Biology Notes PDF - DNA, RNA, Nucleotides, Replication, Transcription

DNA and RNA are polymers of nucleotides By the time Hershey and Chase performed their experiments, much was already known about DNA. Scientists had identified all its atoms and knew how they were covalently bonded to one another. What was not understood was the specific arrangement of atoms that gav...

DNA and RNA are polymers of nucleotides By the time Hershey and Chase performed their experiments, much was already known about DNA. Scientists had identified all its atoms and knew how they were covalently bonded to one another. What was not understood was the specific arrangement of atoms that gave DNA its unique properties-the capacity to store genetic information, copy it, and pass it from generation to generation. However, only one year after Hershey and Chase published their results, scientists figured out the three-dimensional structure of DNA and the basic strategy of how it works. We will examine that momentous discovery in Module 10.3, but first, let's look at the underlying chemical structure of DNA and its chemical cousin RNA. Recall from Module 3.15 that DNA and RNA are nucleic acids, consisting of long chains (polymers) of chemical units (monomers) called nucleotides. Figure 10.2A shows four representations of various parts of the same molecule. At left is a view of a DNA double helix. One of the strands is opened up (center) to show two different views of an individual DNA polynucleotide, a nucleotide polymer (chain). The view on the far right zooms into a single nucleotide from the chain. Each type of DNA nucleotide has a different nitrogen-containing base: adenine (A), cytosine (C), thymine (T), or guanine (G). Because nucleotides can occur in a polynucleotide in any sequence and polynucleotides vary in length from long to very long, the number of possible polynucleotides is enormous. The chain shown in this figure has the sequence ACTGG, only one of many possible arrangements of the four types of nucleotides that make up DNA. Looking more closely at our polynucleotide, we see in the center of Figure 10.2A that each nucleotide consists of three components: a nitrogenous base (in DNA: A, C, T, or G), a sugar (blue), and a phosphate group (yellow). The nucleotides are joined to one another by covalent bonds between the sugar of one nucleotide and the phosphate of the next. This results in a sugar-phosphate backbone, a repeating pattern of sugar-phosphate-sugar-phosphate. The nitrogenous bases are arranged like ribs that project from the backbone. Examining a single nucleotide in even more detail (on the right in Figure 10.2A), you can see the chemical structure of its three components. The phosphate group has a phosphorus atom (P) at its center and is the source of the word acid in nucleic acid. The sugar has five carbon atoms, shown in red here for emphasis- four in its ring and one extending above the ring. The ring also includes an oxygen atom. The sugar is called deoxyribose because, compared with the sugar ribose, it is missing an oxygen atom. (Notice that the Catom in the lower right corner of the ring is bonded to an H atom instead of to an -OH group, as it is in ribose; see Figure 10.2C. Hence, DNA is "deoxy" —which means "without an oxygen" —compared to RNA.) The full name for DNA is deoxyribonucleic acid, with the nucleic portion of the word referring to DNA's location in the nuclei of eukaryotic cells. Each nitrogenous base (thymine, in our example at the right in Figure 10.2A) has a single or double ring consisting of nitrogen and carbon atoms with various functional groups attached. Recall from Module 3.2 that a functional group is a chemical group that affects a molecules function by participating in specific chemical reactions. In the case of DNA, the main role of the functional groups is to determine which other kind of bases each base can hydrogen-bond with. For example, the NH, group hanging off cytosine is capable of forming a hydrogen bond to the C=0 group hanging off guanine, but not with the NH, group protruding from ade-nine. The chemical groups of the bases are therefore responsible for DNA's most important property, which you will learn more about in the next module. In contrast to the acidic phosphate group, nitrogenous bases are basic, hence their name. The four nucleotides found in DNA differ only in the structure of their nitrogenous bases (Figure 10.2B). At this point, the structural details are not as important as the fact that the bases are of two types. Thymine (T) and cytosine (C) are single-ring structures called pyrimidines. Adenine (A) and guanine (G) are larger, double-ring structures called purines. The one-letter abbreviations can be used either for the bases alone or for the nucleotides containing them. What about RNA (Figure 10.2C)? As its name—ribonucleic acid-implies, its sugar is ribose rather than deoxyribose. Notice the ribose in the RNA nucleotide in Figure 10.2C; unlike deoxyribose, the sugar ring has an -OH group attached to the C atom at its lower-right corner. Another difference between RNA and DNA is that instead of thymine, RNA has a nitrogenous base called uracil (U). (You can see the structure of uracil in Figure 10.2C; it is very similar to thymine.) Except for the presence of ribose and uracil, an RNA polynucleotide chain is identical to a DNA polynucleotide chain. Figure 10.2D is a computer graphic of a piece of RNA polynucleotide about 20 nucleotides long. In this 3-D view, each sphere represents an atom, and notice that the color scheme is the same as in the other figures in this module. The yellow phosphate groups and blue ribose sugars make it easy to spot the sugar-phosphate In this module, we reviewed the structure of the nucleic acids DNA and RNA. In the next module, we'll see how two DNA polynucleotides join together in a molecule of DNA. DNA Replication One of biology's overarching themes— the relationship between structure and function-is evident in the double helix. The idea that there is specific pairing of bases in DNA was the flash of inspiration that led Watson and Crick to the correct structure of the double helix. At the same time, they saw the functional significance of the base-pairing rules. They ended their classic 1953 paper with this statement: "It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material." The logic behind the Watson-Crick proposal for how DNA is copied-by specific pairing of complementary bases-is quite simple. You can see this by covering one of the strands in the parental DNA molecule in Figure 10.4A. You can determine the sequence of bases in the covered strand by applying the base-pairing rules to the unmasked strand: A pairs with T (and T with A), and G pairs with C (and C with G). Watson and Crick predicted that a cell applies the same rules when copying its genes. As shown in Figure 10.4A, the two strands of parental DNA (blue) separate. Each then becomes a template for the assembly of a complementary strand from a supply of free nucleotides (gray) that is always available within the nucleus. The nucleotides line up one at a time along the template strand in accordance with the base-pairing rules. Enzymes link the nucleotides to form the new DNA strands. The completed new molecules, identical to the parental molecule, are known as daughter DNA (although no gender should be inferred). Watson and Crick's model predicts that when a double helix replicates, each of the two daughter molecules will have one old strand, which was part of the parental molecule, and one newly created strand. This model for DNA replication is known as the semiconservative model because half of the parental molecule is maintained (conserved) in each daughter molecule. The semiconservative model of replication was confirmed by experiments performed in the 1950s. Although the general mechanism of DNA replication is conceptually simple, the actual process is complex, requiring the coordination of more than a dozen enzymes and other proteins. Some of the complexity arises from the need for the helical DNA molecule to untwist as it replicates and for the two new strands to be made roughly simultaneously (Figure 10.4B). Another challenge is the speed of the process. E. coli, with about 4.6 million DNA base pairs, can copy its entire genome in less than an hour. Humans, with over 6 billion base pairs in 46 diploid chromosomes, require only a few hours. And yet, the process is amazingly accurate; typically, only about one DNA nucleotide per several billion is incorrectly paired. In the next module, we take a closer look at the mechanisms of DNA replication that allow it to proceed with such speed and accuracy. The DNA molecule of a eukaryotic chromosome has many origins where replication can start simultaneously. Thus, hundreds or thousands of bubbles can be present at once, shortening the total time needed for replication. Eventually, all the bubbles fuse, yielding two completed daughter DNA molecules (see the bottom of Figure 10.5A). Figure 10.5B shows the molecular building blocks of a tiny segment of DNA, reminding us that the DNA's sugar-phosphate backbones run in opposite directions. Notice that each strand has a 3' ("three-prime") end and a 5' ("five-prime") end. The primed numbers refer to the carbon atoms of the nucleotide sugars. At one end of each DNA strand, the sugar's 3' carbon atom is attached to an —OH group; at the other end, the sugar's 5' carbon is attached to a phosphate group. The opposite orientation of the strands is important in DNA replication. The enzymes that link DNA nucleotides to a growing G daughter strand, called DNA polymerases, add nucleotides only to the 3' end of the strand, never to the 5' end. Thus, a daughter DNA strand can only grow in OH the 5' → 3' direction. You see the consequences of this enzyme specificity in Figure A Figure 10.5B The opposite orientations of DNA strands 10.5C, where the forked structure represents one side of a replication bubble. One of the daughter strands (shown in gray) can be synthesized in one continuous piece by a DNA polymerase working toward the forking point of the parental DNA. However, to make the other daughter strand, polymerase molecules must work outward from the forking point. The only way this can be accomplished is if the new strand is synthesized in short pieces as the fork opens up. These pieces are called Okazaki fragments, after the Japanese husband-and-wife team of molecular biologists who discovered them. Another enzyme, called DNA ligase, then links, or ligates, the pieces together into a single DNA strand. In addition to their roles in adding nucleotides to a DNA chain, DNA polymerases carry out a proofreading step that quickly removes nucleotides that have base-paired incorrectly during replication. DNA polymerases and DNA ligase are also involved in repairing DNA damaged by harmful radiation, such as ultraviolet light and X-rays, or toxic chemicals in the environment, such as those found in tobacco smoke. DNA replication ensures that all the somatic cells in a multicellular organism carry the same genetic information. It is also the means by which genetic instructions are copied for the next generation of the organism. In the next module, we begin to pursue the connection between DNA instructions and an organism's phenotypic traits. Transcription In eukaryotic cells, transcription, the transfer of genetic information from DNA to RNA, occurs in the nucleus. (The nucleus, after all, contains the DNA; see Figure 10.6A for a review.) An RNA molecule is transcribed from a DNA tem- plate by a process that resembles the synthesis of a DNA strand during DNA replication (see Module 10.4). Figure 10.9A is a close-up view of the process of transcrip- tion. As with replication, the two DNA strands must first separate at the place where the process will start. In transcription, however, only one of the DNA strands serves as a template for the newly forming RNA molecule; the other strand is unused. The nucleotides that make up the new RNA molecule take their place one at a time along the DNA template strand by forming hydrogen bonds with the nucleotide bases there. Notice that the RNA nucleotides follow the same base-pairing rules that govern DNA replication, except that U, rather than T, pairs with A. The RNA nucleotides are linked by the tran-scription enzyme RNA polymerase, symbolized in the figure by the large gray shape. Figure 10.9B is an overview of the transcription of an en- tire prokaryotic gene. (We focus on prokaryotes here; eukary- otic transcription occurs via a similar but more complex process.) Specific sequences of nucleotides along the DNA mark where transcription of a gene begins and ends. The "start transcribing" signal is a nucleotide sequence called a promoter. A promoter is a specific binding site for RNA polymerase and determines which of the two strands of the DNA double helix is used as the template in transcription. 1 The first phase of transcription, called initiation, is the attachment of RNA polymerase to the promoter and the start of RNA synthesis. 2 During a second phase of transcription, elongation, the RNA grows longer. As RNA synthesis contin- ues, the RNA strand peels away from its DNA template, allowing the two separated DNA strands to come back together in the region already transcribed. 3 Finally, in the third phase, termination, the RNA polymerase reaches a se- quence of bases in the DNA template called a terminator. This sequence signals the end of the gene; at that point, the polymerase molecule detaches from the RNA molecule and In addition to producing RNA that encodes amino acid se-quences, transcription makes two other kinds of RNA that are involved in building polypeptides. We discuss these three kinds of RNA-messenger RNA, transfer RNA, and riboso- mal RNA-in the next three modules. Translation The kind of RNA that encodes amino acid sequences is called messenger RNA (mRNA) because it conveys genetic messages from DNA to the translation machinery of the cell. Messenger RNA is transcribed from DNA, and the information in the mRNA is then translated into polypeptides. In prokaryotic cells, which lack nuclei, transcription and translation occur in the same place: the cytoplasm. In eukaryotic cells, however, mRNA molecules must exit the nucleus via the nuclear pores and enter the cytoplasm, where the machinery for polypeptide synthesis is located. Before leaving the nucleus as mRNA, eukaryotic transcripts are modified, or processed, in several ways (Figure 10.10). One kind of RNA processing is the addition of extra nucleotides to the ends of the RNA transcript. These additions include a small cap (a single G nucleotide) at one end and a long tail (a chain of 50 to 250 A nucleotides) at the other end. The cap and tail (yellow in the figure) facilitate the export of the mRNA from the nucleus, protect the mRNA from attack by cellular en-zymes, and help ribosomes bind to the mRNA. The cap and tail themselves are not translated into protein. Another type of RNA processing is made necessary in eukaryotes by noncoding stretches of nucleotides that interrupt the nucleotides that actually code for amino acids. It is as if unintelligible sequences of letters were randomly interspersed in an otherwise intelligible document. Most genes of plants and animals, it turns out, include such internal noncoding regions, which are called introns ("intervening sequences"). The coding regions— the parts of a gene that are expressed-are called exons. As Figure 10.10 shows, both exons (darker color) and introns (lighter color) are transcribed from DNA into RNA. However, before the RNA leaves the nucleus, the introns are removed, and the exons are joined to produce an mRNA molecule with a continuous coding sequence. (The short non-coding regions just inside the cap and tail are considered parts of the first and last exons.) This cutting-and-pasting process is called RNA splicing. In most cases, RNA splicing is catalyzed by a complex of proteins and small RNA molecules, but sometimes the RNA transcript itself catalyzes the process. In other words, RNA can sometimes act as an enzyme that removes its own introns! As we will see in the next chapter (in Module 11.4), RNA splicing also provides a means to produce multiple polypeptides from a single gene. As we have discussed, translation is a conversion between different languages-from the nucleic acid language to the protein language-and it involves more elaborate machinery than transcription. The first important ingredient required for translation is the processed mRNA. Once it is present, the machinery used to translate mRNA requires enzymes and sources of chemical energy, such as ATP. In addition, translation requires two heavy-duty components: ribosomes and a kind of RNA called transfer RNA, the subject of the next module. Translation of any language requires an interpreter, someone or something that can recognize the words of one language and convert them to another. Translation of a genetic message carried in mRNA into the amino acid language of proteins also requires an interpreter. To convert the words of nucleic acids (codons) to the amino acid words of proteins, a cell employs a molecular interpreter, a special type of RNA called transfer RNA (tRNA). A cell that is producing proteins has in its cytoplasm a supply of amino acids, either obtained from food or made from other chemicals. But amino acids themselves cannot recognize the codons in the mRNA. The amino acid tryptophan, for ex-ample, is no more attracted by codons for tryptophan than by any other codons. It is up to the cell's molecular interpreters, tRNA molecules, to match amino acids to the appropriate codons to form the new polypeptide. To perform this task, RNA molecules must carry out two functions: (1) picking up the appropriate amino acids and (2) recognizing the appropriate codons in the mRNA. The unique structure of tRNA molecules enables them to perform both tasks. Each amino acid is joined to the correct tRNA by a specific enzyme. There is a family of 20 versions of these enzymes, one enzyme for each amino acid. Each enzyme specifically binds one type of amino acid to all tRNA molecules that code for that amino acid, using a molecule of ATP as energy to drive the re-action. The resulting amino acid-tRNA complex can then furnish its amino acid to a growing polypeptide chain, a process that we describe in Module 10.12. The computer graphic in Figure 10.11B shows a tRNA molecule (green) and an ATP molecule (purple) bound to the enzyme molecule (blue). (To help you see the two distinct molecules, the tRNA molecule is shown with a stick representation, while the enzyme is shown as space-filling spheres.) In this figure, you can see the proportional sizes of these three molecules. The amino acid that would attach to the tRNA is not shown; it would be less than half the amino acid. Once an amino acid is attached to its appropriate tRNA, it can be incorporated into a growing polypeptide chain. This is accomplished within ribosomes, the cellular structures directly responsible for the synthesis of protein. We examine ribosomes in the next module. Figure 10.11A shows two representations of a tRNA molecule. The structure on the left shows the backbone and bases, with hydrogen bonds between bases shown as dashed magenta lines. The structure on the right is a simplified schematic that emphasizes the most important parts of the structure. Notice from the structure on the left that a tRNA molecule is made of a single strand of RNA-one polynucleotide chain— consisting of about 80 nucleotides. By twisting and folding upon itself, tRNA forms several double-stranded regions in which short stretches of RNA base-pair with other stretches via hydrogen bonds. A single-stranded loop at one end of the folded molecule contains a special triplet of bases called an anticodon. The anticodon triplet is complementary to a codon triplet on mRNA. During translation, the anticodon on tRNA recognizes a particular codon on mRNA by using base-pairing rules. At the other end of the tRNA molecule is a site where one specific kind of amino acid can attach. In the modules that follow, we represent tRNA with the simplified shape shown on the right in Figure 10.11A. This shape emphasizes the two parts of the molecule- the anticodon and the amino acid attachment site— that give ERNA its ability to match a particular nucleic acid word (a codon in mRNA) with its corresponding protein word (an amino acid). Although all tRNA molecules are similar, there is a slightly different variety of tRNA for each amino acid. We have now looked at many of the components a cell needs to carry out translation: instructions in the form of mRNA molecules, tRNA to interpret the instructions, a supply of amino acids and enzymes (for attaching amino acids to tRNA), and ATP for energy. The final components are the ribosomes, structures in the cytoplasm that position mRNA and tRNA close together and catalyze the synthesis of polypeptides. A ribosome consists of two sub-units, each made up of proteins and a kind of RNA called ribosomal RNA (rRNA). In Figure 10.12A, you can see the actual shapes and relative sizes of the ribosomal subunits. You cannalso see where mRNA, tRNA, and the growing polypeptide are located during translation. The ribosomes of prokaryotes and eukaryotes are very similar in function, but those of eukaryotes are slightly larger and different in composition. The differences are medically significant. Certain antibiotic drugs can inactivate prokaryotic ribosomes while leaving eukaryotic ribosomes unaffected. These drugs, such as tetracycline and streptomycin, are used to combat bacterial infections. The simplified drawings in Figures 10.12B and 10.12C indicate how tRNA anticodons and mRNA codons fit together on ribosomes. As Figure 10.12B shows, each ribosome has a binding site for mRNA and the two main binding sites (P and A) for tRNA. Figure 10.12C shows tRNA molecules occupying these two sites. The subunits of the ribosome act like a vise, holding the tRNA and mRNA molecules close together, allowing the amino acids carried by the tRNA to be connected into the polypeptide chain. Translation can be divided into the same three phases as tran-scription: initiation, elongation, and termination. The process of polypeptide initiation brings together the mRNA, a tRNA bearing the first amino acid, and the two subunits of a ribosome. As shown in Figure 10.13A, an mRNA molecule is longer than the genetic message it carries. The light pink nucleotides at either end of the molecule are not part of the message, but help the mRNA to bind to the ribosome. The initiation process establishes exactly where translation will begin, ensuring that the mRNA codons are translated into the correct sequence of amino acids. Initiation occurs in two steps (Figure 10.13B). An mRNA molecule binds to a small ribosomal subunit. A special initiator RNA binds to the specific codon, called the start codon, where translation is to begin on the mRNA molecule. Next, a large ribosomal subunit binds to the small one, creating a functional ribosome. The initiator tRNA fits into one of the two tRNA binding end of the sites on the ribosome. This site, called the P site, will hold the growing polypeptide. The other tRNA binding site, called the A site, is vacant and ready for the next amino acid. Once initiation is complete, amino acids are added one by one to the first amino acid. Each addition occurs in a three-step elongation process (Figure 10.14): Codon recognition. The anticodon of an incoming tRNA molecule, carrying its amino acid, pairs with the mRNA codon in the A site of the ribosome. 2 Peptide bond formation. The polypeptide separates from the tRNA in the P site and attaches by a new peptide bond to the amino acid carried by the tRNA in the A site. The ribosome catalyzes formation of the peptide bond, adding one more amino acid to the growing polypeptide chain. Translocation. The P site tRNA now leaves the ribosome, and the ribosome translocates (moves) the remaining tRNA in the A site, with the growing polypeptide, to the P site. The codon and anticodon remain hydrogen-bonded, and the mRNA and tRNA move as a unit. This movement brings into the A site the next mRNA codon to be translated, and the process can start again with step 1. Elongation continues until a stop codon reaches the ribosomes A site. Stop codons-UAA, UAG, and UGA-do not code for amino acids but instead act as signals to stop transla-tion. This is the termination stage of translation. The completed polypeptide is freed from the last tRNA, and the ribosome splits back into its separate subunits. Mutations Many inherited traits can be understood in molecular terms. For instance, sickle-cell disease (see Module 9.13) can be traced Normal gene through a difference in a protein to one tiny change in a gene. In one of the two kinds of polypeptides in the hemoglobin protein, mRNA Protein an individual with sickle-cell disease has a single different amino acid-wine (Val) instead of glutamate (Glu). This difference is caused by the change of a single nucleotide in the coding strand of DNA (Figure 10.16A). In the double helix, one nucleotide pair Nucleotide substitution Any change in the nucleotide sequence of DNA is called a mut lica. Mutations can involve large regions of a chromosome er just a single nucleotide pair, as in sickle-cell disease. Here we consider how mutations involving only one or a few nucieotide pairs can affect gene translation. Mutations within a gene can be divided into two general categories: nucleotide substitutions, and nucleotide insertions or deletions (Figure 10.16B). A nucleotide substitution is the replacement of one nucleotide and its base-pairing partner with another pair of nucleotides. For example, in the second row in Figure 10.16B, A replaces G in the fourth codon of the mRNA. What effect can a substitution have? Because the genetic code is redundant, some substitution mutations have no effect at all. For example, if a mutation causes an mRNA codon to change from GAA to GAG, no change in the protein product would result because GAA and GAG both code for the same amino acid (Glu; see Figure 10.8A). Such a change is called a silent mutation. Other substitutions, called missense mutations, do change the amino acid coding. For example, if a mutation causes an mRNA codon to change from GGC to AGC, as in the second row of Figure 10.16B. The resulting protein will have a serine (Ser) instead of a glycine (Gly) at this position. Some missense mutations have little or no effect on the shape or function of the resulting protein, but others, as in the case of sickle- cell disease, prevent the protein from performing its normal function. Occasionally, a nucleotide substitution leads to an improved protein that enhances the success of the mutant organism and its descendants. Much more often, though, mutations are harmful. Some substitutions, called nonsense mutations, change an amino acid codon into a stop codon. For example, if an AGA (Arg) codon is mutated to a UGA (stop) codon, the result will be a prematurely terminated pro-tein, which probably will not function properly. Mutations involving the insertion or deletion of one or more nucleotides in a gene often have disastrous effects. Because mRNA is read as a series of nucleotide triplets (codons) during translation, adding or subtracting nucleotides may alter the reading frame (triplet grouping) of the message. All the nucleotides that are "downstream" of the insertion or deletion will be regrouped into different codons (Figure 10.16B, bottom two rows). The result will most likely be a nonfunctional polypeptide. The production of mutations, called mutagenesis, can occur in a number of ways. Spontaneous mutations are due to errors that occur during DNA replication or recombination. Other mutations are caused by physical or chemical agents, called mutagens. High-energy radiation, such as X-rays or ultraviolet light, is a physical mutagen. One class of chemical mutagens consists of chemicals that are similar to normal DNA bases but pair incorrectly or are otherwise disruptive when incorporated into DNA. For example, the anti-AIDS drug AZT works because its structure is similar enough to thymine that viral polymerases incorporate it into newly synthesized DNA, but different enough that the drug blocks further replication. Although mutations are often harmful, they are also extremely useful, both in nature and in the laboratory. It is because of mutations that there is such a rich diversity of genes in the living world, a diversity that makes evolution by natural selection possible. Mutations are also essential tools for geneti-cists. Whether naturally occurring (as in Mendel's peas) or created in the laboratory (Morgan used X-rays to make most of his fruit fly mutants; see Module 9.18), mutations create the different alleles needed for genetic research.