`
`~
`
`Wlonald Voet
`
`Judith, 6. Voet Charlotte W. Pr
`
`Gilead 2010
`I-MAKv. Gilead
`IPR2018-00125
`
`Gilead 2010
`I-MAK v. Gilead
`IPR2018-00125
`
` 1
`
`

`

` Biochemistry
`
`Seattle Pacific University
`
`
` Judith G. Voet
`
`
`Swarthmore College, Emeritus
`
`
`
`
`Donald Voet
`University of Pennsylvania
`
`
`
` 2
`
`

`

`scholar, teacher, friend
`
`This book was set in 10/12 Times Ten by Aptara and printed and bound by
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`and Richard Henderson, MRC Laboratory of Molecular Biology, U.K.; Thomas Steitz, Yale
`University.
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`
`
`
`
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`
`
`Copyright © 2008 by Donald Voet, Judith G. Voet, and Charlotte W.Pratt. All rights
`reserved,
`
`Nopart of this publication may be reproduced, storedin a retrieval system ortransmitted
`in any form or by any means,electronic, mechanical, photocopying, recording, scanning or
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`To order books orfor customerservice, please call 1-800-CALL WILEY (225-5945).
`
`ISBN-13
`
`978-0470-12930-2
`
`Printed in the United States of America
`1098765
`
` 3
`
`

`

`Nucleotides,
`Nucleic Acids,
`and Genetic
`Information
`
`A DNA molecule consists of two strands that wind around a central
`axis, shown here as a glowing wire. [lllustration, Irving Geis. Image
`from the Irving Geis Collection/Howard Hughes Medical Institute.
`Rights owned by HHMI. Reproduction by permission only.]
`
`Aucleic acids DNA and RNA,are the primary players in the storage
`
`MEDIA RESOURCES
`(Available at www.wiley.com/college/voet)
`Guided Exploration 1. Overview of transcription and translation
`Guided Exploration 2. DNA sequence determination by the chain-terminator
`method
`Guided Exploration 3. PCR andsite-directed mutagenesis
`Interactive Exercise 1. Three-dimensional structure of DNA
`Animated Figure 3-26. Construction of a recombinant DNA molecule
`Animated Figure 3-27. Cloning with bacteriophage
`Animated Figure 3-30. Site-directed mutagenesis
`. Kinemage Exercise 2-1. Structure of DNA
`_Kinemage Exercise 2-2. Watson—Crick base pairs
`Bioinformatics Exercises Chapter 3. Databases for the Storage and “Mining” of
`Genome Sequences
`soncnnesininainne
`
`CHAPTER CONTENTS
`
`Nucleotides
`
`Introduction to Nucleic Acid
`Structure
`
`A. Nucleic Acids Are Polymers of Nucleotides
`B. DNA Forms a Double Helix
`Cc. RNA Is a Single-Stranded Nucleic Acid
`Overview of Nucleic Acid Function
`A. DNA Carries Genetic Information
`B. Genes Direct Protein Synthesis
`.
`.
`.
`Nucleic Acid Sequencing
`A. Restriction Endonucleases Cleave DNA at
`Specific Sequences
`. Electrophoresis Separates Nucleic Acids
`According to Size
`DNA Is Sequenced by the Chain-Terminator
`Method
`D. Entire Genomes Have Been Sequenced
`E. Evolution Results from Sequence Mutations
`Manipulating DNA
`A. Cloned DNA Is an Amplified Copy
`B. DNALibraries Are Collections of Cloned
`DNA
`
`C. DNA Is Amplified by the Polymerase Chain
`Reaction
`Dp
`- Recombinant DNA Technology Has
`Numerous Practical Applications
`
`espite obvious differences in lifestyle and macroscopic appear-
`jance, organisms exhibit striking similarity at the molecular level.
`The structures and metabolic activities of all cells rely on a
`mmon set of molecules that includes amino acids, carbohydrates, lipids,
`d nucleotides, as well as their polymeric forms. Each type of compound
`_ be described in terms of its chemical makeup, its interactions with
`et molecules, and its physiological function. We begin our survey of bio-
`lecules with a discussion of the nucleotides and their polymers, the
`
`life.
`ucleotides are involved in nearly every facet of cellular
`ifically, they participate in oxidation—-reduction reactions, energy
`Sfer, intracellular signaling, and biosynthetic reactions. Their polymers,
`
` 4
`
`

`

`
`
`40
`
`i
`
`Chapter 3 Nucleotides, Nucleic Acids, and Genetic Information
`
`a
`
`i
`
`and decoding of genetic information. Nucleotides and nucleic acids also
`perform structural and catalytic roles in cells. No other class of molecules
`participates in such varied functions or in so many functions that are es-
`sential for life.
`Evolutionists postulate that the appearance of nucleotides permitted
`the evolution of organisms that could harvest and store energy from their
`surroundings and, most importantly, could make copies of themselves.
`Although the chemical and biological details of early life-forms are the
`subject of speculation, it is incontrovertible that life as we know it is
`inextricably linked to the chemistry of nucleotides and nucleic acids.
`In this chapter, we briefly examine the structures of nucleotides and the
`nucleic acids DNA and RNA.Wealso consider how the chemistry of these
`molecules allows them to carry biological information in the form of a
`sequence of nucleotides. This information is expressed by the transcription
`of a segment of DNA to yield RNA, which is then translated to form
`protein. Because a cell’s structure and function ultimately depend onits
`genetic makeup, we discuss how genomic sequences provide information
`about evolution, metabolism, and disease. Finally, we consider some of the
`techniques used in manipulating DNA in the laboratory. In later chapters,
`we will examine in greater detail the participation of nucleotides and
`nucleic acids in metabolic processes. Chapter 24 includes additional infor-
`mation about nucleic acid structures, DNA’s interactions with proteins, and
`DNA packaging in cells, as a prelude to several chapters discussing the
`roles of nucleic acids in the storage and expression of genetic information.
`
`
`Nucleotides
`
`LEARNING OBJECTIVE
`
`Nucleotides are ubiquitous molecules with considerable structural diversity.
`ce There are eight common varieties of nucleotides, each composedof a nitroge-
`s Becomefamiliar with the structures and
`nous base linked to a sugar to which at least one phosphate group is also
`nomenclature of the eight common
`attached. The bases of nucleotides are planar, aromatic, heterocyclic mole-
`nucleotides.
`cules that are structural derivatives of either purine or pyrimidine (although
`they are not synthesized in vivo from either of these organic compounds).
`
`6
`
`NK ONS
`2
`1
`9 8
`2
`6
`XA 3
`N
`Xi
`\
`N
`N
`H
`
`4
`
`
`
`Purine
`
`Pyrimidine
`
`The most common purines are adenine (A) and guanine (G), and the
`major pyrimidines are cytosine (C), uracil (U), and thymine (T). The
`purines form bondsto a five-carbon sugar (a pentose) via their N9 atoms,
`whereas pyrimidines do so through their N1 atoms (Table 3-1).
`In ribonucleotides, the pentose is ribose, while in deoxyribonucleotides
`(or just deoxynucleotides), the sugar is 2’-deoxyribose (i.c., the carbon at
`position 2’ lacks a hydroxyl group).
`
`—chy
`era
`
`—Ci 0
`
`H
`
`Ribose
`
`Deoxyribose
`
`
`
`
`
`
`
` 5
`
`

`

`Section 3-1 Nucleotides
`
`Base
`Formula
`
`Base
`(X = BH)
`
`Nucleoside
`(X = ribose
`
`Nucleotide?
`(X = ribose phosphate“)
`
`
`
` ‘Namesand Abbreviations of Nucleic Acid Bases, Nucleosides, and Nucleotides
`
`
`
`
`
`NZ
`N
`Adenine
`Adenosine
`Adenylic acid
`lL
`| S
`’
`Ade
`Ado
`Adenosine monophosphate
`
`
`SS
`
`x
`A
`A
`AMP
`
`
`Guanylic acid
`Guanosine
`Guanine
`~N
`Ay |
`»
`Gua
`Guo
`Guanosine monophosphate
`
`
`
`NUON TY
`G
`G
`GMP
`
`
`
`
`
`NZ
`a |
`ON
`Xx
`
`ov
`
`Cytosine
`Cyt
`Cc
`
`Cytidine
`Cyd
`Cc
`
`Cytidylic acid
`Cytidine monophosphate
`CMP
`
`
`
`
`
`
`BAN
`ow |
`X
`Xx
`
`O
`
`H
`
`CH3
`
`ov
`
`ON
`
`Uracil
`Ura
`U
`
`Thymine
`Thy
`T
`
`Uridine
`Urd
`U
`
`Uridylic acid
`Uridine monophosphate
`UMP
`
`Deoxythymidine
`dThd
`dT
`
`Deoxythymidylic acid
`Deoxythymidine monophosphate
`dTMP
`
`
`
`
`
`
`
`
`
`- “The presence of a 2'-deoxyribose unit in place of ribose, as occurs in DNA,is implied by the prefixes “deoxy” or “d.” For example, the deoxynu-
`cleoside of adenine is deoxyadenosine or dA. However, for thymine-containing residues, which rarely occur in RNA, the prefix is redundant and
`
`
`_ may be dropped. The presence of a ribose unit may be explicitly implied by the prefix “ribo.”
`The position of the phosphate group in a nucleotide may beexplicitly specified as in, for example, 3’-AMP and 5'-GMP.
`
`
`
`
`Note that the “primed” numbers refer to the atoms of the pentose;
`“unprimed” numbersrefer to the atoms of the nitrogenous base.
`
`_
`In a ribonucleotide or a deoxyribonucleotide, one or more phosphate
`groups are bonded to atom C3’ or atom C5’ of the pentose to form
`
`a3’-nucleotide or a 5’-nucleotide, respectively (Fig. 3-1). When the
`Phosphate group is absent, the compound is known as a nucleoside. A
`
`
`“hucleotide can therefore be called a nucleoside-5'-phosphate.
`
`lucleotides most commonly contain one to three phosphate groups at the
`
`S Position and are called nucleoside monophosphates, diphosphates,
`
`(b)
`(a)
`Nd triphosphates.
`5!
`5!
`.
`:
`HO—CH,
`-O
`~"0,PO—CH,
`_O
`
`
`a HU
`ki Bu
`(a) A 5’-ribonucleotide
`{gure 3-1 Chemical structures of nucleotides.
`HY
`ENi
`! () a 3’-deoxynucleotide. The purine or pyrimidine base is linked to Cl’ of
`~0,PO yf
`OH OH
`© pentose and at least one phosphate (red) is also attached. A nucleoside
`
`ONSISts Only of a base and a
`pentose
`
`Miernrnin een P costae 5'-Ribonucleotide 3’-Deoxynucleotide
`
`
`
`
`_
`
`
`
`
`
`
`
`
`
`
`
`
` 6
`
`

`

`
`
`42
`
`Chapter 3 Nucleotides, Nucleic Acids, and Genetic Information
`
`Glucose
`
`ADP
`*
`c
`w
`\
`
`
`HO
`
`|
`
`og
`
`NH,
`CH.OH
`NX
`|g
`LK
`0
`ay
`INI 0
`Sy
`
`i
`E
`|
`li
`O-P-O-P—-O—-CHe 9
`O7
`H
`OH
`6-
`H oH
`H
`HO OH
`In this nucleotide
`#@ Figure 3-2 | ADP-glucose.
`derivative, glucose (blue) is attached to adenosine
`(black) by a diphosphate group (red).
`
`The structures, names, and abbreviations of the common bases, nucleo-
`sides, and nucleotides are given in Table 3-1. Ribonucleotides are
`components of RNA(ribonucleic acid), whereas deoxynucleotides are com-
`ponents of DNA (deoxyribonucleic acid). Adenine, guanine, and cytosine
`occur in both ribonucleotides and deoxynucleotides (accounting for six of
`N.
`S the eight common nucleotides), but uracil primarily occurs in ribonu-
`_cleotides and thymine occurs in deoxynucleotides. Free nucleotides, which
`N’
`are anionic, are almost always associated with the counterion Mg” in cells.
`Nucleotides Participate in Metabolic Reactions. The bulk of the nu-
` cleotides in any cell are found in polymeric forms, as either DNA or RNA,
`whose primary functions are information storage and transfer. However,
`free nucleotides and nucleotide derivatives perform an enormous variety of
`metabolic functions not related to the management of genetic information.
`Perhaps the best known nucleotide is adenosine triphosphate (ATP), a
`nucleotide containing adenine, ribose, and a triphosphate group. ATPis
`often mistakenly referred to as an energy-storage molecule, but it is
`more accurately termed an energy carrier or energy transfer agent. The
`process of photosynthesis or the breakdown of metabolic fuels such as
`carbohydrates and fatty acids leads to the formation of ATP from
`adenosine diphosphate (ADP):
`
`Adenosine
`
`O
`O
`i
`i
`5
`HPOZ + HO—P—O—P—O~CHy oO
`Om
`o7
`H #H
`H
`
`H
`OH OH
`
`.
`
`S
`
`NH,
`
`oO
`
`Nio y
`
`N
`
`+
`
` H,O
`
`typically linked to the nucleotide through a mono- or diphosphate group.
`
`O
`oO
`N
`O
`I
`li
`I
`“O-P—O-P—O- PO CHa O
`O7
`om
`O~
`H #H
`H
`
`H
`OH OH
`
`Adenosine diphosphate (ADP)
`
`Adenosinetriphosphate (ATP)
`
`ATPdiffuses throughout the cell to provide energy for other cellular work,
`such as biosynthetic reactions, ion transport, and cell movement. The
`chemical potential energy of ATP is made available whenit transfers one
`(or two) of its phosphate groupsto another molecule. This process can be
`represented by the reverse of the preceding reaction, namely, the hydro-
`lysis of ATP to ADP. (As weshall see in later chapters, the interconver-
`sion of ATP and ADPinthe cell is not freely reversible, and free phos-
`phate groups are seldom released directly from ATP.) The degree to which
`ATPparticipates in routine cellular activities is illustrated by calculations
`indicating that while the concentration of cellular ATP is relatively
`moderate (~5 mM), humanstypically recycle their own weight of ATP
`each day.
`Nucleotide derivatives participate in a wide variety of metabolic
`processes. For example, starch synthesis in plants proceeds by repeated
`additions of glucose units donated by ADP-glucose (Fig. 3-2). Other
`nucleotide derivatives, as we shall see in later chapters, carry groups that
`undergo oxidation-reduction reactions. The attached group, which may be
`4 small molecule such as glucose (Fig. 3-2) or even another nucleotide, is
`
`
`
`
`CHECK YOUR UNDERSTANDING
`Describe the general structure of a nucleoside
`and a nucleotide.
`Describe the difference between a
`ribonucleotide and a deoxyribonucleotide.
`
`
`
`
`
` 7
`
`

`

`
`
`|
`
`“Dama: Veet, Fundamentalsof Biochi
`ariuaeet
`
`
`Home: © Read, Study & Practice FOR. Gradabons
`
`
`>Open Assignment
`
`
`
`
`stionof lipid bila.
`‘ae eeeeealsesnesPcpoouevasa6 assignment
`
`
` Which ondofthe following statements at
`
`Which of thefollowing describes the anti
`Whatis the primary driving force in tha formationoflipid bilayers and protein tertiary structure?
`trragular toops of secondary structure te:
`
`Which of the following aming acid rasidus
`A dornain is:
`&nergy released when additional ion pairs are formed.
`
`. The exclusion of non-polar substances from aquaous solution.
`SGes
`
`
`The formation of van der Waals interactions between neighbouring groups.
`Where are irregular sacondary structures
` Which of the following statements ts NOT:
`
`
`; Energy released when additional hydragen bondsare formed.
`Whichof the following series of amine ac
`Whichofthefollowingstatementsabout ~~
`
`
`Which of the following statements about
`vgs
`“MWhot is the primary driving force in the farmotion of lield bila...
`Which onaof the following sequencas of
`Compare the alpha-helix with the structe
`|
`Which one of the following statements at
`compensate for the reduced entropy of the side chains involved and the loss of the solvation free energy.
`|
`Whatultimately detarminas the unique t
`Which of thafollowing sarias of amino ac | .
`.
`_.
`.
`;
`Coo.
`;
`;
`a
`Which of thefollowing stabilizes the foldi
`Whatis the primary driving force in the formation oflipid bilayers and protein tertiary structure?
`What BEST distinguishes irregular seconc
`!
`. Yehish. of the folloving stotamants about:
`
` X INCORRECT. The energy that formation of ion pairs might contribute to the folding process does not
`
`©
`
`© The formation of van der Waals interactions between neighbouring groups,
`*, The exclusion of non-polar substances frorn aqueoussolution,
`@) Energy released when additional ian pairs are formed,
`Cy Energy released when additional hydrogen bonds are formed,
`
`987 804708129302
`
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