5 3 Template strand RNA polymerase Transcription UGG A U A AG CU A G U A RNA transcript 5 Translation Protein N aa1 aa2 aa3 aa4 C-terminal ATGGCATGCAAT GCTCATCG TACCGTACGTTATCGA G TAG FI
Trang 1Problems 903
tective caps on the chromosome ends DNA polymerases cannot
repli-cate the extreme 5-ends of chromosomes, but a special polymerase
called telomerase maintains telomere length Telomerase is a
ribo-nucleoprotein, and its RNA component serves as template for telomere
synthesis
28.6 How Are RNA Genomes Replicated? Many viruses have genomes
composed of RNA, not DNA DNA may be an intermediate in the
repli-cation of such viruses; that is, viral RNA serves as the template for DNA
synthesis This reaction is catalyzed by reverse transcriptase, an
RNA-dependent DNA polymerase
28.7 How Is the Genetic Information Shuffled by Genetic
Recombina-tion? Genetic recombination is the exchange (or incorporation) of one
DNA sequence with (or into) another Recombination between very
simi-lar DNA sequences is called homologous recombination Homologous
re-combination proceeds according to the Holliday model The RecBCD
en-zyme complex unwinds dsDNA and cleaves its single strands RecA protein
acts in recombination to catalyze the ATP-dependent DNA strand
ex-change reaction Procession of strand separation and re-pairing into
hy-brid strands along the DNA duplex initiates branch migration, displacing
the homologous DNA strand from the DNA duplex and replacing it with
the ssDNA strand RuvA, RuvB, and RuvC resolve the Holliday junction to
form the recombination products DNA replication is an essential
compo-nent of both DNA recombination and DNA repair processes
Further-more, recombination mechanisms can restart replication forks that have
halted at breaks or other lesions in the DNA strands
Transposons are mobile DNA segments ranging in size from several
hundred base pairs to more than 8 kbp that move enzymatically from
place to place in the genome
28.8 Can DNA Be Repaired? Repair systems correct damage to DNA
in order to maintain its information content The most common forms
of damage are (1) replication errors, (2) deletions or insertions, (3)
UV-induced alterations, (4) DNA strand breaks, and (5) covalent
crosslink-ing of strands Cells have extraordinarily diverse and effective DNA
re-pair systems to deal with these problems, some of which are also
involved in DNA replication and recombination When repair systems
fail, the genome may still be preserved if an “error-prone” mode of replication allows the lesion to be bypassed
28.9 What Is the Molecular Basis of Mutation? Mutations change the sequence of bases in DNA, either by the substitution of one base pair for another (point mutations) or by the insertion or deletion of one or more base pairs Point mutations arise by the pairing of bases with in-appropriate partners during DNA replication, by the introduction of base analogs into DNA, or by chemical mutagens Chemical mutagens are agents that chemically modify bases so that their base-pairing char-acteristics are altered
28.10 Do Proteins Ever Behave as Genetic Agents? DNA is the ge-netic material in organisms, although some viruses have RNA genomes The possibility that proteins carry genetic information was a point of speculation early in the history of molecular biology but was ultimately discounted for lack of evidence Prions (an acronym for proteinaceous infectious particle) may be an exception Prion particles are devoid of nucleic acid, yet they can transmit disease Prion diseases are novel be-cause they may be either inherited (like a genetic agent) or acquired by infection PrP, the prion protein, comes in several forms: PrPc, the nor-mal cellular protein, and a conformational variant of PrPcknown as PrPscor PrPresfound in association with prion diseases The propensity
of the PrPscconformational form to polymerize into cell-destructive ag-gregates is thought to be the basis of prion diseases
Special Focus: Gene Rearrangements and Immunology—Is It Possible
to Generate Protein Diversity Using Genetic Recombination? Animals have evolved a way to exploit genetic recombination in order to generate protein diversity The immunoglobulin genes are a highly evolved system for maximizing protein diversity from a finite amount of genetic infor-mation Cells active in the immune response are capable of gene re-arrangements The antibody diversity found in IgG molecules is a prime example of proteins produced via gene rearrangements IgG L-chain genes are created by combining three separate genes, and H-chain genes
by combining four V–J and V–D–J joining in L- and H-chain gene assem-bly is mediated by RAG proteins
PROBLEMS
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chapter at www.cengage.com/login
1.If15N-labeled E coli DNA has a density of 1.724 g/mL, 14N-labeled
DNA has a density of 1.710 g/mL, and E coli cells grown for many
generations on 14NH4 as a nitrogen source are transferred to media
containing15NH4 as the sole N-source, (a) what will be the density
of the DNA after one generation, assuming replication is
semicon-servative? (b) Supposing replication took place by a dispersive
mech-anism, what would be the density of DNA after one generation?
(c) Design an experiment to distinguish between semiconservative
and dispersive modes of replication
2.(a) What are the respective roles of the 5-exonuclease and
3-exonuclease activities of DNA polymerase I? (b) What might be a
fea-ture of an E coli strain that lacked DNA polymerase I 3-exonuclease
activity?
3.Assuming DNA replication proceeds at a rate of 750 base pairs per
second, calculate how long it will take to replicate the entire E coli
genome Under optimal conditions, E coli cells divide every 20
min-utes What is the minimal number of replication forks per E coli
chromosome in order to sustain such a rate of cell division?
4.On the basis of Figure 28.2, draw a simple diagram illustrating
replication of the circular E coli chromosome (a) at an early stage,
(b) when one-third completed, (c) when two-thirds completed,
and (d) when almost finished, assuming the initiation of
replica-tion at oriC has occurred only once Then, draw a diagram showing
the E coli chromosome in problem 3 where the E coli cell is
dividing every 20 minutes
5. It is estimated that there are forty molecules of DNA polymerase III
per E coli cell Is it likely that the growth rate of E coli is limited by
DNA polymerase III availability?
6. Approximately how many Okazaki fragments are synthesized in the
course of replicating an E coli chromosome? How many in
repli-cating an “average” human chromosome?
7. How do DNA gyrases and helicases differ in their respective func-tions and modes of action?
8. Assuming DNA replication proceeds at a rate of 100 base pairs per second in human cells and origins of replication occur every
300 kbp, how long would it take to replicate the entire diploid human genome? How many molecules of DNA polymerase does each cell need to carry out this task?
9. From the information in Figure 28.17, diagram the recombina-tional event leading to the formation of a heteroduplex DNA re-gion within a bacteriophage chromosome
10. Homologous recombination in E coli leads to the formation of
regions of heteroduplex DNA By definition, such regions con-tain mismatched bases Why doesn’t the mismatch repair system
of E coli eliminate these mismatches?
11. If RecA protein unwinds duplex DNA so that there are about 18.6 bp per turn, what is the change in , the helical twist of DNA,
com-pared to its value in B-DNA?
12. Diagram a Holliday junction between two duplex DNA molecules and show how the action of resolvase might give rise to either patch
or splice recombinant DNA molecules
Trang 2904 Chapter 28 DNA Metabolism: Replication, Recombination, and Repair
13. Show the nucleotide sequence changes that might arise in a dsDNA
(coding strand segment GCTA) upon mutagenesis with (a) HNO2,
(b) bromouracil, and (c) 2-aminopurine
14. Transposons are mutagenic agents Why?
15. Give a plausible explanation for the genetic and infectious
proper-ties of PrPsc
16. Hexameric helicases, such as DnaB, the MCM proteins, and
papil-loma virus E1 helicase (illustrated in Figures 16.23–16.25), unwind
DNA by passing one strand of the DNA duplex through the central
pore, using a mechanism based on ATP-dependent binding
interac-tions with the bases of that strand The genome of E coli K12 consists
of 4,686,137 nucleotides Assuming that DnaB functions like
papil-loma virus E1 helicase, from the information given in Chapter 16 on
ATP-coupled DNA unwinding, calculate how many molecules of ATP
would be needed to completely unwind the E coli K12 chromosome
17. Asako Furukohri, Myron F Goodman, and Hisaji Maki wanted to
dis-cover how the translesion DNA polymerase IV takes over from DNA
polymerase III at a stalled replication fork (see Journal of Biological
Chemistry 283:11260–11269, 2008) They showed that DNA
poly-merase IV could displace DNA polypoly-merase III from a stalled
replica-tion fork formed in an in vitro system containing DNA, DNA
poly-merase III, the -clamp, and SSB Devise your own experiment to
show how such displacement might be demonstrated (Hint: Assume
that you have protein identification tools that allow you to distinguish easily between DNA polymerase III and DNA polymerase IV.)
18.The eukaryotic translesion DNA polymerases fall into the Y family
of DNA polymerases Structural studies reveal that their fingers and thumb domains are small and stubby (see Figure 28.10) In addi-tion, Y-family polymerase active sites are more open and less con-strained where base pairing leads to selection of a dNTP substrate for the polymerase reaction Discuss the relevance of these struc-tural differences Would you expect Y-family polymerases to have 3-exonuclease activity? Explain your answer
Preparing for the MCAT Exam
19.Figure 28.11 depicts the eukaryotic cell cycle Many cell types “exit” the cell cycle and don’t divide for prolonged periods, a state termed
G0; some, for example neurons, never divide again
a In what stage of the cell cycle do you suppose a cell might be when
it exits the cell cycle and enters G0?
b The cell cycle is controlled by checkpoints, cyclins, and CDKs De-scribe how biochemical events involving cyclins and CDKs might control passage of a dividing cell through the cell cycle
20.Figure 28.40 gives some examples of recombination in IgG codons
95 and 96, as specified by the Vand Jgenes List the codon possi-bilities and the amino acids encoded if recombination occurred in codon 97 Which of these possibilities is less desirable?
FURTHER READING
General
Holliday, R., 1964 A mechanism for gene conversion in fungi Genetic
Research 5:282–304 The classic model for the mechanism of DNA
strand exchange during homologous recombination
Kornberg, A., 2005 DNA Replication, 2nd ed., New York: Macmillan A
comprehensive detailed account of the enzymology of DNA
metab-olism, including replication, recombination, repair, and more
Lewin, B., 2007 Genes IX Sudbury, MA: Jones and Bartlett A
contem-porary genetics text that seeks to explain heredity in terms of
mo-lecular structures
Meselson, M., and Stahl, F W., 1958 The replication of DNA in Escherichia
coli Proceedings of the National Academy of Sciences U.S.A 44:671–682.
The classic paper showing that DNA replication is semiconservative
Meselson, M., and Weigle, J J., 1961 Chromosome breakage
accompa-nying genetic recombination in bacteriophage Proceedings of the
National Academy of Sciences U.S.A 47:857–869 The experiments
demonstrating that physical exchange of DNA occurs during
re-combination
Ogawa, T., and Okazaki, T., 1980 Discontinuous DNA replication
An-nual Review of Biochemistry 49:421–457 Okazaki fragments and their
implications for the mechanism of DNA replication
Palmiter, R D., et al., 1982 Dramatic growth of mice that develop from
eggs microinjected with metallothionein-growth hormone fusion
genes Nature 300:611–615.
DNA Replication
Baker, T A., and Bell, S P., 1998 Polymerases and the replisome:
Ma-chines within maMa-chines Cell 92:295–305.
Bell, S P., and Dutta, A., 2002 DNA replication in eukaryotic cells
An-nual Review of Biochemistry 71:333–374.
Blow, J J., and Dutta, A., 2005 Preventing re-replication of
chromoso-mal DNA Nature Reviews Molecular Cell Biology 6:476–486.
Botchan, M., 2007 A switch for S phase Nature 445:272–274.
Cvetic, C A., and Walter, J C., 2006 Getting a grip on licensing:
Mech-anism of stable MCM2-7 loading onto replication origins Cell 21:
143–148
Franklin, M C., Wang, J., and Steitz, T A., 2001 Structure of the
repli-cating complex of a pol family DNA polymerase Cell 105:657–667
Frick, D N., and Richardson, C C., 2001 DNA primases Annual Review
of Biochemistry 70:39–80.
Goodman, M F., 2002 Error-prone repair DNA polymerases in
prokary-otes and eukaryprokary-otes Annual Review of Biochemistry 71:17–50.
Hübscher, U., Maga, G., and Spadari, S., 2002 Eukaryotic DNA
po-lymerases Annual Review of Biochemistry 71:133–163.
Keck, J L., 2000 Structure of the RNA polymerase domain of the E coli
primase Science 287:2482–2486.
Kool, E T., 2002 Active site tightness and substrate fit in DNA
replica-tion Annual Review of Biochemistry 71:191–219.
Leu, F P., Georgescu, R., and O’Donnell, M., 2003 Mechanism of the
E coli processivity switch during lagging-strand synthesis Molecular
Cell 11:315–327.
Machida, Y J., and Dutta, A., 2005 Cellular checkpoint mechanisms
monitoring proper initiation of DNA replication Journal of
Biologi-cal Chemistry 280:6253–6256.
Marians, K J., 2008 Understanding how the replisome works Nature
Structural and Molecular Biology 15:125–127.
McHenry, C., 2003 Chromosomal replicases as asymmetric dimers:
Studies of subunit arrangement and functional consequences
Mo-lecular Microbiology 49:1157–1165.
Pomerantz, R T., and O’Donnell, M., 2007 Replisome mechanics:
In-sights into a twin DNA polymerase machine Trends in Microbiology
15:156–164
Randell, J C W., Bowers, J L., Rodriguez, H K., and Bell, S P., 2006 Sequential ATP hydrolysis by Cdc6 and ORC directs loading of the
Mcm2-7 helicase Molecular Cell 21:29–39.
Rothwell, P J., and Waksman, G., 2005 Structure and mechanism of
DNA polymerases Advances in Protein Chemistry 71:401–440 Steitz, T A., 1998 A mechanism for all polymerases Nature 391:231–232.
Tye, B K., 1999 MCM proteins in DNA replication Annual Review of
Bio-chemistry 68:649–686.
Protein Rings in DNA Metabolism
Hingorani, M M., and O’Donnell, M., 2000 A tale of toroids in DNA
metabolism Nature Reviews Molecular Cell Biology 1:22–30.
Wyman, C., and Botchan, M., 1995 DNA replication: A familiar ring to
DNA polymerase processivity Current Biology 5:334–337.
Telomerase
Blackburn, E H., 1992 Telomerases Annual Review of Biochemistry 61:
113–129
Trang 3Further Reading 905
Collins, K., 1999 Ciliate telomerase biochemistry Annual Review of
Bio-chemistry 68:187–218.
Kim, N W., 1994 Specific association of human telomerase activity with
immortal cells and cancer Science 266:2011–2015.
Nakamura, T M., et al., 1997 Telomerase catalytic subunit homologs
from fission yeast and human Science 277:955–959.
Prions
Cohen, F E., and Prusiner, S B., 1998 Pathological conformations of
prion proteins Annual Review of Biochemistry 67:793–819.
Prusiner, S B., 1996 Molecular biology and pathogenesis of prion
dis-eases Trends in Biochemical Sciences 21:482–487.
Prusiner, S B., 1997 Prion diseases and the BSE crisis Science 278:
245–251
Recombination
Alberts, B., 2003 DNA replication and recombination Nature 421:
431–435
Anderson, D G., and Kowalczykowski, S C., 1997 The translocating
RecBCD enzyme stimulates recombination by directing RecA
pro-tein onto ssDNA in a -regulated manner Cell 90:77–86
Baumann, P., and West, S C., 1998 Role of the human RAD51 protein
in homologous recombination and double-stranded-break repair
Trends in Biochemical Sciences 23:247–252.
Beernink, H T H., and Morrical, S W., 1999 RMPs: Recombination/
replication proteins Trends in Biochemical Sciences 24:385–389.
Chen, Z., Yang, H., and Pavletich, N P., 2008 Mechanism of
homolo-gous recombination from the RecA-ssDNA/dsDNA structures
Na-ture 453:489–494.
Cox, M M., 2007 Motoring along with the bacterial RecA protein
Na-ture Reviews Molecular Cell Biology 8:127–138.
Haber, J E., 1999 DNA recombination: The replication connection
Trends in Biochemical Sciences 24:271–275.
Kowalczykowski, S C., 2000 Initiation of genetic recombination and
recombination-dependent replication Trends in Biochemical Sciences
25:156–165
Krishna, R., Prabu, J R., Manjunath, G P., Datta, S., et al., 2007
Snap-shots of RecA protein involving movement of the C-domain and
dif-ferent conformations of the DNA-binding loops: Crystallographic
and comparative analysis of 11 structures of Mycobacterium smegmatis
RecA Journal of Molecular Biology 367:1130–1144.
Lovett, S T., 2003 Connecting replication and recombination
Molecu-lar Cell 11:554–556.
Lusetti, S L., and Cox, M M., 2002 The bacterial RecA protein and the
recombinational DNA repair of replication forks Annual Review of
Biochemistry 71:71–100.
Rafferty, J B., et al., 1996 Crystal structure of DNA recombination
pro-tein RuvA and a model for its binding to the Holliday junction
Sci-ence 274:415–421.
Roca, A I., and Cox, M M., 1997 RecA protein: Structure, function,
and role in recombinational DNA repair Progress in Nucleic Acid
Re-search and Molecular Biology 56:127–223.
Taylor, A F., and Smith, G R., 2003 RecBCD enzyme is a DNA helicase
with fast and slow motors of opposite polarity Nature 423:889–893.
See also Dillingham, M S., Spies, M., and Kowalczykowski, S C.,
2003 RecBCD is a bipolar DNA helicase Nature 423:893–897.
Wigley, D B., 2007 RecBCD: The supercar of DNA repair Cell 131:
651–653
Yamada, K., Ariyoshi, M., and Morikawa, K., 2004 Three-dimensional structural views of branch migration and resolution in DNA
homolo-gous recombination Current Opinion in Structural Biology 14:130–137.
Transposons
Lambowitz, A M., and Belfort, M., 1993 Introns as mobile genetic
ele-ments Annual Review of Biochemistry 62:587–622.
Stellwagen, A E., and Craig, N L., 1998 Mobile DNA elements:
Control-ling transposition with ATP-dependent molecular switches Trends in
Biochemical Sciences 23:486–490.
V(D)J Recombination and the Immunoglobulin Genes
Gellert, M., 2002 V(D)J recombination: RAG proteins, repair factors,
and regulation Annual Review of Biochemistry 71:101–132.
Hiom, K., and Gellert, M., 1997 A stable RAG1-RAG2-DNA complex
that is active in V(D)J cleavage Cell 88:65–72.
Lewis, S M., and Wu, G E., 1997 The origins of V(D)J recombination
Cell 88:159–162.
Nossal, G J V., 2003 The double helix and immunology Nature 421:
440–444
Transgenic Animals
Morgan, R A., and Anderson, W F., 1993 Human gene therapy Annual
Review of Biochemistry 62:192–217.
Schnieke, A E., et al., 1997 Human factor IX transgenic sheep
pro-duced by transfer of nuclei from transfected fetal fibroblasts Science
278:2130–2133
Wilmut, I., et al., 1997 Viable offspring derived from fetal and adult
mammalian cells Nature 385:810–818 See also Campbell, K H S.,
et al., 1996 Sheep cloned by nuclear transfer from a cultured cell
line Nature 380:64–66.
Repair
Bartek, J., and Lukas, J., 2003 Damage alert Nature 421:486–488 Friedberg, E C., 2003 DNA damage and repair Nature 421:436–440.
Friedberg, E C., Walker, G C., and Siede, W., 1995 DNA Repair and Mu-tagenesis Washington, DC: ASM Press.
Marians, K J., 2000 PriA-directed replication fork restart in Escherichia
coli Trends in Biochemical Sciences 25:185–189.
McCollough, A K., et al., 1999 Initiation of base excision repair:
Gly-cosylase mechanisms and structures Annual Review of Biochemistry
68:255–285
Michel, B., 2000 Replication fork arrest and DNA recombination
Trends in Biochemical Sciences 25:173–178.
Modrich, P., and Lahue, R., 1996 Mismatch repair in replication
fi-delity, genetic recombination, and cancer biology Annual Review of
Biochemistry 65:101–133.
Mol, C D., et al., 1999 DNA repair mechanisms for the recognition and
removal of damaged DNA bases Annual Review of Biophysics and
Bio-molecular Structure 28:101–128.
Morgan, A R., 1993 Base mismatches and mutagenesis: How important
is tautomerism? Trends in Biochemical Sciences 18:160–163.
Parikh, S S., et al., 1999 Envisioning the molecular choreography of
DNA base excision repair Current Opinion in Structural Biology 9:37–47 Sancar, A., 1994 Mechanisms of DNA excision repair Science 266:
1954–1956 (Science named the extended family of DNA repair
en-zymes its “Molecules of the Year” in 1994 See the 23 December
1994 issue of Science for additional readings.)
Trang 4British Museum, UK/Bridgeman Art Library
and the Regulation
of Gene Expression
All cells contain three major classes of RNA—mRNA, ribosomal RNA (rRNA), and transfer RNA (tRNA) and all participate in protein synthesis (see Chapters
10 and 30) Further, all RNAs are synthesized from DNA templates by
DNA-dependent RNA polymerases in the process known as transcription However,
only mRNAs direct the synthesis of proteins Protein synthesis occurs via the
process of translation, wherein the instructions encoded in the sequence of bases
in mRNA are translated into a specific amino acid sequence by ribosomes, the
“workbenches” of polypeptide synthesis (see Chapter 30).
Transcription is tightly regulated in all cells In prokaryotes, only 3% or so of the genes are undergoing transcription at any given time The metabolic condi-tions and the growth status of the cell dictate which gene products are needed at any moment Similarly, differentiated eukaryotic cells express only a small per-centage of their genes in fulfilling their biological functions, not the full genetic potential encoded in their chromosomes.
29.1 How Are Genes Transcribed in Prokaryotes?
In prokaryotes, virtually all RNA is synthesized by a single species of DNA-dependent RNA polymerase The only exception is the short RNA primers formed by primase during DNA replication Like DNA polymerases, RNA po-lymerase links ribonucleoside 5-triphosphates (ATP, GTP, CTP, and UTP, repre-sented generically as NTPs) in an order specified by base pairing with a DNA template:
The enzyme moves along a DNA strand in the 3→5 direction, joining the 5-phosphate of an incoming ribonucleotide to the 3-OH of the previous residue Thus, the RNA chain grows 5→3 during transcription, just as DNA chains do dur-ing replication Subsequent hydrolysis of PPito inorganic phosphate by the pyro-phosphatases present in all cells removes the product PPi, thus making the poly-merase reaction thermodynamically favorable.
Prokaryotic RNA Polymerases Use Their Sigma Subunits to Identify Sites Where Transcription Begins
Transcription is initiated in prokaryotes by RNA polymerase holoenzyme, a complex
multimeric protein (about 400 kD) large enough to be visible in the electron micro-scope Its subunit composition is 2 After two -subunits (35 kD each) dimerize,
one recruits and the other to form the clawlike core polymerase structure
(Fig-ure 29.1) The two largest subunits, (171 kD) and (124 kD), perform most of the
enzymatic functions The -subunit forms most of the upper jaw of the claw and
con-tains the catalytic Mg2-binding site; forms the lower jaw DNA passes through a
2.7-nm channel between the jaws of the claw Nucleotide substrates reach the
The Rosetta stone, inscribed in 196 B.C The writing
on the Rosetta stone is in three forms: hieroglyphs,
Demotic (the conventional Egyptian script of the
time), and Greek (the Greeks ruled Egypt in 196 B.C.)
The Rosetta stone represents the transcription of
hieroglyphic symbols into two living languages
Shown here is part of the interface where hieroglyphs
and Demotic meet
Now that we have all this useful information, it
would be nice to do something with it
From the Unix Programmer’s Manual
KEY QUESTIONS
29.1 How Are Genes Transcribed in Prokaryotes?
29.2 How Is Transcription Regulated in
Prokaryotes?
29.3 How Are Genes Transcribed in Eukaryotes?
29.4 How Do Gene Regulatory Proteins
Recognize Specific DNA Sequences?
29.5 How Are Eukaryotic Transcripts Processed
and Delivered to the Ribosomes for
Translation?
29.6 Can We Propose a Unified Theory of Gene
Expression?
ESSENTIAL QUESTION
Expression of the information encoded in DNA depends on transcription of that infor-mation into RNA.
How are the genes of prokaryotes and eukaryotes transcribed to form RNA products that can be translated into proteins?
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Trang 529.1 How Are Genes Transcribed in Prokaryotes? 907
catalytic center through a secondary channel entering on the back side of the
en-zyme Binding of the -subunit to allows the RNA polymerase to recognize
differ-ent DNA sequences that act as promoters A number of related proteins, the sigma
identify the location of transcription start sites, where transcription begins Both and
contribute to formation of the catalytic site for RNA synthesis Dissociation of the
tran-scribe DNA into RNA but is unable to recognize promoters and initiate transcription.
Bacteriophage T7 expresses a simpler (monomeric) RNA polymerase (Figure 29.2)
that shares the functional characteristics of prokaryotic RNA polymerases.
A DEEPER LOOK
Conventions Used in Expressing the Sequences of Nucleic Acids and Proteins
Certain conventions are useful in tracing the course of
infor-mation transfer from DNA to protein The strand of duplex
DNA that is read by RNA polymerase is termed the template
strand Thus, the strand that is not read is the nontemplate
strand.Because the template strand is read by the RNA
poly-merase moving 3→5 along it, the RNA product, called the
transcript, grows in the 5→3 direction (see accompanying
figure) Note that the nontemplate strand has a nucleotide
se-quence and direction identical to those of the RNA transcript,
except that the transcript has U residues in place of T Portions
of the RNA transcript will eventually be translated into the
amino acid sequence of a protein (see Chapter 30) by a
process in which successive triplets of bases (termed codons),
read 5→3, specify a particular amino acid Polypeptide chains
are synthesized in the N⎯→C direction, and the 5-end of
mRNA encodes the N-terminus of the protein
By convention, when the order of nucleotides in DNA is
shown as a single strand, it is the 5→3 sequence of
nucleo-tides in the nontemplate strand that is presented
Conse-quently, if convention is followed, DNA sequences are written
in terms that correspond directly to mRNA sequences, which
correspond in turn to the amino acid sequences of proteins as
read beginning with the N-terminus
5
3
Template strand RNA polymerase
Transcription
UGG
A
U A
AG
CU
A G U
A
RNA transcript 5
Translation
Protein N aa1 aa2 aa3 aa4 C-terminal
ATGGCATGCAAT GCTCATCG TACCGTACGTTATCGA
G TAG
FIGURE 29.1 Structure of the Thermus thermophilus core
RNA polymerase 2 (pdb id 2O5I).The template
DNA strand is shown in green, the nontemplate DNA strand in blue, and the RNA transcript in hot pink The two chains are orange, the chain is cyan, and the
chain is yellow.The active-site Mg2 is shown as a red sphere
Trang 6908 Chapter 29 Transcription and the Regulation of Gene Expression
The Process of Transcription Has Four Stages
Transcription can be divided into four stages: (1) binding of RNA polymerase holoenzyme at promoter sites, (2) initiation of polymerization, (3) chain elonga-tion, and (4) chain termination.
Binding of RNA Polymerase to Template DNA The process of transcription begins when the -subunit of RNA polymerase recognizes a promoter sequence (Figure
29.3), and RNA polymerase holoenzyme and the promoter form a closed promoter
complex (Figure 29.3, Step 2) This stage in RNA polymerase⬊DNA interaction is
re-ferred to as the closed promoter complex because the dsDNA has not yet been
“opened” (unwound) so that the RNA polymerase can read the base sequence of the DNA template strand and transcribe it into a complementary RNA sequence.
Once the closed promoter complex is established, the RNA polymerase holoen-zyme unwinds about 14 base pairs of DNA (base pairs located at positions 12 to 2,
relative to the transcription start site; see later discussion), forming the very stable open
promoter complex (Figure 29.3, Step 3) Promoter sequences can be identified in vitro
by DNA footprinting: RNA polymerase holoenzyme is bound to a putative promoter
sequence in a DNA duplex, and the DNA⬊protein complex is treated with DNase I DNase I cleaves the DNA at sites not protected by bound protein, and the set of DNA fragments left after DNase I digestion reveals the promoter (by definition, the pro-moter is the RNA polymerase holoenzyme binding site).
RNA polymerase binding typically protects a nucleotide sequence spanning the region from 70 to 20, where the 1 position is defined as the transcription start
site: that base in the nontemplate DNA strand that is identical with the first base in the RNA transcript The next base, 2, specifies the second base in the transcript Nontemplate strand bases in the 5, or “minus,” direction from the transcript start site are numbered 1, 2, and so on (Note that there is no zero.) Nontemplate
nu-cleotides in the “minus” direction are said to lie upstream of the transcription start
site, whereas nucleotides in the 3, or “plus,” direction are downstream of the tran-scription start site The transcript start site on the template strand is usually a pyrimi-dine, so most transcripts begin with a purine RNA polymerase binding protects 90 bp
of DNA, equivalent to a distance of 30 nm along B-DNA Because RNA polymerase is only 16 nm in its longest dimension, the DNA must be wrapped around the enzyme.
Properties of Prokaryotic Promoters Promoters recognized by the principal factor,
70, serve as the paradigm for prokaryotic promoters These promoters vary in size from 20 to 200 bp but typically consist of a 40-bp region located on the 5-side
of the transcription start site Within the promoter are two consensus sequence
elements These two elements are the Pribnow box1near 10, whose consensus se-quence is the hexameric TATAAT, and a sese-quence in the ⴚ35 region containing the hexameric consensus TTGACA (Figure 29.4) The Pribnow box and the 35 region are separated by about 17 bp of nonconserved sequence RNA polymerase
holo-FIGURE 29.2 Bacteriophage T7 RNA polymerase (pdb id
1MSW) in the act of transcription.T7 RNA polymerase
is a 99-kD monomeric protein The DNA is shown
enter-ing the enzyme from the upper right The template
strand is green, the nontemplate strand is blue, the RNA
transcript is hot pink
1Named for David Pribnow, who, along with David Hogness, first recognized the importance of this se-quence element in transcription
A consensus sequence can be defined as the
bases that appear with highest frequency at each
position when a series of sequences believed to
have common function is compared
Trang 7Recognition of promoter
by; binding of polymerase
holoenzyme to DNA;
migration to promoter
5
DNA template
Formation of an RNA
polymerase:closed promoter
complex
5
Unwinding of DNA at
promoter and formation
of open promoter complex
5
RNA polymerase initiates
mRNA synthesis, usually
with a purine
5
Purine NTP
Pu P P
NTPs
RNA polymerase
holoenzyme-catalyzed elongation of
mRNA by about 4 more
nucleotides
5
3
P
N N
P P
5
3
5
Release of -subunit as core
RNA polymerase proceeds
down the template, elongating
RNA transcript
Promoter
P P
5
Pu
Pu
RNA pol
Step 1
Step 2
Step 3
Step 4
Step 5
Step 6
ACTIVE FIGURE 29.3 Sequence of events in the initiation and elongation phases of tran-scription as it occurs in prokaryotes Nucleotides in this region are numbered with reference to the base at the transcription start site, which is designated 1 Test yourself on the concepts in this figure at www cengage.com/login.
G
Initiation site (+1)
Pribnow box (–10 region) –35 region
Gene
Consensus
sequence: T C T T G A C A T
42 38 82 84 79 64 53 45 41
[11–15 bp] T A T A A T
79 95 44 59 51 96
[5–8 bp]
42 48 55 51 A G
Initiation site
araBAD
araC
bioA
bioB
galP2
lac
lacI
rrnA1
rrnD1
rrnE1
tRNATyr
trp
G A T C C T A C C T G A C G C T T T T T A T C G C A A C T C T C T A C T G T T T C T C C A T A C C C G T T T T T
G C C G T G A T T A T A G A C ACT T T T G T T A C G C G T T T T T G T C A T G G C T T T G G T C C C G C T T T G
T T C C A A A A C G T G T T T TTT G T T G T T A A T T C G G T G T A G A C T T G T A A A C C T A A A T C T T T T
C A T A A T C G A C T T G T A AAC C A A A T T G A A A A G A T T T A G G T T T A C A A G T C T A C A C C G A A T
A T T T A T T C C A T G T C A CAC T T T T C G C A T C T T T G T T A T G C T A T G G T T A T T T C A T A C C A T
A C C C C A G G C T T T A C A CTT T A T G C T T C C G G C T C G T A T G T T G T G T G G A A T T G T G A G C G G
C C A T C G A A T G G C G C A AAA C C T T T C G C G G T A T G G C A T G A T A G C G C C C G G A A G A G A G T C
A A A A T A A A T G C T T G A CTC T G T A G C G G G A A G G C G T A T TAT C A C A C C C C C G C G C C G C T G
C A A A A A A A T A C T T G T GCA A A A A A T T G G G A T C C C T A T AAT G C G C C T C C G T T G A G A C G A
C A A T T T T T C T A T T G C GGC C T G C G G A G A A C T C C C T A T AAT G C G C C T C C A T C G A C A C G G
C A A C G T A A C A C T T T A CAG C G G C G C G T C A T T T G A T A TGA T G C G C C C C G C T T C C C G A TA
A A A T G A G C T G T T G A C AAT T A A T C A T C G A A C T A G T T AAC T A G T A C G C A A G T T C A C G TA
FIGURE 29.4 The nucleotide sequences of representative E coli promoters (In accordance with convention,
these sequences are those of the nontemplate strand where RNA polymerase binds.) Consensus sequences
for the 35 region, the Pribnow box, and the initiation site are shown at the bottom.The numbers represent
the percent occurrence of the indicated base (Note: The 35 region is only roughly 35 nucleotides from the
transcription start site; the Pribnow box [the 10 region] likewise is located at approximately position 10.)
In this figure, sequences are aligned relative to the Pribnow box
Trang 8910 Chapter 29 Transcription and the Regulation of Gene Expression
enzyme uses its -subunit to bind to the conserved sequences, and the more closely
the 35 region sequence corresponds to its consensus sequence, the greater is the
efficiency of transcription of the gene The highly expressed rrn genes in E coli that
encode ribosomal RNA (rRNA) have a third sequence element in their promoters,
the upstream element (UP element), located about 20 bp immediately upstream of
the 35 region (Transcription from the rrn genes accounts for more than 60% of total RNA synthesis in rapidly growing E coli cells.) Whereas the -subunit
recog-nizes the 10 and 35 elements, the C-terminal domains (CTD) of the -subunits
of RNA polymerase recognize and bind the UP element.
In order for transcription to begin, the DNA duplex must be “opened” so that RNA polymerase has access to single-stranded template The efficiency of initiation
is inversely proportional to the melting temperature, Tm, in the Pribnow box, sug-gesting that the A⬊T-rich nature of this region is aptly suited for easy “melting” of the DNA duplex and creation of the open promoter complex (see Figure 29.3) Nega-tive supercoiling facilitates transcription initiation by favoring DNA unwinding The RNA polymerase -subunit is directly involved in melting the dsDNA
Inter-action of the -subunit with the nontemplate strand maintains the open complex
formed between RNA polymerase and promoter DNA, with the -subunit acting as a
A DEEPER LOOK
DNA Footprinting—Identifying the Nucleotide Sequence in DNA Where
a Protein Binds
DNA footprinting is a widely used technique to
identify the nucleotide sequence within DNA
where a specific protein binds, such as the
promotersequence(s) bound by RNA polymerase
holoenzyme In this technique, the protein is
in-cubated with a labeled (*) DNA fragment
con-taining the nucleotide sequence where the
pro-tein is believed to bind (The DNA fragment is
labeled at only one end.) Then, a DNA cleaving
agent, such as DNase I, is added to the solution
containing the DNA⬊protein complex DNase I
cleaves the DNA backbone in exposed regions—
that is, wherever the presence of the DNA-binding
protein does not prevent DNase I from binding A
control solution containing naked DNA (a sample
of the same labeled DNA fragment with no
DNA-binding protein added) is also treated with DNase
I When these DNase I digests are analyzed by gel
electrophoresis, a difference is found between the
set of labeled fragments from the DNA⬊protein
complex and the set from naked DNA The
ab-sence of certain fragments in the digest of the
DNA⬊protein complex reveals the location of the
protein-binding site on the DNA (see
accompany-ing figure)
Adapted from Rhodes, D., and Fairall, L., 1997 Analysis
of sequence-specific DNA-binding proteins In Protein
Function: A Practical Approach, Creighton, T E., ed.,
Oxford: IRL Press at Oxford University Press
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
DNA:protein complex
protein
Gel electrophoresis
Naked DNA
( arrows indicate DNase I cleavage sites)
Denature dsDNA:
sets of labeled fragments
Protein binding site
Trang 929.1 How Are Genes Transcribed in Prokaryotes? 911
sequence-specific single-stranded DNA-binding protein Association of the -subunit
with the nontemplate strand stabilizes the open promoter complex and leaves the
bases along the template strand available to the catalytic site of the RNA polymerase.
Initiation of Polymerization RNA polymerase has two binding sites for NTPs: the
initiation site and the elongation site The first nucleotide binds at the initiation
site, base-pairing with the 1 base exposed within the open promoter complex (see
Figure 29.3, Step 4) The second incoming nucleotide binds at the elongation site,
base-pairing with the 2 base The ribonucleotides are then united when the 3-O
of the first nucleotide makes a phosphoester bond with the -phosphorus atom of
the second nucleotide, and PPiis eliminated Note that the 5-end of the transcript
starts out with a triphosphate attached to it Movement of RNA polymerase along
the template strand (translocation) to the next base prepares the RNA polymerase to
add the next nucleotide (see Figure 29.3, Step 5) Once an oligonucleotide 9 to
12 residues long has been formed, the -subunit dissociates from RNA polymerase,
signaling the completion of initiation (see Figure 29.3, Step 6) The core RNA
po-lymerase is highly processive and goes on to synthesize the remainder of the mRNA.
As the core RNA polymerase progresses, advancing the 3-end of the RNA chain,
the DNA duplex is unwound just ahead of it About 12 base pairs of the growing
RNA remain base-paired to the DNA template at any time, with the RNA strand
becoming displaced as the DNA duplex rewinds behind the advancing RNA
polymerase.
Chain Elongation Elongation of the RNA transcript is catalyzed by the core
po-lymerase, because once a short oligonucleotide chain has been synthesized, the
-subunit dissociates The accuracy of transcription is such that about once every
104 nucleotides, an error is made and the wrong base is inserted Because many
transcripts are made per gene and most transcripts are smaller than 10 kb, this
error rate is acceptable.
Two possibilities can be envisioned for the course of the new RNA chain In one,
the RNA chain is wrapped around the DNA as the RNA polymerase follows the
tem-plate strand around the axis of the DNA duplex, but this possibility seems unlikely
due to its potential for tangling the nucleic acid strands (Figure 29.5a) In reality,
transcription involves supercoiling of the DNA, so positive supercoils are created
RNA
(a)
RNA polymerase
RNA polymerase
Gyrase introducing negative supercoils
removing negative supercoils
FIGURE 29.5 Supercoiling versus transcription (a) If the RNA polymerase followed the template strand around
the axis of the DNA duplex, no supercoiling of the DNA would occur but the RNA chain would be wrapped
around the double helix once every 10 bp This possibility seems unlikely because it would be difficult to
un-tangle the transcript from the DNA duplex (b) Alternatively, gyrases and topoisomerases could remove the
torsional stresses induced by transcription
Trang 10912 Chapter 29 Transcription and the Regulation of Gene Expression
ahead of the transcription bubble and negative supercoils are created behind it (Figure 29.5b) To prevent torsional stress from inhibiting transcription, gyrases in-troduce negative supercoils (and thereby remove positive supercoils) ahead of RNA polymerase, and topoisomerases remove negative supercoils behind the DNA seg-ment undergoing transcription (Figure 29.5b).
Chain Termination Two types of transcription termination mechanisms operate in
bacteria: one that is dependent on a specific protein called rho termination factor
(for the Greek symbol, ) and another, intrinsic termination, that is not In
intrin-sic termination, termination is determined by specific sequences in the DNA called
termination sites. These sites are not indicated by particular bases showing where transcription halts Instead, these sites consist of three structural features whose base-pairing possibilities lead to termination:
1 Inverted repeats, which are typically G⬊C-rich, so a stable stem-loop structure can form in the transcript via intrachain base-pairing (Figure 29.6)
2 A nonrepeating segment that punctuates the inverted repeats
3 A run of 6 to 8 As in the DNA template, coding for Us in the transcript Termination then occurs as follows: A G⬊C-rich, stem-loop structure, or “hairpin,” forms in the transcript The hairpin apparently causes the RNA polymerase to pause, whereupon the A⬊U base pairs between the transcript and the DNA template strand are displaced through formation of somewhat more stable A⬊T base pairs be-tween the template and nontemplate strands of the DNA The result is spontaneous dissociation of the nascent transcript from DNA.
The alternative mechanism of termination—factor-dependent termination—is less common and mechanistically more complex Rho factor is an ATP-dependent helicase (hexamer of 50-kD subunits) that catalyzes the unwinding of RNA⬊DNA hybrid duplexes (or RNA⬊RNA duplexes) The rho factor recognizes and binds to C-rich regions in the RNA transcript These regions must be unoccupied by trans-lating ribosomes for rho factor to bind Once bound, rho factor advances in the
5 →3 direction until it reaches the transcription bubble (Figure 29.7) There it cat-alyzes the unwinding of the transcript and template, releasing the nascent RNA chain It is likely that the RNA polymerase stalls in a G ⬊C-rich termination region, al-lowing rho factor to overtake it.
29.2 How Is Transcription Regulated in Prokaryotes?
In bacteria, genes encoding the enzymes of a particular metabolic pathway are often grouped adjacent to one another in a cluster on the chromosome This pattern of or-ganization allows all of the genes in the group to be expressed in a coordinated
fash-ion through transcriptfash-ion into a single polycistronic mRNA encoding all the enzymes
of the metabolic pathway.2A regulatory sequence lying adjacent to the DNA being
C C U C G G A A A
T
T A
T A
A T
A T
A T
G C
G C
C G
T A
C G
C G
T A
T A
T A
T A
G C
G C
A T
G C
C G
C G
T A
T A
T A
T A
T A
T A
T A
T A
5
Inverted repeat Inverted repeat Direction of transcription
G–C rich
G–C rich
A–T rich Last base transcribed
Transcription
Sense strand
mRNA terminus
G G A G C C U U U
U U U U
3 terminus
U U U U U
A U U
FIGURE 29.6 The termination site for the E coli trp
operon (the trp operon encodes the enzymes of
trypto-phan biosynthesis) The inverted repeats give rise to a
stem-loop, or “hairpin,” structure ending in a series of U
residues
2A polycistronic mRNA is a single RNA transcript that encodes more than one polypeptide Cistron is a
genetic term for a DNA region representing a protein: Cistron and gene are essentially equivalent terms.