Biochemistry, 4th Edition P99 ppsx

944 Chapter 29 Transcription and the Regulation of Gene Expressionbinds U2 snRNP, and then the triple snRNP complex of U4/U6U5 replaces U1 at the 5-splice site.. This realization led Geo

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put (for example, as ATP) is needed The lariat product is unstable; the 2-5

phos-phodiester branch is quickly cleaved to give a linear excised intron that is rapidly

de-graded in the nucleus.

Splicing Depends on snRNPs

The hnRNA (pre-mRNA) substrate is not the only RNP complex involved in the

splic-ing process Splicsplic-ing also depends on a unique set of small nuclear ribonucleoprotein

particles, so-called snRNPs (pronounced “snurps”) In higher eukaryotes, each

snRNP consists of a small RNA molecule 100 to 200 nucleotides long and a set of

about 10 different proteins Some of the different proteins form a “core” set common

to all snRNPs, whereas others are unique to a speciﬁc snRNP The major snRNP

species are very abundant, present at greater than 100,000 copies per nucleus The

RNAs of snRNPs are typically rich in uridine, hence the classiﬁcation of particular

snRNPs as U1, U2, and so on The prominent snRNPs are given in Table 29.6 U1

snRNA folds into a secondary structure that leaves the 11 nucleotides at its 5-end

single-stranded The 5-end of U1 snRNA is complementary to the consensus sequence

at the 5-splice junction of the pre-mRNA (Figure 29.43), as is a region at the 5-end of

U6 snRNA U2 snRNA is complementary to the consensus branch site sequence.

snRNPs Form the Spliceosome

Splicing occurs when the various snRNPs come together with the pre-mRNA to

form a multicomponent complex called the spliceosome The spliceosome is a

large complex, roughly equivalent to a ribosome in size, and its assembly requires

ATP Assembly of the spliceosome begins with the binding of U1 snRNP at the

5-splice site of the pre-mRNA (Figure 29.44) Each subsequent step in spliceosome

assembly requires ATP-dependent RNA rearrangements catalyzed by spliceosomal

DEAD-box ATPases/helicases The branch-point sequence (UACUAAC in yeast)

3

OH

AGp

Exon 1

pre-mRNA 5

5

GUA

3-splice site

UG AAU pGA G

Y N Y R Y

Branch site 5-splice site

A

pG

3

5

3

5

3

C

Yn A G

Y N Y R Y

Exon 2

A

2OH

A G OH

U G A

A

U G A

A

pG NC

Y n A G

A G

Y N Y R Y

Exon 2

A

NC

Yn A G

FIGURE 29.42 Splicing of mRNA precur-sors A representative precursor mRNA is depicted Exon 1 and Exon 2 indicate two exons separated by an intervening sequence (or intron) with consensus 5,

3, and branch sites.The fate of the phosphates at the 5- and 3-splice sites can be followed by tracing the fate of

the respective ps The products of the

splicing reaction, the lariat form of the excised intron and the united exons, are shown at the bottom of the ﬁgure

snRNP Length (nt) Splicing Target

U4 145 5 splice, recruitment

TABLE 29.6 The snRNPs Found in Spliceosomes

⎫

⎬

⎭

⎧

⎨

⎩

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944 Chapter 29 Transcription and the Regulation of Gene Expression

binds U2 snRNP, and then the triple snRNP complex of U4/U6U5 replaces U1 at the 5-splice site The substitution of base-pairing interactions between U1 and the pre-mRNA 5-splice site by base-pairing between U6 and the 5-splice site is just one

of the many RNA rearrangements that accompany the splicing reaction Base-pairings between U6 and U2 RNA bring the 5-splice site and the branch point RNA sequences into proximity Interactions between U2 and U6 lead to release of U4 snRNP The spliceosome is now activated for catalysis: A transesteriﬁcation reaction involving the 2-O of the invariant A residue in the branch-point sequence displaces the 5-exon from the intron, creating the lariat intermediate The free 3-O of the 5-exon now triggers a second transesteriﬁcation reaction through attack on the

P atom at the 3-exon splice site This second reaction joins the two exons and re-leases the intron as a lariat structure In addition to the snRNPs, a number of pro-teins with annealing functions as well as propro-teins with ATP-dependent RNA-unwinding activity participate in spliceosome function The spliceosome is thus a dynamic structure that uses the pre-mRNA as a template for assembly, carries out its transesteriﬁcation reactions, and then disassembles when the splicing reaction

is over.

Alternative RNA Splicing Creates Protein Isoforms

In one mode of splicing, every intron is removed and every exon is incorporated

into the mature RNA without exception This type of splicing, termed constitutive splicing, results in a single form of mature mRNA from the primary transcript However, many eukaryotic genes can give rise to multiple forms of mature RNA transcripts The mechanisms for production of multiple transcripts from a single gene include use of different promoters, selection of different polyadenylation

sites, alternative splicing of the primary transcript, or even a combination of the

three.

Different transcripts from a single gene make possible a set of related

polypep-tides, or protein isoforms, each with slightly altered functional capability Such

vari-ation serves as a useful mechanism for increasing the apparent coding capacity of the genome Furthermore, alternative splicing offers another level at which regula-tion of gene expression can operate For example, mRNAs unique to particular cells, tissues, or developmental stages could be formed from a single gene by choos-ing different 5- or 3-splice sites or by omittchoos-ing entire exons Translation of these mature mRNAs produces cell-speciﬁc protein isoforms that display properties tai-lored to the needs of the particular cell Such regulated expression of distinct pro-tein isoforms is a fundamental characteristic of eukaryotic cell differentiation and development.

C C A U U C A

A G G U A A G U C

U

U1

Branch point

3 5'

3

A G Y

3-splice site region

3-splice site

5-splice site

5 exon

5-splice site region

FIGURE 29.43 Mammalian U1 snRNA can be arranged in

a secondary structure where its 5-end is single-stranded

and can base-pair with the consensus 5-splice site of

the intron.(Adapted from Figure 1 in Rosbash, M., and Seraphin,

B., 1991 Who’s on ﬁrst? The U1 snRNP-5 splice site interaction

and splicing Trends in Biochemical Sciences 16:187.)

ATP

ATP U2

U2

U4

U5

U1

U6

U1

U5

U1

U5

U6

U5

U6

U4 U6 U5 U4 U6

U5 U6

U4 U5 U6

FIGURE 29.44 Events in spliceosome assembly U1

snRNP binds at the 5-splice site, followed by the

associ-ation of U2 snRNP with the UACUAA*C branch-point

sequence The triple U4/U6-U5 snRNP complex replaces

U1 at the 5-splice site and directs the juxtaposition of

the branch-point sequence with the 5-splice site,

whereupon U4 snRNP is released Lariat formation

oc-curs, freeing the 3-end of the 5-exon to join with the

5-end of the 3-exon, followed by exon ligation U2, U5,

and U6 snRNPs dissociate from the lariat following exon

ligation

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Fast Skeletal Muscle Troponin T Isoforms Are an Example

of Alternative Splicing

In addition to many other instances, alternative splicing is a prevalent mechanism

for generating protein isoforms from the genes encoding muscle proteins (see

Chapter 16), allowing distinctive isoforms aptly suited to the function of each

mus-cle An impressive manifestation of alternative splicing is seen in the expression

possibilities for the rat fast skeletal muscle troponin T gene (Figure 29.45) This

gene consists of 18 exons, 11 of which are found in all mature mRNAs (exons 1

through 3, 9 through 15, and 18) and thus are constitutive Five exons, those

num-bered 4 through 8, are combinatorial in that they may be individually included or

excluded, in any combination, in the mature mRNA Two exons, 16 and 17, are

mutually exclusive: One or the other is always present, but never both Sixty-four

different mature mRNAs can be generated from the primary transcript of this gene

by alternative splicing Because each exon represents a cassette of genetic

infor-mation encoding a segment of protein, alternative splicing is a versatile way to

in-troduce functional variation within a common protein theme.

RNA Editing: Another Mechanism That Increases the Diversity

of Genomic Information

RNA editing is a process that changes one or more nucleotides in an RNA transcript

by deaminating a base, either A→I (adenine to inosine, through deamination at the

6-position in a purine ring) or C⎯ →U (cytosine to uracil, through deamination at

the 4-position in a pyrimidine ring) These changes alter the coding possibilities

in a transcript, because I will pair with G (not U as A does) and U will pair with A

(not G as C does) RNA editing has the potential to increase protein diversity by

(1) altering amino acid coding possibilities, (2) introducing premature stop codons,

or (3) changing splice sites in a transcript If RNA splicing is cutting-and-pasting,

then these single-base changes are aptly termed RNA editing.

A-to-I editing is carried out by adenosine deaminases that act on RNA (the ADAR

family of RNA-editing enzymes) ADARs act only on double-stranded regions of

RNA Typically, such regions form when an exon region containing an A to be

edited base-pairs with a complementary base sequence in an intron known as the

editing site complementary sequence, or ECS ADARs are abundant in the nervous system

of animals A prominent example of RNA editing occurs in transcripts encoding

mammalian glutamate receptors (GluRs; see Chapter 32) Deamination of the

Fast skeletal troponin T gene and spliced mRNAs

TATA

5

UT

DNA

mRNAs

Any of the 32 possible combinations with zero, one, two, three, four, or all five of exons 4 through 8

UT

( )

5 UT

1–

6

7–

10

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

11–

16

17–

22

23–

27

28–

31

32–

36

37–

43

44–

58

199–

228

243–

259

AATAAA

59–

97

98–

123

124–

161

162–

198

229–

242

229–

242

( )

17 16

FIGURE 29.45 Organization of the fast skeletal muscle troponin T gene and the 64 possible mRNAs that can be

generated from it Exons are constitutive (yellow), combinatorial (green), or mutually exclusive (blue or orange)

Exon 1 is composed of 5-untranslated (UT) sequences, and Exon 18 includes the polyadenylation site (AATAAA)

and 3-UT sequences.The TATA box indicates the transcription start site.The amino acid residues encoded by

each exon are indicated below Many exon⬊intron junctions fall between codons.The ”sawtooth”exon

bound-aries indicate that the splice site falls between the ﬁrst and second nucleotides of a codon, the “concave/convex”

exon boundaries indicate that the splice site falls between the second and third nucleotides of a codon, and ﬂush

boundaries between codons signify that the splice site falls between intact codons Each mRNA includes all

con-stitutive exons, 1 of the 32 possible combinations of Exons 4 to 8, and either Exon 16 or 17

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GluR-B gene transcript changes a glutamine codon CAG to CIG, which is read by the translational machinery as an arginine codon (CGG), dramatically altering the conductance properties of the membrane receptor produced from the edited tran-script, as compared to the receptor produced from the unedited transcript.

C-to-I editing is carried out on single-stranded regions of transcripts by an

edito-some core structure consisting of a cytosine deaminase and an adapter protein that

brings the deaminase and the transcript together A prominent example of C-to-I editing targets a single C residue in a 14-kb transcript encoding the 4536-residue apolipoprotein B100 protein (see Chapter 24) ApoB RNA editing changes codon

2153 (a CAA [glutamine] codon) to a UAA stop codon, which leads to a shortened protein product, ApoB48, consisting of the N-terminal 48% of apoB100 In humans, apoB100 is made in the liver and found in liver-derived VLDL serum lipoprotein complexes In contrast, apoB48 is made in intestinal cells and found in intestinal-de-rived lipid complexes.

The stages of eukaryotic gene expression—from transcriptional activation, tran-scription, transcript processing, nuclear export of mRNA, to translation—have tra-ditionally been presented as a linear series of events, that is, as a pathway of discrete, independent steps However, it now is clear that each stage is part of a continuous process, with physical and functional connections between the various transcrip-tional and processing machineries This realization led George Orphanides and Danny Reinberg to propose a “uniﬁed theory of gene expression.” The principal tenet of this theory is that eukaryotic gene expression is a continuous process, from transcription through processing and protein synthesis: DNA→RNA→protein (Fig-ure 29.46) Furthermore, regulation occurs at multiple levels in this continuous process, in a coordinated fashion Tom Maniatis and Robin Reed provide additional support for this theory by pointing out that eukaryotic gene expression depends on

an interacting network of multicomponent protein machines—nucleosomes, HATs, and the remodeling apparatus; RNA polymerase II and its associated factors, which include capping, splicing, and polyadenylylation enzymes; and the proteins involved

in mRNA export to the cytoplasm for translation on ribosomes, the topic of the next chapter Translation is not inevitable If a noncoding RNA base-pairs with the mRNA, translation will be thwarted For example, base pairing with a microRNA will result

in gene silencing (see Chapter 10) Base pairing with a small interfering RNA (siRNA, Chapter 10) leads to gene knockdown by RNAi through mRNA destruction

by the RISC protein complex (see Figure 12.20) Recall that gene silencing is a post-transcriptional regulatory mechanism that prevents translation of an mRNA, whereas RNAi carries out post-transcriptional destruction of mRNAs.

It should be mentioned that eukaryotic cells have elaborate systems for mRNA surveillance; these systems destroy any messages containing errors, such as the

nonsense mediated decay (NMD) system NMD degrades any message that has premature nonsense, or “stop,” codons Furthermore, the regulation of mRNA levels through selective destruction provides another mechanism for the post-transcriptional regulation of gene expression.

RNA Degradation

The amount of speciﬁc mRNAs or proteins present in a cell at any time represents a balance between the rates of macromolecular synthesis and degradation Regulated degradation of mRNAs (discussed here) and proteins (discussed in Chapter 31) is a rapid and effective way to control the cellular levels of these macromolecules Be-cause indiscriminate degradation of RNAs and proteins could have detrimental con-sequences within the cell, such degradation typically is compartmentalized Targeted degradation of RNAs and proteins is enclosed within ringlike or cylindrical

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macro-molecular complexes—the exosome for RNA and the proteasome for proteins (see

Chapter 31) The catalytically active component of an exosome is an RNase PH

family member that processively degrades RNA in the 3→5 direction.

Exosomes have a fundamental structural pattern: a ring of six subunits

sur-rounding a central cavity, with one or more of the subunits having RNase PH

ac-tivity (Figure 29.47) RNAs to be degraded are threaded into the central cavity The

architecture of the exosome restricts substrate access and compartmentalizes the

RNase activity so that indiscriminate degradation of cellular RNase is avoided Only

RNAs targeted to the exosome are destroyed.

RNAPII

GpppX

5 5

Coupled transcription and mRNA processing Transcription factor

*

Chromatin

decompaction

Chromatin Coupled initiationand 5 capping

GpppX 5 5

GpppX

5 5

GpppX5 5

GpppX 5 5

Cleavage and 3 polyadenylation Splicing

Nuclear pore NUCLEUS

CYTOPLASM

mRNA packaging mRNA export

Protein folding

Protein

GpppX5 5

AAA2003

Translation

Mediator

IIEIIH

IIB TBP TATA

A

RNAPII IIF

RNAPII

G/ L

RISC

miRNA

Gene silencing

FIGURE 29.46 A uniﬁed theory of gene expression Each step in gene expression, from transcription to

transla-tion, is but a stage within a continuous process Each stage is physically and functionally connected to the

next, ensuring that all steps proceed in an appropriate fashion and overall regulation of gene expression is

tightly integrated.(Adapted from Figure 2 in Orphanides, G., and Reinberg, D., 2002 A uniﬁed theory of gene expression Cell

108:439–451.)

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29.1 How Are Genes Transcribed in Prokaryotes? In prokaryotes,

vir-tually all RNA synthesis is carried out by a single species of

DNA-dependent RNA polymerase RNA polymerase links ribonucleoside

5-triphosphates in an order speciﬁed by base pairing with a DNA

tem-plate The enzyme reads 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, so the RNA chain grows 5→3 during transcription

Transcription begins when the -subunit of RNA polymerase recognizes

a promoter and forms a complex with it Next, the RNA polymerase

holoenzyme unwinds about 14 base pairs of DNA, and transcription

com-mences Once an oligonucleotide 9 to 12 residues long has been formed,

and goes on to synthesize the remainder of the mRNA Prokaryotes have

two types of transcription termination mechanisms: one that is

depen-dent on  termination factor protein and another that depends on

disso-ciation of the mRNA through reestablishment of DNA base pairs

29.2 How Is Transcription Regulated in Prokaryotes? Bacterial genes

encoding a common metabolic pathway are often grouped adjacent to

one another in an operon, allowing all of the genes to be expressed in

a coordinated fashion The operator, a regulatory sequence adjacent to

the structural genes, determines whether transcription takes place The

operator is located next to a promoter Interaction of a regulatory

pro-tein with the operator controls transcription Small molecules act as

sig-nals of the nutritional or environmental conditions These small

mole-cules interact with operator-binding regulatory proteins and determine

whether transcription occurs Induction is the increased synthesis of

en-zymes in response to a small molecule called a co-inducer Repression is

the decreased transcription in response to a speciﬁc metabolite termed

a co-repressor The lac operon provides an example of induction, and

the trp operon, repression Operon regulation depends on the

interac-tion of sequence-speciﬁc DNA-binding proteins with regulatory

se-quences along the DNA DNA looping increases the regulatory input

available to a speciﬁc gene

29.3 How Are Genes Transcribed in Eukaryotes? Transcription is

more complicated in eukaryotes because eukaryotic DNA is wrapped

around histones to form nucleosomes, and nucleosomes repress gene

expression by limiting access of the transcriptional apparatus to genes

Two classes of transcriptional co-regulators are necessary to overcome

nucleosome repression: (1) histone-modifying enzymes, such as HATs,

and (2) ATP-dependent chromatin-remodeling complexes Gene

acti-vation also requires interaction of RNA polymerase with the promoter

RNA polymerase II consists of 12 different polypeptides The largest,

RPB1, has a C-terminal domain (CTD) containing multiple repeats of

the heptapeptide sequence Y SPTSPS; 5 of these 7 residues can be phos-phorylated by protein kinases The CTD orchestrates events in the tran-scription process RNA polymerase II promoters commonly consist of the core element, near the transcription start site, where general tran-scription factors bind, and more distantly located regulatory elements, known as enhancers or silencers These regulatory sequences are rec-ognized by speciﬁc DNA-binding proteins that activate transcription above basal levels A eukaryotic transcription initiation complex consists

of RNA polymerase II, ﬁve general transcription factors, and Mediator The CTD of RNA polymerase II anchors Mediator to the polymerase and allows RNA polymerase II to communicate with transcriptional activators bound at sites distal from the promoter

29.4 How Do Gene Regulatory Proteins Recognize Specific DNA Sequences? Proteins that recognize nucleic acids present a three-dimensional shape or contour that is structurally and chemically comple-mentary to the surface of a DNA sequence Nucleotide sequence– specific recognition by the protein involves a set of atomic contacts with the bases and the sugar–phosphate backbone Protein contacts with the bases of DNA usually occur within the major groove (but not always) Roughly 80% of DNA-binding regulatory proteins fall into one of three principal classes based on distinctive structural motifs: the helix-turn-helix (or HTH), the zinc finger (or Zn-finger), and the leucine

zipper-basic region (or bZIP) In addition to their DNA-binding domains, these

proteins also have protein⬊protein recognition domains essential to oligomerization, DNA looping, transcriptional activation, and signal re-ception (effector binding)

29.5 How Are Eukaryotic Transcripts Processed and Delivered to the Ribosomes for Translation? In eukaryotes, primary transcripts must be processed to form mature messenger RNAs and exported from the nu-cleus to the cytosol for translation Shortly after transcription initiation, the 5-end of the growing transcript is capped with a guanylyl residue that

is then methylated at the 7-position Additional methylations may occur at the 2-O positions of the next two nucleotides and at the 6-amino group

of a ﬁrst adenine Transcription termination does not normally occur until RNA polymerase II has transcribed past the polyadenylation signal Most eukaryotic mRNAs have a poly(A) tails consisting of 100 to 200 ade-nine residues at their 3-end, added post-transcriptionally by poly(A) polymerase Most eukaryotic genes are split genes, subdivided into cod-ing regions, called exons, and noncodcod-ing regions, called introns Intron excision and exon ligation, a process called splicing, also occurs in the nucleus Splicing is mediated by the spliceosome, which is assembled from a set of small nuclear ribonucleoprotein particles called snRNPs Splicing requires precise cleavage at the 5- and 3-ends of introns and

Cavity

FIGURE 29.47 Structure of the human exosome core

(pdb id 2NN6).The exosome core is composed of

nine different polypeptide chains A hexameric ring of

polypeptides (each a different color in this image)

sur-rounds a central cavity This cavity is capped by a set of

three other proteins (all colored hot pink here) The

hu-man exosome core is catalytic inactive, but it serves as a

platform for the association of additional subunits that

have 3-exonuclease activity Evidence suggests that the

human exosome core selects RNAs for degradation, and

associated 3-exonucleases then degrade them

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Preparing for an exam? Create your own study path for this

chapter at www.cengage.com/login

1.The 5-end of an mRNA has the sequence

…AGAUCCGUAUGGCGAUCUCGACGAAGACUC-CUAGGGAAUCC…

What is the nucleotide sequence of the DNA template strand from

which it was transcribed? If this mRNA is translated beginning with

the ﬁrst AUG codon in its sequence, what is the N-terminal amino

acid sequence of the protein it encodes? (See Table 30.1 for the

genetic code.)

2.Describe the sequence of events involved in the initiation of

tran-scription by E coli RNA polymerase Include in your detran-scription

those features a gene must have for proper recognition and

tran-scription by RNA polymerase

3.RNA polymerase has two binding sites for ribonucleoside

triphos-phates: the initiation site and the elongation site The initiation site

has a greater K mfor NTPs than the elongation site Suggest what

possible signiﬁcance this fact might have for the control of

tran-scription in cells

4.Make a list of the ways that transcription in eukaryotes differs from

transcription in prokaryotes

5.DNA-binding proteins may recognize speciﬁc DNA regions either

by reading the base sequence or by “indirect readout.” How do

these two modes of protein⬊DNA recognition differ?

6.(Integrates with Chapter 11.) The metallothionein promoter is

illustrated in Figure 29.27 How long is this promoter, in nm? How

many turns of B-DNA are found in this length of DNA? How many

nucleosomes (approximately) would be bound to this much DNA?

(Consult Chapter 11 to review the properties of nucleosomes.)

7.Describe why the ability of bZIP proteins to form heterodimers

increases the repertoire of genes whose transcription might be

re-sponsive to regulation by these proteins

8.Suppose exon 17 were deleted from the fast skeletal muscle

tro-ponin T gene (Figure 29.45) How many different mRNAs could

now be generated by alternative splicing? Suppose that exon 7 in a

wild-type troponin T gene were duplicated How many different

mRNAs might be generated from a transcript of this new gene by

al-ternative splicing?

9.Figure 29.30 illustrates some of the various covalent modiﬁcations

that occur on histone tails How might each of these modiﬁcations

inﬂuence DNA⬊histone interactions?

10.(Integrates with Chapter 15.) Predict from Figure 29.12 whether

the interaction of lac repressor with inducer might be cooperative.

Would it be advantageous for inducer to show cooperative binding

to lac repressor? Why?

11.What might be the advantages of capping, methylation, and

poly-adenylylation of eukaryotic mRNAs?

12.(Integrates with Chapter 28.) Figure 29.24 shows only one Mg2ion

in the RNA polymerase II active site; more recent studies reveal the

presence of two Why is the presence of two Mg2ions signiﬁcant?

13. (Integrates with Chapter 11.) The SWI/SNF chromatin-remodeling complex peels about 50 bp from the nucleosome Assuming B-form DNA, how long is this DNA segment? In forming nucleosomes, DNA is wrapped in turns about the histone core octamer What frac-tion of a DNA turn around the core octamer does 50 bp of DNA comprise? How does 50 bp of DNA compare to the typical size of eu-karyotic promoter modules and response elements?

14. Draw the structures that comprise the lariat branch point formed during mRNA splicing: the invariant A, its 5-R neighbor, its 3-Y neighbor, and its 2-G neighbor

15. (Integrates with Chapters 6 and 11.) The -helices in HTH

(helix-turn-helix motif) DNA-binding proteins are formed from 7– or 8–amino acid residues What is the overall length of these -helices?

How does their length compare with the diameter of B-form DNA?

16. Bacteriophage T7 RNA polymerase bound to two DNA strands and

an RNA strand, as shown in pdb 1MSW, provides a glimpse of

tran-scription View this structure at www.pdb.org to visualize how the

template DNA strand is separated from the nontemplate strand and transcribed into an RNA strand Which Phe residue of the enzyme plays a signiﬁcant role in DNA strand separation? In which domain

of the polymerase is this Phe located? (You might wish to consult Yin, Y W., and Steitz, T A., 2002 Structural basis for the transition from initiation to elongation transcription in T7 RNA polymerase

Science 298:1387–1395 to conﬁrm your answer.)

17. RNA polymerase II is inhibited by -amanitin This

mushroom-derived toxin has no effect on the enzyme’s afﬁnity for NTP sub-strates, but it dramatically slows polymerase translocation along the

DNA Go to www.pdb.org to view pdb ﬁle 1K83, which is the structure

of RNA polymerase II with bound -amanitin Locate -amanitin

within this structure and discuss why its position is consistent with its mode of inhibition

18. C/EBP is a bZIP transcription factor in neuronal differentiation,

learning and memory process, and other neuronal and glial

func-tions The structure of the bZIP domain of C/EBP bound to DNA

is shown in pdb ﬁle 1GU4 Explore this structure to discover the leucine zipper dimerization domain and the DNA-binding basic

re-gions On the left side of the www.pdb.org 1GU4 page under “Display

Files,” click “pdb file” to see the atom-by-atom coordinates in the three-dimensional structure (scroll down past “Remarks” to find this information) Toward the end of this series, find the amino acid sequence of the C/EBP domain used in this study Within this

amino acid sequence, ﬁnd the leucine residues of the leucine zip-per and the basic residues in the DNA-binding basic region

Preparing for the MCAT Exam

19. Figure 29.15 highlights in red the DNA phosphate oxygen atoms Some of them interact with catabolite activator protein (CAP) What kind of interactions do you suppose predominate and what kinds of CAP amino acid side chains might be involved in these in-teractions?

20. Chromatin decompaction is a preliminary step in gene expression (Figure 29.46) How is chromatin decompacted?

the accurate joining of the two exons Exon/intron junctions are deﬁned

by consensus sequences recognized by the spliceosome In addition, a

con-served sequence within the intron, the branch site, is also essential to

splic-ing The splicing reaction involves formation of a lariat intermediate

through attachment of the 5-phosphate group of the intron’s invariant

5-G to the 2-OH at the invariant branch site A to form a 2-5

phos-phodiester bond The lariat structure is excised when the exons are

ligated In constitutive splicing, every intron is removed and every exon

is incorporated into the mature RNA without exception However,

alter-native splicing can give rise to different transcripts from a single gene,

making possible a set of protein isoforms, each with slightly altered

func-tional capability Fast skeletal muscle troponin T isoforms are an exam-ple of alternative splicing

29.6 Can We Propose a Uniﬁed Theory of Gene Expression? Each stage in eukaryotic transcription is part of a continuous process, with physical and functional connections between the various transcriptional and processing machineries These multicomponent protein machines are organized into an interacting network, and regulation occurs in a coordinated fashion at multiple levels in the continuous process Eu-karyotic cells also have elaborate systems for mRNA surveillance Not all protein-coding transcripts are translated Gene silencing or RNAi may intervene to prevent translation of mature RNAs

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FURTHER READING

Transcription in Prokaryotes

Busby, S., and Ebright, R H., 1994 Promoter structure, promoter

recog-nition, and transcription activation in prokaryotes Cell 79:743–746.

Campbell, E A., Pavlova, O., Zenkin, N., Leon, F., Irschik, H., Jansen,

R., Severinov, K., and Darst, S A., 2005 Structural, functional, and

genetic analysis of sorangicin inhibition of bacterial RNA

polym-erase EMBO Journal 24:674–682.

Yin, Y W., and Steitz, T A., 2002 Structural basis for the transition from

initiation to elongation transcription in T7 RNA polymerase Science

298:1387–1395

Regulation of Transcription in Prokaryotes

Berg, O G., and von Hippel, P H., 1988 Selection of DNA binding sites

by regulatory proteins Trends in Biochemical Sciences 13:207–211.

Dover, S L., et al., 1997 Activation of prokaryotic transcription through

arbitrary protein–protein contacts Nature 386:627–630.

Jacob, F., and Monod, J., 1961 Genetic regulatory mechanisms in the

synthesis of proteins Journal of Molecular Biology 3:318–356.

Matthews, K S., 1992 DNA looping Microbiological Reviews 56:123–136.

Platt, T., 1998 RNA structure in transcription elongation, termination,

and antitermination In RNA Structure and Function, Simons, R W.,

and Grunberg-Monago, M., eds., pp 541–574 Cold Spring Harbor,

NY: Cold Spring Harbor Press

Schleif, R., 1992 DNA looping Annual Review of Biochemistry 61:199–223.

Transcription in Eukaryotes

Burley, S., 1998 X-ray crystallographic studies of eukaryotic

transcrip-tion factors Cold Spring Harbor Symposium on Quantitative Biology

LXIII:33–40

Burley, S K., and Roeder, R G., 1996 Biochemistry and structural

biol-ogy of transcription factor IID (TFIID) Annual Review of

Biochem-istry 65:769–799.

Conaway, R C., and Conaway, J W., 1999 Transcription elongation and

human disease Annual Review of Biochemistry 68:301–319.

Cramer, D., 2006 Recent structural studies of RNA polymerases II and

III Biochemical Society Transactions 34:1058–1061.

Haag, J R., Pikaard, C S., 2007 RNA polymerase I: A multifunctional

molecular machine Cell 131:1224–1225.

Kettenberger, H., Armache, K J., and Cramer, P., 2004 Complete RNA

polymerase II elongation complex structure and its interactions

with NTP and TFIIS Molecular Cell 16:955–965.

Kornberg, R D., 1998 Mechanism and regulation of yeast RNA

polym-erase II transcription Cold Spring Harbor Symposium on Quantitative

Biology LXIII:229–232.

Reinberg, D., et al., 1998 The RNA polymerase II general transcription

factors: Past, present, and future Cold Spring Harbor Symposium on

Quantitative Biology LXIII:83-103.

Saunders, A., Core, L J., and Lis, J T., 2006 Breaking barriers to

tran-scription elongation Nature Reviews Molecular Cell Biology 7:557–567.

Wang, D., Bushnell, D A., Westover, K D., Kaplan, C D., and Kornberg,

R D., 2006 Structural basis of transcription: Role of the trigger

loop in substrate speciﬁcity and catalysis Cell 127:941–954.

Westover, K D., Bushnell, D A., and Kornberg, R D., 2004 Structural

basis of transcription: Nucleotide selection by rotation in the RNA

polymerase II active center Cell 119:481–489.

Westover, K D., Bushnell, D A., and Kornberg, R D., 2004 Structural

basis of transcription: Separation of RNA from DNA by RNA

po-lymerase II Science 303:1014–1016.

Regulation of Transcription in Eukaryotes

Amaral, P P., Dinger, M E., Mercer, T R., and Mattick, J S., 2008 The

eukaryotic genome as an RNA machine Science 319:1787–1789.

Amoutzias, G D., Robertson, D L., Van de Peer, Y., and Oliver, S G.,

2008 Choose your partners: Dimerization in eukaryotic

transcrip-tion factors Trends in Biochemical Sciences 33:220–229.

Bailey, C H., Bartsch, D., and Kandel, E R., 1996 Toward a molecular

deﬁnition of long-term memory storage Proceedings of the National

Academy of Sciences U.S.A 93:13445–13452.

Björklund, S., et al., 1999 Global transcription regulators of eukaryotes

Cell 96:759–767.

Carey, M., and Smale, S T., 2000 Transcriptional Regulation in Eukaryotes:

Concepts, Strategies, and Techniques New York: Cold Spring Harbor

Laboratory Press

Core, L J., and Lis, J T., 2008 Transcriptional regulation through

promoter-proximal pausing of RNA polymerase II Science 319:

1791–1792

Hobart, O., 2004 Common logic of transcription factor and microRNA

action Trends in Biological Sciences 29:462–468.

Makeyev, E V., and Maniatis, T., 2008 Multilevel regulation of gene

ex-pression by microRNAs Science 319:1789–1790.

Margaritis, T., and Holstege, F C P., 2008 Poised RNA polymerase II

gives pause for thought Cell 133:581–584.

Maston, G A., Evans, S K., and Green, M R., 2006 Transcriptional

reg-ulatory elements in the human genome Annual Review of Genomics

and Human Genetics 7:29–59.

Moore, M J., 2002 Nuclear RNA turnover Cell 108:431–434.

Severinov, K., 2000 RNA polymerase structure-function: Insights into

points of transcriptional regulation Current Opinion in Microbiology

3:118–125

Shamovsky, I., and Nudler, E., 2008 Modular RNA heats up Molecular

Cell 29:415–417.

Struhl, K., 1999 Fundamentally different logic of gene regulation in

prokaryotes and eukaryotes Cell 98:1–4.

Tuch, B B., Ki, H., and Johnson, A D., 2008 Evolution of eukaryotic

transcription circuits Science 319:1797–1799.

Tully, T., 1997 Regulation of gene expression and its role in long-term

memory and synaptic plasticity Proceedings of the National Academy of

Sciences U.S.A 94:4239–4241.

Utley, R T., et al., 1998 Transcriptional activators direct histone

acetyl-transferase complexes to promoters Nature 394:498–502.

Mediator

Biddick, R., and Young, E T., 2005 Yeast Mediator and its role in

tran-scription regulation Comptes Rendus Biologies 328:773–782.

Björklund, S., and Gustafson, C M., 2005 The yeast Mediator complex

and its regulation Trends in Biochemical Sciences 30:240–244.

Kornberg, R D., 2005 Mediator and the mechanism of transcription

ac-tivation Trends in Biochemical Sciences 30:235–239 This article

intro-duces a series of reviews on Mediators that highlights the May 2005

issue of this journal (Trends in Biochemical Sciences 30:235–271).

The Histone Code

Allis, D C., Jenuwin, T., Reinberg, D., and Caparros, M-L., 2006

Epi-genetics New York: Cold Spring Harbor Laboratory Press.

Eisenberg, J C., and Elgin, C R., 2005 Antagonizing the neighbors

Na-ture 438:1090–1091.

Goldberg, A D., Allis, C D., and Bernstein, E., 2007 Epigenetics: A

landscape takes shape Cell 128:635–638.

Shahbazian, M D., and Grunstein, M., 2007 Functions of site-speciﬁc

histone acetylation and deacetylation Annual Review of Biochemistry

76:75–100

Nucleosome Structure and Gene Expression

Armstrong, J A., 2007 Negotiating the nucleosome: Factors that allow

RNA polymerase II to elongate through chromatin Biochemistry and

Cell Biology 85:426–434.

Boeger, H., et al., 2003 Nucleosomes unfold completely at a

transcrip-tionally active promoter Molecular Cell 11:1587–1598.

Brown, C E., et al., 2000 The many HATs of transcriptional

coactiva-tors Trends in Biochemical Sciences 25:15–19.

Fan, H Y., et al., 2003 Distinct strategies to make nucleosomal DNA

ac-cessible Molecular Cell 11:1311–1322.

Felsenfeld, G., and Groudine, M., 2003 Controlling the double helix

Nature 421:448–453.

Hampsey, M., and Reinberg, D., 2003 Tails of intrigue: Phosphorylation of

RNA polymerase II mediates histone methylation Cell 113:429–432.

Trang 9

Iñigues-Llhî, J A., 2006 For a healthy histone code, a little SUMO in the

tail keeps the acetyl away ACS Chemical Biology 1:204–206.

Kassabov, S R., et al., 2003 SWI/SNF unwraps, slides, and rewraps the

nucleosome Molecular Cell 11:391–403.

Kornberg, R D., and Lorch, Y., 1999 Twenty-ﬁve years of the

nucleo-some, fundamental particle of the eukaryotic chromosome Cell 98:

285–294

Ng, H H., and Bird, A., 2000 Histone deacylases: Silencers for hire

Trends in Biochemical Sciences 25:121–126.

Osley, M A., 2006 Regulation of histone H2A and H2B ubiquitylation

Brieﬁngs in Functional Genomics and Proteomics 5:179–189.

Reinberg, D., and Sims, R J III, 2006 deFACTo nucleosome dynamics

Journal of Biological Chemistry 281:23297–23301.

Van Vugt, J J F A., Ranes, M., Campsteijn, C., and Logie, C., 2007 The

ins and outs of ATP-dependent chromatin remodeling in budding

yeast: Biophysical and proteomic perspectives Biochimica Biophysica

Acta 1769:153–171.

Workman, J L., ed., 2003 Protein Complexes That Modify Chromatin New

York: Springer

Wu, C., et al., 1998 ATP-dependent remodeling of chromatin Cold

Spring Harbor Symposium on Quantitative Biology LXIII:525–534.

Zaman, Z., et al., 1998 Gene transcription by recruitment Cold Spring

Harbor Symposium on Quantitative Biology LXIII:167–171.

Zlatanova, J., et al., 2000 Linker histone binding and displacement:

Ver-satile mechanism for transcriptional regulation Faseb Journal 14:

1697–1704.

DNA-Binding Gene Regulatory Proteins

Berg, J M., and Shi, Y., 1996 The galvanization of biology: A growing

appreciation for the roles of zinc Science 271:1081–1085.

Edmondson, D G., and Olson, E N., 1993 Helix-loop-helix proteins as

regulators of muscle-speciﬁc transcription Journal of Biological

Chem-istry 268:755–758.

Glover, J N M., and Harrison, S C., 1995 Crystal structure of the

het-erodimeric bZIP transcription factor c-Fos-c-Jun bound to DNA.

Nature 373:257–261.

Landschulz, W H., Johnson, P F., and McKnight, S L., 1988 The

leucine zipper: A hypothetical structure common to a new class of

DNA-binding proteins Science 240:1759–1764.

Pabo, C O., and Sauer, R T., 1992 Transcription factors: Structural

families and principles of DNA recognition Annual Review of

Bio-chemistry 61:1053–1095.

Patikoglou, G., and Burley, S K., 1997 Eukaryotic transcription factor–

DNA complexes Annual Review of Biophysics and Biomolecular Structure

26:289–325

Vinson, C R., Sigler, P B., and McKnight, S L., 1989 Scissors-grip

model for DNA recognition by a family of leucine zipper proteins.

Science 246:911–916.

von Hippel, P H., 2007 From “simple” DNA–protein interactions to the

macromolecular machines of gene expression Annual Review of

Bio-physics and Structural Biology 36:79–105.

Processing of Eukaryotic Transcripts

Breitbart, R E., Andreadis, A., and Nadal-Ginard, B., 1987 Alternative

splicing: A ubiquitous mechanism for the generation of multiple

protein isoforms from single genes Annual Review of Biochemistry

56:467–495

Kramer, A., 1996 The structure and function of proteins involved in

mammalian pre-mRNA splicing Annual Review of Biochemistry 65:

367–409

Leff, S E., Rosenfeld, M G., and Evans, R M., 1986 Complex tran-scriptional units: Diversity in gene expression by alternative RNA

processing Annual Review of Biochemistry 55:1091–1117.

Maeder, C., and Guthrei, C., 2008 Modiﬁcations target spliceosome

dy-namics Nature Structural Biology 15:426–428.

Sachs, A., and Wahle, E., 1993 Poly (A) tail metabolism and function in

eukaryotes Journal of Biological Chemistry 268:22955–22958 Sharp, P A., 1987 Splicing of messenger RNA precursors Science 235:

766–771

Sims, R J III, Milhouse, S., Chen, C.-F., Lewis, B A., et al., 2007 Recog-nition of trimethylated histone H3 lysine 4 facilitates the recruit-ment of transcription post-initiation factors and pre-mRNA splicing

Molecular Cell 28:665–676.

Staley, J P., and Guthrie, C., 1998 Mechanical devices of the

spliceo-some: Motors, clocks, springs, and things Cell 92:315–326.

RNA Editing

Blanc, V., and Davidson, N O., 2003 C-to-U RNA editing: Mechanisms

leading to genetic diversity Journal of Biological Chemistry 278:

1395–1398

Hoopengardner, B., 2006 Adenosine-to-inosine RNA editing:

Perspec-tives and predictions Mini-Reviews in Medicinal Chemistry 6:1213–1216.

Maas, S., Rich, A., and Nishikura, K., 2003 A-to-I RNA editing: Recent

news and residual mysteries Journal of Biological Chemistry 278:

1391–1394

Samuel, C E., 2003 RNA editing minireview series Journal of Biological

Chemistry 278:1389–1390.

A Uniﬁed Theory of Gene Expression

Maniatis, T., and Reed, R., 2002 An extensive network of coupling

among gene expression machines Nature 416:499–506.

Narliker, G J., Fan, H.-Y., and Kingston, R E., 2002 Cooperation be-tween complexes that regulate chromatin structure and

transcrip-tion Cell 108:475–487.

Orphanides, G., and Reinberg, D., 2002 A uniﬁed theory of gene

ex-pression Cell 108:439–451.

Schreiber, S L., and Bernstein, B E., 2002 Signaling network model of

chromatin Cell 111:771–778.

Woychik, N A., and Hampsey, M., 2002 The RNA polymerase II

ma-chinery: Structure illuminates function Cell 108:439–451.

RNA Degradation

Lorentzen, E., and Conti, E., 2006 The exosome and proteasome:

Nanocompartments for degradation Cell 125:651–654.

Pruijn, G., 2005 Doughnuts dealing with RNA Nature Structural and

Molecular Biology 12:562–564.

Schmid, M., and Jensen, T H., 2008 The exosome: A multipurpose

RNA-decay machine Trends in Biochemical Sciences 33:501–510.

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George Holton/Photo Researchers, Inc.

We turn now to the problem of how the sequence of nucleotides in an mRNA mol-ecule is translated into the speciﬁc amino acid sequence of a protein The problem

raises both informational and mechanical questions First, what is the genetic code

that allows the information speciﬁed in a sequence of bases to be translated into the amino acid sequence of a polypeptide? That is, how is the 4-letter language of nu-cleic acids translated into the 20-letter language of proteins? Implicit in this question

is a mechanistic problem: It is easy to see how base pairing establishes a one-to-one correspondence that allows the template-directed synthesis of polynucleotide chains

in the processes of replication and transcription However, there is no obvious chem-ical afﬁnity between the purine and pyrimidine bases and the 20 different amino acids Nor is there any obvious structural or stereochemical connection between polynucleotides and amino acids that might guide the translation of information.

Francis Crick reasoned that adapter molecules must bridge this information gap.

These adapter molecules must interact speciﬁcally with both nucleic acids (mRNAs) and amino acids At least 20 different adapter molecules would be needed, at least one for each amino acid The various adapter molecules would be able to read the genetic code in an mRNA template and align the amino acids according to the tem-plate’s directions so that they could be polymerized into a unique polypeptide Transfer RNAs (tRNAs; Figure 30.1) are the adapter molecules (see Chapter 10) Amino acids are attached to the 3-OH at the 3-CCA end of tRNAs as aminoacyl

es-ters The formation of these aminoacyl-tRNAs, so-called charged tRNAs, is catalyzed

by speciﬁc aminoacyl-tRNA synthetases There is one of these enzymes for each of

the 20 amino acids and each aminoacyl-tRNA synthetase loads its amino acid only onto tRNAs designed to carry it In turn, these tRNAs speciﬁcally recognize unique sequences of bases in the mRNA through complementary base pairing.

Once it was realized that the sequence of bases in a gene speciﬁed the sequence of amino acids in a protein, various possibilities for such a genetic code were consid-ered How many bases are necessary to specify each amino acid? Is the code over-lapping or nonoverover-lapping (Figure 30.2)? Is the code punctuated or continuous? Mathematical considerations favored a triplet of bases as the minimal code word, or

codon, for each amino acid: A doublet code based on pairs of the four possible bases, A, C, G, and U, has 42 16 unique arrangements, an insufﬁcient number to encode the 20 amino acids A triplet code of four bases has 43 64 possible code words, more than enough for the task.

The Genetic Code Is a Triplet Code

The genetic code is a triplet code read continuously from a fixed starting point in each mRNA Specifically, it is defined by the following:

1 A group of three bases codes for one amino acid.

2 The code is not overlapping.

The Maya encoded their history in hieroglyphs carved

on stelae and temples like these ruins in Tikal,

Guatemala

We are a spectacular, splendid manifestation

of life We have language and can build

metaphors as skillfully and precisely as

ribosomes make proteins We have affection.

We have genes for usefulness, and usefulness

is about as close to a “common goal” of

nature as I can guess at And ﬁnally, and

perhaps best of all, we have music.

Lewis Thomas (1913–1994)

“The Youngest and Brightest Thing Around”

in The Medusa and the Snail (1979)

KEY QUESTIONS

30.1 What Is the Genetic Code?

30.2 How Is an Amino Acid Matched with Its

Proper tRNA?

30.3 What Are the Rules in Codon–Anticodon

Pairing?

30.4 What Is the Structure of Ribosomes, and

How Are They Assembled?

30.5 What Are the Mechanics of mRNA

Translation?

30.6 How Are Proteins Synthesized in Eukaryotic

Cells?

ESSENTIAL QUESTION

Ribosomes synthesize proteins by reading the nucleotide sequence of mRNAs and polymerizing amino acids in an N ⎯ →C direction.

How is the nucleotide sequence of an mRNA molecule translated into the amino acid sequence of a protein molecule?

Create your own study path for

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