life and Death of Microorganisms 179 FIGURE 7.3 Three Functional Types of Protein-encoding gene rRNA gene tRNA gene RNA Molecules The different functional types of RNA—messenger RNA (mRNA), ribosomal RNA (rRNA), and transfer RNA DNA Transcription (tRNA)—are transcribed from different genes. The mRNA is translated, and the tRNA and rRNA fold into characteristic three- Messenger RNA (mRNA) Ribosomal RNA (rRNA) Transfer RNA (tRNA) dimensional structures that each play a role in protein synthesis. Translation ? Ribosomal RNA is a component of ribosomes. What are ribosomes? Protein These genes encode either ribosomal RNA (rRNA) or for translation. If it is then turned “off,” the number of tran- transfer RNA (tRNA), each of which plays a different but scripts will rapidly decline. By simply regulating the synthe- critical role in protein synthesis. sis of mRNA molecules, a cell can quickly change the levels of protein production. Regulating Gene Expression MicroAssessment 7.1 Although a cell’s DNA can encode thousands of different pro- Replication is the process of duplicating double-stranded DNA. teins, not all of them are needed at the same time or in equal Transcription is the process of copying the information encoded in quantities (figure 7.4). Because of this, cells require mecha- DNA into RNA. Translation is the process in which the information nisms to regulate the expression of certain genes. carried by mRNA is used to synthesize the encoded protein. A fundamental aspect of gene regulation is the cell’s 1. How does the 59 end of a DNA strand differ from the 39 end? ability to quickly destroy mRNA. Within minutes of being 2. What are the base-pairing rules? produced, transcripts are degraded by cellular enzymes. 3. If the nucleotide sequence of one strand of DNA is Although this might seem wasteful, it actually provides cells 59 ACGTTGCA 39, what is the sequence of the with an important regulatory mechanism. If transcription of a complementary strand? + gene is turned “on,” transcripts will continue to be available Gene A Gene B Gene C Low levels of gene A No transcription of gene B Continuous transcription of transcription generates leads to no synthesis of gene C generates many some transcripts of protein B. transcripts of the gene. the gene. Translation of each of the gene A transcripts generates some protein A. Translation of each of the gene C transcripts generates many molecules of protein C. FIGURE 7.4 The Level of Gene Expression Can Be Controlled ? How does the fact that mRNA is quickly degraded help a cell control gene expression? 180 Chapter 7 The Blueprint of Life, from DNA to Protein 7.2 DNA Replication a chromosome to be replicated in half the time it would take if the process were unidirectional. The progression of bidi- Learning Outcome rectional replication around a circular DNA molecule creates 3. Describe the DNA replication process, including its initiation two advancing forks where DNA synthesis is occurring. These and the events that occur at the replication fork. regions, called replication forks, ultimately meet at a termi- nating site when the process is complete. binary fission, p. 93 DNA is replicated so that each of the two cells generated Each of the two DNA molecules created through replica- during binary fission can receive one complete copy of the tion contain one of the original strands paired with a newly genome. The replication process is generally bidirectional, synthesized strand. Because one strand of the original mol- meaning it proceeds in both directions from a specific starting ecule is conserved in each molecule, replication is said to be point called the origin of replication (figure 7.5). This allows semiconservative. Original strand DNA Replication New strand Origin of replication Original strand Replication forks Site where replication New ends strand Replication of chromosomal Bidirectional replication creates DNA starts at the origin of two advancing forks where DNA replication and then proceeds synthesis is occurring. The replication forks ultimately in both directions. meet at a terminating site. DNA replication is semiconservative, meaning that each of the two (a) molecules created contains one of the original strands paired with a Original newly synthesized strand. double-stranded molecule FIGURE 7.5 Replication of a Bacterial Chromosome (a) Process of bidirectional replication. (b) Partially replicated chromosome; an electron micrograph and a diagrammatic depiction. (b) Replication forks Replication forks ? What is a replication fork?  Part I Life and Death of Microorganisms 181 Initiation of DNA Replication TABLE 7.1 Components of DNA Replication To initiate replication of a DNA molecule, specific proteins in Bacteria recognize and bind to the origin of replication. Bacterial chro- Component Comment mosomes and plasmids typically contain only one of these ini- DNA gyrase Enzyme that temporarily breaks the strands of DNA, tiating sites, whereas archaeal and eukaryotic chromosomes relieving the tension caused by unwinding the two have multiple sites. A molecule that lacks this sequence will strands of the DNA helix. not be replicated. The proteins that bind to the origin of repli- DNA ligase Enzyme that joins two DNA fragments together by cation include DNA gyrase and helicases, which temporarily forming a covalent bond between the sugar and break and unwind the DNA helix at that site. This exposes phosphate residues of adjacent nucleotides. single-stranded regions that can act as templates. Enzymes DNA Enzymes that synthesize DNA; they use one strand of called primases then synthesize short stretches of RNA polymerases DNA as a template to make the complementary strand. complementary to the exposed templates. These small frag- Nucleotides can be added only to the 39 end of an existing fragment—therefore, synthesis always occurs ments, called primers, are critical in the steps of replication in the 59 to 39 direction. described next. Helicases Enzymes that unwind the DNA helix at the replication fork. Origin of Distinct region of a DNA molecule at which replication The Process of DNA Replication replication is initiated. The process of DNA replication requires the coordinated Primase Enzyme that synthesizes small fragments of RNA to action of many different enzymes and other proteins. The serve as primers for DNA synthesis. most critical of these exist together in DNA-synthesizing Primer Fragment of nucleic acid to which DNA polymerase can “assembly lines” called replisomes (table 7.1). add nucleotides (the enzyme can add nucleotides only Enzymes called DNA polymerases synthesize DNA to an existing fragment). in the 59 to 39 direction, using one parent strand as a tem- Replisome The complex of enzymes and other proteins that plate to make the complement (figure 7.6). To do this, DNA synthesize DNA. polymerase adds nucleotides onto the 39 end of the new Okazaki Nucleic acid fragment produced during discontinuous strand, powering the reaction with the energy released when a fragment synthesis of the lagging strand of DNA. Template strand 5' 3' T A C G G T A C T A G T A G T A G T C G A T T C G A A DNA Replication T C A T C A T C A G C T A A G C T T Direction 3' 3' 5' of synthesis A DNA polymerase New strand P P P O O O FIGURE 7.6 The Process of DNA Synthesis DNA polymerase T A G synthesizes a new strand by adding A T C one nucleotide at a time to the 39 end of the elongating strand. The base- pairing rules determine the specific O O O nucleotides that are added. OH OH ? Considering that DNA is synthesized in P~ the 59 to 39 direction, which direction P~ P P P must DNA polymerase travel along the template strand: 59 to 39 or 39 to 59? 182 Chapter 7 The Blueprint of Life, from DNA to Protein high-energy phosphate bond of the incoming nucleo- tide is hydrolyzed. DNA polymerases add nucleotides only onto an existing nucleotide strand, so they cannot Replication forks initiate synthesis. This explains why RNA primers are required at the origin of replication—they pro- vide the DNA polymerase with a molecule to which it 3' can add additional nucleotides. high-energy phosphate 5' bond, p. 42 1 A helicase “unzips” the two strands of DNA. In order for replication to progress, the heli- Leading cases must progressively “unzip” the DNA strands strand at each replication fork to reveal additional template 5' 5' RNA primer sequences (figure 7.7 1 ). 2 Synthesis of one new 3' Helicase strand proceeds continuously as fresh template is exposed, because DNA polymerase simply adds DNA polymerase adds nucleotides to the 39 end. This strand is called the nucleotides onto the 3' end of the strand. 5' leading strand. 3 Synthesis of the other strand, the lagging strand, is more complicated. This is because 3' DNA polymerases cannot add nucleotides to the 59 2 Synthesis of the leading strand 5' proceeds continuously as fresh end of a nucleotide chain so as additional template template is exposed. is exposed, synthesis must be reinitiated. Each time synthesis is reinitiated, another RNA primer must be 5' made first. The result is a series of small fragments, 3' 5' each of which has a short stretch of RNA at its 59 Okazaki fragment end. These fragments are called Okazaki fragments. of the lagging strand 4 As DNA polymerase adds nucleotides to the 39 Primase synthesizes the RNA primer. end of one Okazaki fragment, it eventually reaches the 59 end of another. A different type of DNA poly- 3 Synthesis of the lagging strand must be reinitiated merase then removes the RNA primer nucleotides and as more template is exposed. Each time synthesis 3' simultaneously replaces them with deoxynucleotides. is reinitiated, a new RNA primer must be made. 5' 5 The enzyme DNA ligase then seals the gaps Discontinuous synthesis generates Okazaki fragments. between fragments by forming a covalent bond between the adjacent nucleotides. When a circular bacterial chromosome is replicated, the two replication forks eventually meet at a site opposite the origin of replication. Two complete DNA molecules have been produced at this point, and these can be passed on to the two 5' daughter cells. 3' 5' It takes approximately 40 minutes for the E. coli 5 DNA ligase seals chromosome to be replicated. How, then, can the the gaps between organism have a generation time of only 20 minutes? Okazaki fragments by forming a covalent This can happen because, under favorable growing bond between them. conditions, a cell initiates a new round of replication before the preceding round of replication is complete. 4 As DNA polymerase adds nucleotides to the 3' end of one Okazaki fragment, In this way, each of the two daughter cells will get one it encounters the 5' end of another. DNA ligase complete chromosome that has already started another A different type of DNA polymerase then removes the RNA primer round of replication. generation time, p. 93 nucleotides and simultaneously 3' replaces them with deoxynucleotides. 5' MicroByte Antibacterial medications called fluoroquinolones target FIGURE 7.7 The Replication Fork This diagram is simplified to the enzyme DNA gyrase, a component of the bacterial highlight the key differences between synthesis of the leading and lagging replisome. strands. Both strands are synthesized simultaneously. ? Synthesis of which strand requires the repeated action of DNA ligase?  Part I Life and Death of Microorganisms 183 MicroAssessment 7.2 TABLE 7.2 Components of Transcription DNA replication begins at the origin of replication and then in Bacteria proceeds bidirectionally, creating two replication forks. DNA Component Comment polymerases synthesize DNA in the 59 to 39 direction, using one strand as a template to generate the complementary strand. (2) strand Strand of DNA that serves as the template for New DNA is synthesized in continuous leading and fragmented RNA synthesis; the resulting RNA molecule is complementary to this strand. lagging strands. Okazaki fragments formed during lagging strand synthesis are joined together by DNA ligase. (1) strand Strand of DNA complementary to the one that serves as the template for RNA synthesis; the nucleotide 4. Why is a primer required for DNA synthesis? sequence of the RNA molecule is the same as this 5. How does synthesis of the lagging strand differ from that of strand, except it has uracil rather than thymine. the leading strand? Promoter Nucleotide sequence to which RNA polymerase 6. Eukaryotic chromosomes have multiple origins of binds to initiate transcription. replication. Why would this be the case? + RNA polymerase Enzyme that synthesizes RNA using single-stranded DNA as a template; synthesis always occurs in the 59 to 39 direction. 7.3 Gene Expression in Bacteria Sigma (s) factor Component of RNA polymerase that recognizes the promoter regions. A cell can have different types Learning Outcomes of s factors that recognize different promoters, allowing the cell to transcribe specialized sets of 4. Describe the process of transcription, focusing on the role of genes as needed. RNA polymerase, sigma (s) factors, promoters, and terminators. Terminator Nucleotide sequence at which RNA synthesis stops; 5. Describe the process of translation, focusing on the role of the RNA polymerase falls off the DNA template and mRNA, ribosomes, ribosome-binding sites, rRNAs, tRNAs, releases the newly synthesized RNA. and codons. Recall that gene expression involves two separate but interre- called a promoter; one that stops the process is a terminator. lated processes: transcription and translation. Transcription is Like DNA polymerase, RNA polymerase can add nucleotides the process of synthesizing RNA from a DNA template. During only to the 39 end of a chain and therefore synthesizes RNA translation, information encoded by an mRNA transcript is used in the 59 to 39 direction. Unlike DNA polymerase, however, to synthesize a protein. “Polypeptide” would be a more accu- RNA polymerase can start synthesis without a primer. rate term but the word “protein” is often used in this context for The RNA sequence made during transcription is comple- simplicity. The distinction between these two words is subtle—a mentary and antiparallel to the DNA template (figure 7.9). The polypeptide is simply a chain of amino acids, whereas a protein DNA strand that serves as the template for transcription is called is a functional molecule made up of one or more polypeptides. the minus (2) strand, and its complement is called the plus (1) strand (table 7.2). Because the RNA is complementary to the (2) DNA strand, its nucleotide sequence is the same as the Transcription (1) DNA strand, except it contains uracil rather than thymine. In transcription, the enzyme RNA polymerase synthesizes single-stranded RNA molecules from a DNA template. Nucle- FIGURE 7.9 RNA Is Complementary Gene Expression and Antiparallel to the DNA Template otide sequences in the DNA direct the polymerase where to The DNA strand that serves as a start and where to end (figure 7.8). The DNA sequence to Transcription template for RNA synthesis is called the which RNA polymerase can bind and initiate transcription is (2) strand of DNA. The complement to that is the (1) strand. Translation Region transcribed ? How does the nucleotide sequence of the (1) DNA strand differ from that of the RNA transcript? DNA Promoter Transcription Terminator 5' 3' Plus (+) 5' 3' G C T G A T G A T C C G C G T A GG T G C T strand C G A C T A C T A GG C G C A T C C A C G A of DNA RNA 3' 5' Minus (–) FIGURE 7.8 Nucleotide Sequences in DNA Direct Transcription strand The promoter is a DNA sequence to which RNA polymerase can bind of DNA in order to initiate transcription. The terminator is a sequence at which transcription stops. 5' 3' RNA ? In which direction is RNA synthesized: 59 to 39 or 39 to 59? G C U G A U G A U C C G C G U A GG U G C U 184 Chapter 7 The Blueprint of Life, from DNA to Protein In prokaryotes, mRNA molecules can carry the information Promoters identify the regions of a DNA molecule that will for one or multiple genes. A transcript that carries one gene is be transcribed into RNA. In doing so, they also orient the direc- called monocistronic (a cistron is synonymous with a gene). tion of the RNA polymerase on the DNA molecule, thereby dic- One that carries multiple genes is called polycistronic. The pro- tating which strand will be used as a template (figure 7.11). The teins encoded on a polycistronic message generally have related direction of polymerase movement can be likened to the flow of functions, allowing a cell to express related genes as one unit. a river. Because of this, the words “upstream” and “downstream” are used to describe relative positions of other sequences. As an Initiation of RNA Synthesis example, promoters are upstream of the genes they control. Transcription is initiated when RNA polymerase binds to a promoter (figure 7.10). The binding denatures (melts) a short Elongation of the RNA Transcript stretch of DNA, creating a region of exposed nucleotides that In the elongation phase, RNA polymerase moves along DNA, serves as a template for RNA synthesis. using the (2) strand as a template to synthesizea single- The part of RNA polymerase that recognizes the promoter stranded RNA molecule (see figure 7.10). As with DNA rep- is a loosely attached subunit called sigma (s) factor. A cell lication, nucleotides are added only to the 39 end; the reaction can produce various types of s factors, each recognizing differ- is fueled by hydrolyzing a high-energy phosphate bond of ent promoters. By controlling which s factors are made, cells the incoming nucleotide. When RNA polymerase advances, can transcribe specialized sets of genes as needed. The RNA it denatures a new stretch of DNA and allows the previous polymerases of eukaryotic cells and archaea use proteins called portion to renature (close). This exposes a new region of the transcription factors to recognize promoters. template so elongation can continue. Gene Expression FIGURE 7.10 The Process of RNA Synthesis Bacterial RNA polymerases have a sigma subunit that Transcription allows them to recognize a promoter (as illustrated); the RNA polymerases of eukaryotic cells and archaea use transcription factors to recognize promoters. Translation ? Which component of the bacterial RNA polymerase recognizes the promoter? 5' 3' 3' 5' Promoter Terminator RNA polymerase 1 Initiation 5' 3' RNA polymerase binds to the 3' 5' promoter and melts a short stretch of DNA. Template Sigma strand 2 Elongation Sigma factor dissociates from RNA 5' 3' polymerase, leaving the core 3' 5' enzyme to complete transcription. RNA is synthesized in the 5' to 3' Promoter direction as the enzyme adds 5' nucleotides to the 3' end of RNA the growing chain. GA C U G C T GAC 3 Termination 5' 3' When RNA polymerase encounters a terminator, it falls 3' 5' off the template and releases Promoter the newly synthesized RNA. 5' RNA polymerase dissociates from template.  Part I Life and Death of Microorganisms 185 The orientation of the promoter dictates the direction of transcription, and this determines Terminator Template which strand is used as a template. Terminator strand 5' 3' DNA 3' 5' Promoter 1 Promoter 2 Template strand RNA 3' 5' 5' 3' RNA FIGURE 7.11 The Promoter Orients RNA Polymerase The orientation of RNA polymerase determines which strand will be used as the template. In this diagram, the color of the RNA molecules indicates which DNA strand was used as the template. The light blue RNA was transcribed from the red DNA strand (and is therefore analogous in sequence to the blue DNA strand), whereas the pink RNA was transcribed from the blue DNA strand (and is therefore analogous in sequence to the red DNA strand). ? The light blue RNA strand is complementary to which DNA strand in this figure? To which DNA strand is the pink RNA strand complementary? Once elongation has proceeded far enough for RNA poly- The Role of mRNA merase to move beyond the promoter, another molecule of the The mRNA is a temporary copy of the information in DNA; enzyme can bind, initiating a new round of transcription. Thus, it carries encoded instructions for synthesis of a specific pro- a single gene can be transcribed repeatedly very quickly. tein, or in the case of a polycistronic message, a specific group of proteins. Recall that mRNA is composed of nucleotides, Termination of Transcription whereas proteins are composed of amino acids. The informa- Just as an initiation of transcription occurs at a distinct site tion in mRNA must be decoded into amino acids. This is done on the DNA, so does termination. When RNA polymerase using the genetic code, which correlates a series of three nucle- encounters a sequence called a terminator, it falls off the DNA otides, a codon, with one amino acid (table 7.4). The genetic template and releases the newly synthesized RNA. code is practically universal, meaning that with the exception of a few minor changes, it is used by all living things. Because a codon is a triplet of any combination of the four Translation nucleotides, there are 64 different codons (43). Three are stop Translation is the process of decoding the information carried codons, which will be discussed later. The remaining 61 trans- in the mRNA to synthesize the specified protein. The process late to the 20 different amino acids. This means that more than requires three major structures—mRNA, ribosomes (which one codon can code for a specific amino acid. For example, contain rRNA), and tRNAs—in addition to various other both ACA and ACG encode the amino acid threonine. Because components (table 7.3). of this redundancy, the genetic code is said to be degenerate. TABLE 7.3 Components of Translation in Bacteria Component Comment Anticodon Sequence of three nucleotides in a tRNA molecule that is complementary to a particular codon in mRNA. The anticodon allows the tRNA to recognize and bind to the appropriate codon. mRNA Type of RNA molecule that contains the genetic information decoded during translation. Polyribosome (polysome) Multiple ribosomes attached to a single mRNA molecule. Reading frame Grouping of a stretch of nucleotides into sequential triplets that code for amino acids; an mRNA molecule has three potential reading frames, but only one is typically used in translation. Ribosome Structure that facilitates the joining of amino acids during the process of translation; composed of protein and ribosomal RNA. The prokaryotic ribosome (70S) consists of a 30S and 50S subunit. Ribosome-binding site Sequence of nucleotides in mRNA to which a ribosome binds; the first time the codon for methionine (AUG) appears after that site, translation generally begins. rRNA Type of RNA molecule present in ribosomes. Start codon Codon at which translation is initiated; it is typically the first AUG after a ribosome-binding site. Stop codon Codon that terminates translation, signaling the end of the protein; there are three stop codons. tRNA Type of RNA molecule involved in interpreting the genetic code; each tRNA molecule carries a specific amino acid dictated by its anticodon. 186 Chapter 7 The Blueprint of Life, from DNA to Protein TABLE 7.4 The Genetic Code Second Letter First Third Letter U C A G Letter U UUU UCU UAU Tyr Tyrosine UGU U Phe Phenylalanine Cys Cysteine UUC UCC UAC UGC C Ser Serine UUA UCA UAA “Stop” UGA “Stop” A Leu Leucine UUG UCG UAG “Stop” UGG Trp Trytophan G C CUU CCU CAU CGU U His Histidine CUC CCC CAC CGC C Leu Leucine Pro Proline Arg Arginine CUA CCA CAA CGA A Gln Glutamine CUG CCG CAG CGG G A AUU ACU AAU AGU U Asn Asparagine Ser Serine AUC Ile Isoleucine ACC AAC AGC C Thr Threonine AUA ACA AAA AGA A Lys Lysine Arg Arginine AUG Met Methionine; “Start” ACG AAG AGG G G GUU GCU GAU GGU U Asp Aspartate GUC GCC GAC GGC C Val Valine Ala Alanine Gly Glycine GUA GCA GAA GGA A Glu Glutamate GUG GCG GAG GGG G The genetic code correlates a codon (a series of three nucleotides) to an amino acid, as shown in the table. In some cases, the codon encodes a “stop” signal instead of an amino acid. Many amino acids are specified by more than one codon. For example, threonine is specified by four codons, which differ only in the third nucleotide (ACU, ACC, ACA, and ACG). The nucleotide sequence of mRNA indicates where FIGURE 7.12 Nucleotide Sequences in mRNA the coding region begins and ends (figure 7.12). Gene Expression Direct Translation The ribosome begins to The site at which it begins is particularly critical assemble at the ribosome-binding site and starts Transcription translating at the start codon. Translation ends at the because the translation “ machinery” reads the stop codon. mRNA in groups of three nucleotides. As a con- sequence, any given sequence has three possible Translation ? In which direction does the ribosome move along RNA? reading frames, or ways in which triplets can be grouped (figure 7.13). If translation begins in the Region translated wrong reading frame, a very different, and generally non-functional, protein is synthesized. 5' 3' mRNA The Role of Ribosomes Ribosome- Start Stop binding site codon Translation codon Ribosomes serve as translation “machines”—structures that string amino acids together to make a polypeptide. A ribosome does this by aligning two amino acids and catalyzing the forma- tion of a peptide bond between them. ribosomes, p. 75 Protein Ribosomes also locate key punctuation sequences on the mRNA molecule, such as the points at which protein synthe- sis should begin. The ribosome then moves along the mRNA in the 59 to 39 direction, “presenting” each codon in a sequen- Phe Ser His Cys Glu Val Gly Tyr Tyr tial order for decoding while maintaining the correct reading Ser Pro Leu Ala Gln Ser Met frame.  Part I Life and Death of Microorganisms 187 Reading frame #1 C U G G C A U U G C C U U A U Pro Leu Ala Leu Pro Tyr amino acid tRNA Reading frame #2 C U G G C A U U G C C U U A U hydrogen bond Trp His Cys Leu Anticodon Reading frame #3 C U G G C A U U G C C U U A U G G C C C G Gly Ile Ala Leu 5' 3' mRNA FIGURE 7.13 Reading Frames A nucleotide sequence has three Codon potential reading frames, but only one is typically used for translation. FIGURE 7.14 The Structure of Transfer RNA (tRNA) Three- ? Why is it important that the correct reading frame is used? dimensional illustration of tRNA. The amino acid that the tRNA carries is dictated by its anticodon. The tRNA that recognizes the mRNA codon CCG carries the amino acid proline—as specified by the genetic Prokaryotic ribosomes are composed of a 30S subunit code. and a 50S subunit, each made up of protein and ribosomal RNA (rRNA) (see figures 3.42 and 10.8); the “S” stands for ? A tRNA that has the anticodon GAG carries which amino acid? Svedberg unit, which is a measure of size. Svedberg units are not additive, which is why the 70S ribosome can have 30S that site usually serves as the start codon. The complete ribo- and 50S subunits. ribosomal subunits, p. 265 some assembles there, joined by an initiating tRNA that car- MicroByte ries a chemically altered form of the amino acid methionine Several types of antibiotics, including tetracycline and azithromycin, (N-formylmethionine, or f-Met). The position of the first AUG interfere with the function of the bacterial 70S ribosome. is critical, as it determines the reading frame used for transla- tion of the remainder of that protein. Note that AUG functions The Role of Transfer RNAs as a start codon only when preceded by a ribosome-binding A tRNA is an RNA molecule that carries an amino acid to be site; at other sites, it simply encodes methionine. used in translation. Each tRNA is folded into a complex three- dimensional shape held together by hydrogen bonds, and a Elongation of the Polypeptide Chain specific amino acid is attached at one end (figure 7.14). When The ribosome has two sites to which amino acid–carrying a tRNA recognizes and base-pairs with a given codon in an tRNAs can bind: the P-site and the A-site. At the start of trans- mRNA molecule being translated, the ribosome transfers the lation, the initiating tRNA carrying the f-Met occupies the amino acid carried by that tRNA onto the end of the newly P-site (figure 7.15). A tRNA that recognizes the next codon on forming polypeptide. The recognition between the tRNA and the mRNA then fills the unoccupied A-site. Once both sites are the mRNA occurs because each tRNA has an anticodon— filled, an rRNA creates a peptide bond between the two amino a group of three nucleotides complementary to a codon in acids carried by the tRNAs. This transfers the amino acid from the mRNA. Thus, the codon sequence in mRNA determines the initiating tRNA to the amino acid carried by the incoming the sequence of amino acids in the protein, according to the tRNA. genetic code (table 7.4). After the initiating tRNA has donated its amino acid to Once a tRNA molecule has delivered its amino acid dur- the tRNA in the A-site, the ribosome advances a distance of ing translation, it can be recycled. An enzyme in the cyto- one codon, moving along the mRNA in a 59 to 39 direction. As plasm recognizes the tRNA and then attaches the appropriate this happens, the initiating tRNA is released through a region amino acid. called the E-site. The remaining tRNA, which now carries both amino acids, occupies the P-site. The A-site is tempo- Initiation of Translation rarily empty. A tRNA that recognizes the codon in the A-site In prokaryotes, translation begins as the mRNA molecule is still quickly attaches there, and the process repeats. being synthesized. Part of the ribosome binds to a sequence in Once translation of a gene has progressed far enough mRNA called the ribosome-binding site; the first AUG after for the ribosome to clear the initiating sequences, another 188 Chapter 7 The Blueprint of Life, from DNA to Protein f-Met P-site Initiation The initiating tRNA, carrying the amino E-site A-site acid f-Met, base-pairs with the start codon and occupies the P-site. U A C A U G C C G U A C G A A G A U U A C U A G G A U 5' 3' mRNA f-Met Pro A tRNA that recognizes the next codon then fills the unoccupied A-site. U A C G G C A U G C C G U A C G A A G A U U A C U A G G A U 5' 3' Peptide bond f-Met Pro The ribosome catalyzes the joining of the amino acid carried by the tRNA in the P-site to the one carried by the tRNA in the A-site. U A C G G C A U G C C G U A C G A A G A U U A C U A G G A U 5' 3' (a) FIGURE 7.15 The Process of Translation (a) Initiation. (b) Elongation. (c) Termination. ? What is the importance of the anticodon on the tRNA?  Ty f-Met r Pro Elongation The ribosome advances a distance of P-site one codon. The tRNA that occupied the P-site exits through the E-site and the C E-site A-site tRNA that was in the A-site occupies the A U P-site. A tRNA that recognizes the next A codon quickly fills the empty A-site. U G G G C A U G C C G U A C G A A G A U U A C U A G G A U 5' 3' Ribosome moves along mRNA. f-Met G lu Pro Tyr The ribosome continues advancing along C the mRNA in the 5' to 3' direction, moving G G one codon at a time. C U U A U G A U G C C G U A C G A A G A U U A C U A G G A U 5' 3' (b) f-Met Pro Tyr Glu Asp Tyr Termination A Translation continues until a stop codon U C is reached, signaling the end of the process. No tRNA molecules recognize a stop codon. A U G A U G C C G U A C G A A G A U U A C U A G G A U 5' 3' The components disassemble releasing the newly formed polypeptide. Pro Tyr f-Met Glu Tyr Asp (c) 190 Chapter 7 The Blueprint of Life, from DNA to Protein Gene Expression Transcription Translation RNA polymerase 5' 3' 3' 5' DNA mRNA strand Ribosome Promoter Terminator 5' Polypeptide Direction of ribosome movement Ribosome- Start binding site codon FIGURE 7.16 In Prokaryotes, Translation Begins as the mRNA Molecule Is Still Being Synthesized Ribosomes begin translating the 59 end of mRNA before transcription is complete. More than one ribosome can be translating the same mRNA molecule. ? Why is the position of the first AUG after the ribosome-binding site critical? ribosome can bind. Thus, at any one time, multiple ribosomes MicroAssessment 7.3 can be translating a single mRNA molecule (figure 7.16). This Gene expression involves transcription and translation. In allows maximal protein synthesis from a single mRNA tem- transcription, RNA polymerase synthesizes RNA in the 59 plate. The assembly of multiple ribosomes attached to a single to 39 direction, using one strand of DNA as a template. In mRNA molecule is called a polyribosome, or a polysome. translation, ribosomes synthesize proteins, using the nucleotide sequence of mRNA to determine the amino acid sequence of Termination of Translation the encoded protein. The correct amino acid for a given codon Elongation of the polypeptide terminates when the ribosome is delivered by tRNAs. After synthesis, many proteins are reaches a stop codon, a codon not recognized by a tRNA. modified in some way. At this point, enzymes free the polypeptide by breaking the 7. How does a promoter dictate which DNA strand is used as covalent bond that joins it to the tRNA. The ribosome falls the template? off the mRNA, dissociating into its two component subunits 8. What is the role of tRNA in translation? (30S and 50S). The subunits can then be reused to initiate 9. Could two mRNAs have different nucleotide sequences and translation at other sites. yet code for the same protein? Explain your answer. + Post-Translational Modification Polypeptides must often be modified after they are synthe- sized in order to become functional. For example, some must be folded into a specific three-dimensional structure, a process 7.4 Differences Between that requires the assistance of proteins called chaperones. Eukaryotic and Prokaryotic Polypeptides destined for transport through the cytoplas- mic membrane also must be modified. These have a signal Gene Expression sequence, a characteristic series of hydrophobic amino acids Learning Outcome at their amino terminal end, which “tags” them for transport 6. Describe four differences between prokaryotic and eukaryotic (see figure 3.30). The signal sequence must be removed by gene expression. proteins in the membrane.  Part I Life and Death of Microorganisms 191 TABLE 7.5 Major Differences Between Prokaryotic and Eukaryotic Transcription and Translation Prokaryotes Eukaryotes mRNA is not processed. A cap is added to the 59 end of mRNA, and a poly A tail is added to the 39 end. mRNA does not contain introns. mRNA contains introns, which are removed by splicing. Translation of mRNA begins as it is being transcribed. The mRNA transcript is transported out of the nucleus so that it can be translated in the cytoplasm. mRNA is often polycistronic; translation usually begins at the first AUG mRNA is monocistronic; translation begins at the first AUG. codon that follows a ribosome-binding site. Eukaryotes differ significantly from prokaryotes in several MicroAssessment 7.4 aspects of transcription and translation (table 7.5). Eukary- Eukaryotic pre-mRNA must be processed, which involves otic mRNA, for example, is synthesized in a precursor capping, polyadenylation, and splicing. In eukaryotic cells, the form, called pre-mRNA. The pre-mRNA must be processed mRNA must be transported out of the nucleus before it can be (altered) both during and after transcription to form mature translated in the cytoplasm. Eukaryotic mRNA is monocistronic. mRNA. Shortly after transcription begins, the 59 end of the 10. What is an intron? pre-mRNA is capped by adding a methylated guanine deriva- 11. Would a deletion of two base pairs have a greater tive. This cap binds specific proteins that stabilize the tran- consequence if it occurred in an intron rather than in an script and enhance translation. The 39 end of the molecule is exon? + also modified, even before transcription has been terminated. This process, polyadenylation, cleaves the transcript at a spe- cific sequence and then adds about 200 adenine derivatives to the new 39 end. This creates a poly A tail, which is thought to stabilize the transcript as well as enhance translation. Another Eukaryotic DNA contains important modification is splicing, which removes spe- introns, which interrupt coding regions (exons). cific segments of the transcript (figure 7.17). Splic- ing is necessary because eukaryotic genes are often Exon Intron Exon Intron Exon interrupted by non-coding sequences. These interven- ing sequences, introns, are transcribed along with the Eukaryotic DNA expressed regions, exons, and must be removed from Transcription generates pre-mRNA to create functional mRNA. pre-mRNA (precursor mRNA) The mRNA in eukaryotic cells must be transported that contains introns. A cap and poly A tail are then added. out of the nucleus before it can be translated in the cyto- plasm. Thus, unlike in prokaryotes, the same mRNA Cap Poly A tail molecule cannot be synthesized and translated at the same time or even in the same cellular location. The mRNA of eukaryotes is generally monocistronic,