29.5 How Are Eukaryotic Transcripts Processed and Delivered to the Ribosomes for Translation? 943 put (for example, as ATP) is needed. The lariat product is unstable; the 2Ј-5Ј 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. Splicing 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 5Ј-end single-stranded. The 5Ј-end of U1 snRNA is complementary to the consensus sequence at the 5Ј-splice junction of the pre-mRNA (Figure 29.43), as is a region at the 5Ј-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 5Ј-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) 3Ј OH AGp Exon 1 pre-mRNA 5Ј 5Ј GUA 3Ј-splice site AG pGNCY n AGU U G A AU p GAG YNYR Y Branch site 5Ј-splice site Intron Exon 2 A pGN3Ј 3Ј 3Ј5Ј 3Ј5Ј 3Ј CY n AGYNYR Y Exon 2 A 2ЈOH AG OH U G A A U G p U G A A U G p pGNCY n AG AG YNYR Y Exon 2 A NCY n AGYNYR YA pG 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 5Ј, 3Ј, and branch sites.The fate of the phosphates at the 5Ј- and 3Ј-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 U1 165 5Ј splice U2 189 Branch U4 145 5Ј splice, recruitment U5 115 of branch point to U6 106 5Ј-splice site TABLE 29.6 The snRNPs Found in Spliceosomes ⎫ ⎬ ⎭ ⎧ ⎨ ⎩ 944 Chapter 29 Transcription and the Regulation of Gene Expression binds U2 snRNP, and then the triple snRNP complex of U4/U6иU5 replaces U1 at the 5Ј-splice site. The substitution of base-pairing interactions between U1 and the pre-mRNA 5Ј-splice site by base-pairing between U6 and the 5Ј-splice site is just one of the many RNA rearrangements that accompany the splicing reaction. Base- pairings between U6 and U2 RNA bring the 5Ј-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 2Ј-O of the invariant A residue in the branch-point sequence displaces the 5Ј-exon from the intron, creating the lariat intermediate. The free 3Ј-O of the 5Ј-exon now triggers a second transesterification reaction through attack on the P atom at the 3Ј-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 RNA-annealing functions as well as proteins 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 5Ј- or 3Ј-splice sites or by omitting 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. CCAUUCA AGGUAAGUC U U1 G U A (3meG cap) 5 Ј Branch point 3Ј5' 3Ј AGY 3Ј-splice site region Intron 3Ј exon 3Ј-splice site 5Ј-splice site 5Ј exon 5Ј-splice site region FIGURE 29.43 Mammalian U1 snRNA can be arranged in a secondary structure where its 5Ј-end is single-stranded and can base-pair with the consensus 5Ј-splice site of the intron. (Adapted from Figure 1 in Rosbash, M., and Seraphin, B., 1991.Who’s on first? The U1 snRNP-5 Ј splice site interaction and splicing. Trends in Biochemical Sciences 16:187.) ATP ATP ATP ATP ATP ATP ATP ATP U2 U2 U2 U2 U2 U2 U2 U4 U5 U1 U6 U1 U5 U1 U5 U6 U5 U6 U6 U4 U5 U6 U4 U6 U5 U6 U4 U5 U6 FIGURE 29.44 Events in spliceosome assembly. U1 snRNP binds at the 5Ј-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 5Ј-splice site and directs the juxtaposition of the branch-point sequence with the 5Ј-splice site, whereupon U4 snRNP is released. Lariat formation oc- curs, freeing the 3Ј-end of the 5Ј-exon to join with the 5Ј-end of the 3Ј-exon, followed by exon ligation. U2, U5, and U6 snRNPs dissociate from the lariat following exon ligation. 29.5 How Are Eukaryotic Transcripts Processed and Delivered to the Ribosomes for Translation? 945 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 5Ј UT DNA mRNAs Any of the 32 possible combinations with zero, one, two, three, four, or all five of exons 4 through 8 () 3Ј UT () 5Ј UT 1– 6 7– 10 12 3 4 5 6 7 8 9101112131415 1617 18 1 2 3 9 10 11 12 13 14 15 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 5Ј-untranslated (UT) sequences, and Exon 18 includes the polyadenylation site (AATAAA) and 3Ј-UT sequences.The TATA box indicates the transcription start site.The amino acid residues encoded by each exon are indicated below. Many exonϺintron 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. 946 Chapter 29 Transcription and the Regulation of Gene Expression 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. 29.6 Can We Propose a Unified Theory of Gene Expression? 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 throu gh 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 macro- 29.6 Can We Propose a Unified Theory of Gene Expression? 947 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 3Ј→5Ј 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 A A A 2 0 0 3 Ј G p p p X 5 Ј 5 Ј Coupled transcription and mRNA processing Transcription factor * Chromatin decompaction Chromatin Coupled initiation and 5Ј capping GpppX 5Ј 5Ј GpppX 5Ј 5Ј GpppX 5Ј 5Ј GpppX 5Ј 5Ј Cleavage and 3Ј polyadenylation Splicing Nuclear pore NUCLEUS CYTOPLASM mRNA packaging mRNA export Protein folding Protein GpppX 5Ј 5Ј AAA 200 3Ј Translation Mediator IIE IIH IIB TBP TATA A A U A A RNAPII IIF RNAPII G / L RNAPII RNAPII RNAi siRNA 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.) SUMMARY 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 5Ј-triphosphates in an order specified by base pairing with a DNA tem- plate. The enzyme reads along a DNA strand in the 3Ј→5Ј direction, joining the 5Ј-phosphate of an incoming ribonucleotide to the 3Ј-OH of the previous residue, so the RNA chain grows 5Ј→3Ј 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, the -subunit dissociates. The core RNA polymerase is highly processive 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 YSPTSPS; 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 proteinϺprotein 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 5Ј-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 2Ј-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 3Ј-end, added post-transcriptionally by poly(A) polymerase. Most eukaryotic genes are split genes, subdivided into cod- ing regions, called exons, and noncoding 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 5Ј- and 3Ј-ends of introns and 948 Chapter 29 Transcription and the Regulation of Gene Expression Cavity FIGURE 29.47 Structure of the human exosome core (pdb id ϭ 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 3Ј-exonuclease activity. Evidence suggests that the human exosome core selects RNAs for degradation, and associated 3Ј-exonucleases then degrade them. Problems 949 PROBLEMS Preparing for an exam? Create your own study path for this chapter at www.cengage.com/login. 1. The 5Ј-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 description 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 m for 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 proteinϺDNA 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 DNAϺhistone interactions? 10. (Integrates with Chapter 15.) Predict from Figure 29.12 whether the interaction of lac repressor with inducer mig ht 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 Mg 2ϩ ion in the RNA polymerase II active site; more recent studies reveal the presence of two. Why is the presence of two Mg 2ϩ ions 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 5Ј-R neighbor, its 3Ј-Y neighbor, and its 2Ј-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 5Ј-phosphate group of the intron’s invariant 5Ј-G to the 2Ј-OH at the invariant branch site A to form a 2Ј-5Ј 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 splicin g 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. 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. 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Nature Structural and Molecular Biology 12:562–564. Schmid, M., and Jensen, T. H., 2008. The exosome: A multipurpose RNA-decay machine. T rends in Biochemical Sciences 33:501–510. George Holton/Photo Researchers, Inc. 30 Protein Synthesis 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 3Ј-OH at the 3Ј-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. 30.1 What Is the Genetic Code? 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 nonoverlapping (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 4 2 ϭ 16 unique arrangements, an insufficient number to encode the 20 amino acids. A triplet code of four bases has 4 3 ϭ 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 this chapter with tutorials, simulations, animations, and Active Figures at www.cengage.com/login.