Chapter 29: Transcription and the Regulation of Gene Expression PDF

Reginald H. Garrett Charles M. Grisham www.cengage.com/chemistry/garrett Chapter 29 Transcription and the Regulation of Gene Expression © 2017 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part, except for use as permitted in a license distributed w...

Reginald H. Garrett Charles M. Grisham www.cengage.com/chemistry/garrett Chapter 29 Transcription and the Regulation of Gene Expression © 2017 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part, except for use as permitted in a license distributed with a certain product or service or otherwise on a password-protected website for classroom use. Chapter 29 “Now that we have all this useful information, it would be nice to do something with it.” From the Unix Programmer’s Manual A portion of the Rosetta Stone, inscribed in 196 B.C. Essential Question How are the genes of prokaryotes and eukaryotes transcribed to form RNA products that can be translated into proteins? Outline 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 gene expression be regulated once the transcript has been synthesized? 29.7 Can we propose a unified theory of gene expression? All Cells Contain Three Major Classes of RNA – mRNA, rRNA, and tRNA All three forms participate in protein synthesis All RNAs are synthesized from DNA templates by DNAdependent RNA polymerases This process is called transcription Only mRNAs direct the synthesis of proteins Transcription is tightly regulated in all cells Only 3% of genes in a typical eukaryotic cell are undergoing transcription at any given moment The metabolic conditions and growth status of the cell dictate which gene products are needed at any moment 29.1 How Are Genes Transcribed in Bacteria? In bacteria, virtually all RNA is synthesized by a single species of DNA-dependent RNA polymerase RNA polymerases link NTPs (ATP, GTP, CTP, and UTP) in the order specified by base-pairing with a DNA template The polymerase moves along the DNA strand in the 3'-5' direction Thus, the RNA chain grows 5'-3' during transcription Subsequent hydrolysis of PPi to inorganic phosphate by pyrophosphatases makes the polymerase reaction thermodynamically favorable Sigma Subunits of Bacterial RNA Polymerases Identify Transcription Start Sites Transcription is initiated in bacteria by RNA polymerase holoenzyme, with the subunit composition α2ββ'σ The core polymerase is α2ββ' (see Figure 29.1) Binding of the σ subunit allows the polymerase to recognize DNA sequences that act as promoters Promoters are nucleotide sequences that identify the location of transcription start sites, where transcription begins Without σ bound, core polymerase can transcribe DNA into RNA, but cannot initiate transcription at specific sites Structure of the Core RNA Polymerase From Thermus thermophilus The template DNA strand is green, the nontemplate strand is blue, and the RNA transcript is hot pink. The 2 α chains are orange, the β chain is cyan, the β' chain is yellow. The Process of Transcription Has Four Stages Transcription can be divided into four stages: 1) Binding of RNA polymerase holoenzyme to template DNA at promoter sites 2) Initiation of polymerization 3) Chain elongation 4) Chain termination Binding to Promoter Sites RNA synthesis is normally initiated only at specific sites on the DNA template. RNAP binds non-specifically to the DNA strand and migrates, looking for an initiation site. RNAP binds to its initiation sites through base sequences know as promoters that are recognized by the corresponding σ factor. W. H. Freeman & Company Promoter Binding of Polymerase to Template DNA Polymerase binds nonspecifically to DNA with low affinity and migrates along it, looking for promoter σ-subunit recognizes promoter sequence RNA polymerase holoenzyme and promoter form a closed promoter complex (in which the DNA is not unwound) - Kd = 10-6 to 10-9 M Polymerase then unwinds about 12 pairs to form "open promoter complex" - Kd = 10-14 M RNA polymerase binding protects a nucleotide sequence spanning the region from -70 to +20, where +1 is defined as the transcription start site Transcription Initiation and Elongation Figure 29.3 Sequence of events in the initiation and elongation phases of transcription as it occurs in bacteria. Nucleotides in this region are numbered with reference to the base at the transcription start site, which is designated +1. Mindtap Conventions Used in Expressing the Sequences of Nucleic Acids & Proteins Certain conventions are used in describing information transfer from DNA to protein: o The strand of duplex DNA that is read by RNA polymerase is termed the template stand o The strand not read is the nontemplate strand o The template is read by the RNA polymerase moving 3'-5' along the template, so the RNA product, the transcript, grows in the 5'-3' direction o By convention, when the order of nucleotides in DNA is shown as a single strand, it is the 5'-3' sequence of nucleotides in the nontemplate strand that is shown Strand Designations Unlike replication in which both strands are copied, during transcription, only one strand is copied. Anti-sense or non-coding strand – The DNA strand that serves as the template during transcription, complementary to the transcript Sense or coding strand – The other DNA strand, which has the same sequence as the transcribed RNA 5’ dCGCTATAGCGTTT 3’ 3’ dGCGATATCGCAAA 5’ non-template, sense, coding template, anti-sense, non-coding 5’ rCGCUAUAGCGUUU 3’ transcript Properties of Bacterial Promoters Promoters recognized by the σ factor typically consist of a 40-bp region on the 5'-side of the transcription start site Within the promoter are two consensus sequence elements: o The Pribnow box near -10, with consensus TATAAT This region is ideal for unwinding - why? (It is rich in As and Ts, which only form two H bonds per base pair) o The -35 region, with consensus TTGACA - The σ-subunit binds here. The more the -35 region sequence corresponds to the consensus sequence, the better the σ-subunit binds, and the greater is the efficiency of gene transcription The Nucleotide Sequences of Representative E. coli Promoters Figure 29.4 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. In this figure, sequences are aligned relative to the Pribnow box. Initiation of Polymerization RNA polymerase has two binding sites for NTPs The initiation site prefers to bind ATP and GTP (most RNAs begin with a purine at the 5'-end) The elongation site binds the second incoming NTP 3'-OH of the first nucleotide bound attacks α-P of the second to form a new phosphoester bond (eliminating PPi) When 6-10 unit oligonucleotide has been made, the σ subunit dissociates, signaling the completion of "initiation" Chain Elongation The core polymerase (without σ) is the elongation enzyme RNA polymerase is accurate - only about 1 error in 10,000 bases Even this error rate is OK, since many transcripts are made from each gene Elongation rate is 20-50 bases per second - slower in G/C-rich regions (why??) and faster elsewhere o G-C base pairs share three H bonds, whereas A-T base pairs, with two H bonds, are less stable Chain Elongation Topoisomerases precede and follow polymerase to relieve supercoiling 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 untangle the transcript from the DNA duplex. (b) Instead, gyrases and topoisomerases act to remove the torsional stresses induced by transcription. Chain Elongation Saunders College Publishing Transcription is Rapid Once RNAP has initiated transcription and moved away from the promoter, another RNAP can follow. RNAs needed in large quantities (rRNA) are initiated as often as sterically possible, about one per second. Chain Termination Two types of transcription termination mechanisms operate in bacteria: One depends on rho termination factor o Rho is an ATP-dependent helicase o It moves along the RNA transcript, finds the “transcription bubble," unwinds the DNA:RNA hybrid, and releases the RNA chain o It is likely that the RNA polymerase stalls in a G:C-rich termination region, allowing rho factor to overtake it Figure 29.7 Transcription Termination by Rho Factor Intrinsic Termination The second termination mechanism is termed intrinsic termination Here, termination is determined by specific sequences in the DNA – called termination sites Termination sites consist of three structural features o Inverted repeats, rich in G:C, which form a stable stemloop structure in the RNA transcript o A nonrepeating segment that punctuates the inverted repeats o A run of six-eight As in the DNA template, coding for Us in the transcript Intrinsic Termination Site for the E. coli trp Operon The intrinsic termination site for the E. coli trp operon. The inverted repeats give rise to a step-loop, or “hairpin,” structure ending in a series of U residues ρ-Independent Transcription Termination Saunders College Publishing RNAP pauses when it reaches a termination site. The pause may give the hairpin structure time to fold. The fold disrupts important interactions between the RNAP and its RNA product. The U-rich RNA can dissociate from the template. The complex is now disrupted, and elongation is terminated. Outline 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 gene expression be regulated once the transcript has been synthesized? 29.7 Can we propose a unified theory of gene expression? 29.2 – How Is Transcription Regulated in Bacteria? Genes for enzymes in pathways are grouped in clusters on the chromosome - called operons This allows coordinated expression through transcription into a single polycistronic mRNA Regulatory sequences adjacent to such a unit determine whether it is transcribed – these regulatory sequences are the promoter and the operator Regulatory proteins work with operators and promoters to control transcription of the genes The General Organization of Operons Figure 29.8 Operons consist of transcriptional control regions and a set of related structural genes, all organized in a contiguous linear array along the chromosome. The transcriptional control regions are the promoter and the operator, which lie next to, or overlap, each other, upstream from the structural genes they control. Operators may lie at various positions relative to the promoter, either upstream or downstream. Expression of the operon is determined by access of RNA polymerase to the promoter, and occupancy of the operator by regulatory proteins influences this access. Induction activates transcription from the promoter; repression prevents it. Transcription of Operons Is Controlled by Induction and Repression Increased synthesis of enzymes in response to the presence of a metabolite is induction Decreased synthesis in response to a metabolite is repression Some substrates induce enzyme synthesis even though the enzymes can’t metabolize the substrate - these are gratuitous inducers - such as IPTG (isopropyl β-thiogalactoside) Lactose Is an Inducer of the lac Operon Figure 29.9 The structure of lactose, a β-galactoside. Metabolism of lactose depends on hydrolysis into its component sugars, glucose and galactose, by the enzyme βgalactosidase. Lactose availability induces the synthesis of this enzyme by activating transcription of the lac operon. Bacteria Adapt Bacteria adapt to their environments by producing enzymes that metabolize certain nutrients only when those substances are available. Glucose is E. coli’s metabolic fuel of choice. Adequate amounts of glucose prevent the expression of genes responsible for metabolizing lactose. When the level of glucose is low but lactose is available, cells increase the rate at which they synthesize the lactose metabolizing proteins by ~1000 fold. This allows the cell to limit the synthesis of unnecessary enzymes. The lac Operon Figure 29.11 The operon consists of two transcription units. In one unit, there are three structural genes, lacZ, lacY, and lacA, under control of the promoter, plac, and the operator O. In the other unit, there is a regulator gene, lacI, with its own promoter, placI. Mindtap Ways to Regulate Transcription Negative Regulation (repression): A negative regulatory factor (repressor) blocks the ability of RNAP to bind to and initiate transcription at a strong promoter. RNAP Repressor -35 -10 Example: lac repressor The lac Operon Serves as a Paradigm of Operons lacI mutants constitutively express the genes needed for lactose metabolism The structural genes of the lac operon are controlled by negative regulation lac repressor, a tetrameric protein, is the lacI gene product The lac operator is a palindromic DNA segment lac repressor has a DNA-binding domain on its Nterminus; the C-terminus binds inducer, forms tetramer. The lac Repressor Protein Has a domain structure: N-terminal = DNA binding C-terminal = forms multi-mers. C C C-terminal domain subunit-subunit interactions Hinge region flexible connector N N N-terminal domain DNA binding The Mode of Action of lac Repressor Figure 29.12 The structure of the lac repressor tetramer with bound IPTG (purple) is also shown. Mindtap The lac Operon Is Also Controlled by CAP If both lactose and glucose are present, the lac operon is NOT transcribed. lac repressor is not binding the operator so why is the lac operon not transcribed? Because the lac promoter is weak Polymerase needs CAP to transcribe the lac operon. Ways to Regulate Transcription Positive Regulation (Activation): A positive regulatory factor (activator) improves the ability of RNAP to bind to and initiate transcription at a weak promoter. RNAP Activator -35 Activator binding site Example: CAP -10 +1 Catabolite Activator Protein Provides Positive Control of the lac Operon Some promoters require an accessory protein to activate transcription Catabolite activator protein or CAP is one such protein CAP is a dimer of 22.5-kD peptides N-terminus binds cAMP; C-terminus binds DNA Binding of CAP-(cAMP)2 to DNA assists formation of closed promoter complex Catabolite repression ensures that the operons necessary for metabolism of alternative energy sources (the lac and gal operons) remain repressed until the supply of glucose is exhausted. lac Promotor Is Weak Example: Plac 5’ TTACAC spacer TTGACA 3’ -10 box -35 box 5’ TATGTT spacer TATAAT 3’ Consensus RNAP can’t bind too efficiently on its own. CAP can contact RNAP and stabilize the formation of a closed complex on weak promoters (by 50-fold). CAP Structure CAP is a dimer made up of two identical subunits. Each subunit has a modular, two-domain structure. N N cAMP cAMP N-terminal domain subunit-subunit interactions cAMP binding positive regulation of transcription Hinge region flexible connector C-terminal domain DNA binding cAMP-Induced Structural Transition in CAP cAMP induces a conformational change -- an “allosteric transition”-- in CAP. This involves a change in the subunit-subunit orientation and in the domain-domain orientation. N N N N cAMP cAMP + cAMP Without cAMP Binds DNA with low affinity but has no sequencespecificity With cAMP High-affinity sequencespecific DNA binding + interaction with RNAP What Is the Connection Between CAP and Glucose? CAP is a glucose sensor. When [glucose] is low, [cAMP] increases: - CAP is active in DNA binding. - Transcription is activated. When [glucose] is high, [cAMP] decreases: - CAP does not bind DNA - Transcription is not activated (poor promoter). Transcription of lac operon is activated when lactose is present AND [glucose] is low. Proteins Bound at lac Operon Regulatory Region N N C cAMP C N C N C cAMP RNAP C C -72 -51 CAP binding site N -35 -10 promoter -5 N to Olac +21 Situation I: Glucose Is Sole Carbon Source CAP C RNAP C lac repressor C N N N N [glucose] high [cAMP] low CAP does not bind. C [lactose] low repressor binds -72 -51 CAP binding site -35 -10 promoter -5 N N to +21 Olac lac operon not transcribed, blocked by repressor Situation II: Lactose and Glucose Are Present N lactose C CAP C lactose lactose C N [lactose] high Repressor does not bind. [glucose] high [cAMP] low CAP does not bind. C lactose RNAP -72 -51 CAP binding site -35 -10 promoter -5 to Olac +21 lac operon is not transcribed, poor promoter Situation III: Lactose Is the Sole Carbon Source C CAP N cAMP lactose N cAMP RNAP C C -72 -51 lactose C [glucose] low [cAMP] high CAP binds. -35 CAP binding site -10 promoter -5 to Olac lac operon is transcribed. +21 lactose C [lactose] high Repressor does not bind. C lactose Negative and Positive Control Systems Are Fundamentally Different Negative and positive control systems operate in fundamentally different ways Genes under negative control are transcribed unless they are turned off by the presence of a repressor protein Often, transcription activation is merely the release from negative control In contrast, genes under positive control are expressed only if an active regulator protein is present trp Operon Is Regulated Through a CoRepressor-Mediated Negative Control Circuit The trp operon encodes a leader sequence and five proteins (trpE through TrpA) that synthesize tryptophan Expression of the trp operon is under control of Trp repressor Trp repressor binding excludes RNA polymerase from the promoter When Trp is plentiful, Trp repressor binds two molecules of Trp and associates with the trp operator that is located within the trp promoter trp Operon Is Regulated Through a CoRepressor-Mediated Negative Control Circuit Trp repressor binding excludes RNA polymerase from the promoter, preventing transcription of the trp operon. When Trp becomes limiting, repression is lifted because Trp repressor lacking bound Trp has a lowered affinity for the trp promoter. trp Operon of E. coli Figure 29.18 The trp operon of E. coli. Attenuation Is a Prokaryotic Mechanism for Post-Transcriptional Regulation of Expression In addition to repression, expression of the trp operon is controlled by transcription attenuation Unlike the mechanisms discussed thus far, attenuation regulates transcription after it has begun Attenuation is any regulatory mechanism that manipulates transcription termination or transcription pausing to regulate gene transcription downstream In prokaryotes, transcription and translation are coupled, and the translating ribosome is affected by the formation and persistence of secondary structure in the mRNA Attenuation Is a Prokaryotic Mechanism for Post-Transcriptional Regulation of Expression In many operons encoding enzymes of amino acid biosynthesis, a transcribed leader region lies between the promoter and the first major structural gene These regions encode a short leader peptide containing multiple codons for the pertinent amino acid For example, the leader peptide of the leu operon has four leucine codons, the trp operon has two tandem tryptophan codons, and so forth (Fig. 29.19) Translations of these codons depends on availability of the amino acid trp Operon of E. coli Figure 29.18 The trp operon of E. coli. Sequences of Leader Peptides in Various Amino Acid Biosynthetic Operons Figure 29.19 Amino acid sequences of leader peptides in various amino acid biosynthetic operons regulated by attenuation. Color indicates amino acids synthesized in the pathway catalyzed by the operon’s gene products. The ilv operon encodes enzymes of isoleucine, leucine, and valine biosynthesis. Attenuation Is a Prokaryotic Mechanism for Post-Transcriptional Regulation of Expression When tryptophan is scarce, the entire trp operon is transcribed to give a polycistronic mRNA But as [Trp] increases, more and more of the trp transcripts consist of only a 140-nucleotide fragment corresponding to the 5'-end of trpL Tryptophan availability is causing premature cessation of trp transcription This is transcription attenuation The secondary structure of the 160-bp leader region transcript is the principal control element in transcription attenuation (Figure 29.20) Alternative Secondary Structures for the Leader Region of the Trp Operon Transcript Figure 29.20 Alternative secondary structures for the leader region of the trp operon transcript. Alternative Secondary Structures for the Leader Region of the trp Operon Transcript 1. Transcription begins and progresses until position 92 is reached. 2. The 1:2 hairpin is formed, causing RNA polymerase to pause in its elongation cycle. 3. While RNA polymerase is paused, a ribosome begins to translate the leader region of the transcript. Alternative Secondary Structures for the Leader Region of the trp Operon Transcript, Con’t 4. As long as Trp is plentiful enough that Trp-tRNA is not limiting, the ribosome is not delayed at the two Trp codons. Alternative Secondary Structures for the Leader Region of the trp Operon Transcript, Con’t 5. Translation by the ribosome releases the paused RNA polymerase and transcription continues. The ribosome follows closely behind RNAP, translating the message soon after it is transcribed. Alternative Secondary Structures for the Leader Region of the trp Operon Transcript, Con’t X 6. The presence of the ribosome atop segment 2 blocks formation of the 2:3 antiterminator hairpin, allowing the alternative 3:4 terminator hairpin to form. Alternative Secondary Structures for the Leader Region of the trp Operon Transcript, Con’t 7. Stable hairpin Fig. 29-19, p. 958 structures followed by a run of Us are features typical of rhoindependent transcription termination signals, so the RNA polymerase perceives this hairpin as a transcription stop signal and transcription is terminated at this point. Alternative Secondary Structures for the Leader Region of the trp Operon Transcript, Con’t 8. On the other hand, low levels of Trp and hence low availability of TrptRNA causes the ribosome to stall on segment 1. 9. This leaves segment 2 free to pair with segment 3 and to form the 2:3 anti-terminator hairpin in the transcript. Alternative Secondary Structures for the Leader Region of the trp Operon Transcript, Con’t X 10. Because this hairpin precludes formation of the 3:4 terminator, termination is prevented, and the entire operon is transcribed. Outline 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 gene expression be regulated once the transcript has been synthesized? 29.7 Can we propose a unified theory of gene expression? 29.3 How Are Genes Transcribed in Eukaryotes? Three classes of RNA polymerases (I, II, and III) transcribe rRNA, mRNA, and tRNA genes, respectively Pol III transcribes a few other RNAs as well All three are big, multimeric proteins (500-700 kD) All have two large subunits with sequences similar to β and β' in E. coli RNA polymerase, so the catalytic site is evolutionarily conserved Pol II is most sensitive to α−amanitin, an octapeptide from Amanita phalloides ("destroying angel” mushroom) Pol III is less so, and Pol I is insensitive Sensitivity to α-Amanitin Distinguishes the Three Classes of RNA Polymerase Figure 29.23 The structure of αamanitin, one of a series of toxic compounds known as amatoxins that are found in the mushroom Amanita phalloides. 29.3 How Are Genes Transcribed in Eukaryotes? With three categories of polymerases acting on three sets of genes, there are also at least three categories of promoters that maintain specificity Eukaryotic promoters are very different from bacterial promoters All three eukaryotic RNA polymerases interact with their promoters via transcription factors Transcription factors are DNA-binding proteins that recognize and accurately initiate transcription at specific promoter sequences RNA Polymerase II Transcribes ProteinCoding Genes RNA pol II must be capable of transcribing a great diversity of genes but must also function at any moment only on the genes whose products are appropriate to the needs of the cell The RNA pol II enzymes from yeast and humans are homologous The structure of RNA pol II from yeast is known (Figure 29.24) and consists of 12 polypeptides RNA polymerases adopt a claw-like structure, to grasp the DNA duplex The Regulation of Gene Expression Is More Complex in Eukaryotes Pol II promoters consist of two separate sequence features: o The core promoter, within which the transcription start site (TSS) lies, where general transcription factors bind o More distantly located regulatory elements (known as enhancers and silencers) The two most common core promoters in proteinencoding genes are the TATA box and the Inr (Initiator) element The Regulation of Gene Expression Is More Complex in Eukaryotes In addition to promoters, eukaryotic genes have enhancers, also known as upstream activation sequences, which may lie far from the promoter DNA looping permits multiple proteins to bind to DNA sequences Transcription Initiation by RNA Polymerase II Requires TBP & the GTFs Transcription initiation in eukaryotes proceeds through assembly of a preinitiation complex (PIC) The PIC consists of: o RNA polymerase II o General transcription factors (TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, and TFIIS) Transcription Initiation by RNA Polymerase II Requires TBP & the GTFs The PIC consists of: o A complex called Mediator (Srb/Med)  The CTD of pol II anchors Mediator  Mediator allows Pol II to communicate with transcriptional activators bound at sites distant from the promoter Chromatin-Remodeling Complexes Alleviate Repression Due to Nucleosomes The central structural unit of nucleosomes, the histone “core octamer,” is constructed from the eight histone-fold protein domains of the eight various histone monomers comprising the octamer Interactions between histone tails contributed by core histones in adjacent nucleosomes are an important influence in establishing higher orders of chromatin organization Activation of eukaryotic transcription depends on: o Relief from repression imposed by chromatin structure o Interaction of RNA polymerase II with promoter and transcription regulatory proteins Chromatin-Remodeling Complexes Alleviate Repression Due to Nucleosomes Two sets of factors are important o Chromatin-remodeling complexes that mediate ATP-dependent conformational changes in nucleosome structure o Histone-modifying enzymes that introduce covalent modifications into the N-terminal tails of the histone core octamer Chromatin remodeling and histone modification are closely linked processes Chromatin-Remodeling Complexes Alleviate Repression Due to Nucleosomes Chromatin-remodeling complexes are huge (one megadalton) These assemblies serve to loosen the DNA:protein interactions in nucleosomes by sliding, ejecting, inserting, or otherwise restructuring core octamers Outline 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 gene expression be regulated once the transcript has been synthesized? 29.7 Can we propose a unified theory of gene expression? 29.4 How Do Gene Regulatory Proteins Recognize Specific DNA Sequences? Proteins that recognize nucleic acids do so by the basic rule of macromolecular recognition They present a three-dimensional shape that is structurally and chemically complementary to the surface of a DNA sequence Protein contacts with the bases of DNA usually occur within the major groove of the DNA (but not always) Protein contacts with DNA involve H bonding and salt bridges with electronegative oxygen atoms of the phosphodiester linkages 29.4 How Do Gene Regulatory Proteins Recognize Specific DNA Sequences? 80% of DNA-binding proteins belong to one of three principal classes based on their structures: o The helix-turn-helix (HTH) motif o The zinc-finger (or Zn-finger) motif o The leucine zipper-basic region (or bZIP) Alpha Helices and DNA A perfect fit A recurring feature of DNA-binding proteins is the presence of α-helical segments that fit directly into the major groove of B-form DNA Diameter of a protein α-helix is 1.2 nm Major groove of DNA is about 1.2 nm wide and 0.6 to 0.8 nm deep Proteins can recognize specific sites in DNA Proteins With the Helix-Turn-Helix Motif Use One Helix to Recognize DNA The HTH motif is a protein structural domain consisting of two successive α-helices separated by a sharp β-turn (Figure 29.32) The C-terminal helix (denoted helix 3) fits in major groove of DNA; the N-terminal helix (helix 2) locks helix 3 into its DNA interface Proteins with HTH domains bind to DNA as dimers, with the two helix 3 cylinders antiparallel to each other Recognition of DNA sequence involves the sides of base pairs that face the major groove Proteins With the Helix-Turn-Helix Motif Use One Helix to Recognize DNA Figure 29.32 An HTH motif protein: Antp monomer bound to DNA. Helix 3 (yellow) is locked into the major groove of the DNA by helix 2 (magenta). Some Proteins Bind to DNA via ZnFinger Motifs First discovered in TFIIIA from Xenopus laevis, the African clawed toad Now known to exist in nearly all organisms Two main classes: C2H2 and Cx C2H2 domains consist of Cys-x2-Cys and His-x3-His domains separated by at least seven-eight amino acids This motif can be repeated as many as 13 times over the primary structure of a Zn-finger protein Zn-Finger Motif of the C2H2 Type Figure 29.33 The Zn-finger motif of the C2H2 type showing (a) the coordination of Cys and His residues to Zn and (b) the secondary structure. Structure of a Classic C2H2 Zn-Finger Protein (c) Structure of a classic C2H2 zinc finger protein with three zinc fingers bound to DNA. Some Proteins Bind to DNA via ZnFinger Motifs, Con’t Cx domains consist of four, five, or six Cys residues separated by various numbers of other residues The Cx proteins have a variable number of Cys residues available for Zn chelation Some DNA-Binding Proteins Use a Basic Region Leucine Zipper (bZIP) Motif First found in C/EBP, a DNA-binding protein isolated from rat liver nuclei Now found in nearly all organisms Characteristic features: a 28-residue sequence with Leu every seventh position (thus a total of four Leu residues) and a "basic region" What do you know by now about seven-residue repeats? This suggests amphipathic α−helices and a coiled-coil dimer (see A Deeper Look, The Coiled-Coil Motif in Proteins, Chapter 6, page 161) Model for a Dimeric bZIP Protein Figure 29.34 BR-A and BR-B are basic regions A and B. The Structure of the Leucine Zipper In complex with DNA Leucine zipper proteins (aka bZIP proteins) dimerize, either as homo- or hetero-dimers The basic region is the DNA recognition site The basic region is often modeled as a pair of helices that can wrap around the major groove Homodimers recognize dyad-symmetric DNA Heterodimers recognize non-symmetric DNA Fos and Jun are classic bZIPs Structure of a Leucine Zipper:DNA Complex Figure 29.35 Model for the heterodimeric bZIP transcription factor c-Fos:c-Jun bound to a DNA oligomer containing the AP-1 consensus target sequence TGACTCA. Outline 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 gene expression be regulated once the transcript has been synthesized? 29.7 Can we propose a unified theory of gene expression? How Are Eukaryotic Transcripts Processed & Delivered to the Ribosomes for Translation? In prokaryotes, transcription and translation are concomitant processes In eukaryotes, the two processes are spatially separated: transcription occurs on DNA in the nucleus, and translation occurs on ribosomes in the cytoplasm Thus, transcripts must be transported from the nucleus to the cytosol to be translated How Are Eukaryotic Transcripts Processed & Delivered to the Ribosomes for Translation? On the way, these transcripts undergo processing o Alterations that convert the newly synthesized RNAs (primary transcripts) into mature mRNAs And unlike prokaryotes, eukaryotic mRNAs encode only one polypeptide; i.e., they are monocistronic Eukaryotic Genes Are Split Genes Introns (non-coding regions) intervene between exons (protein-coding regions) Examples: actin gene has 309-bp intron between first three amino acids and the other 350 or so o But chicken pro α-2 collagen gene is 40-kbp long, with 51 exons of only 5 kbp total o In these cases, the exons range in size from 45 to 249 bases The mechanism by which introns are excised and exons are spliced together is complex and must be precise Eukaryotic Genes Are Split Genes Figure 29.36 The organization of split eukaryotic genes. Mindtap mRNA Processing Involves Capping, Methylation, Polyadenylylation, & Splicing Primary transcripts (aka pre-mRNAs or heterogeneous nuclear RNA [hnRNA]) are usually capped by addition of a guanylyl group The reaction is catalyzed by guanylyl transferase The cap G residue is methylated at the 7-position Additional methylations occur at the 2'-O positions of the next two residues and at 6-amino of the first adenine The Capping of Eukaryotic pre-mRNAs Figure 29.38 Guanylyl transferase catalyzes the addition of a guanylyl residue derived from GTP to the 5'-end of the growing transcript, which has a 5-triphosphate group already there. In the process, pyrophosphate (pp) is liberated from GTP and the terminal phosphate (p) is removed from the transcript: Gppp + pppApNpNpNp.. → GpppApNpNpNp… + pp + p (A is often the initial nucleotide in the primary transcript.) Methylation at Several Sites Is Essential to mRNA Maturation Figure 29.39 A cap bearing only a single –CH3 on the guanyl is termed cap O. This methylation occurs in all eukaryotic mRNAs. If a methyl is also added to the 2'-O position of the first nucleotide after the cap, a cap 1 structure is generated. This is the predominant cap form in RNA from all multicellular eukaryotes. 3'-Polyadenylylation of Eukaryotic mRNAs Termination of transcription occurs only after RNA polymerase has transcribed past a consensus AAUAAA sequence - the poly(A) addition site 10-35 nucleotides past this site, a string of 100 to 200 adenine residues are added to the mRNA transcript - the poly(A) tail Poly(A) polymerase adds these A residues Poly(A) tail enhances mRNA stability Nuclear Pre-mRNA Splicing Within the nucleus, hnRNA forms ribonucleoprotein particles (RNPs) through association with a characteristic set of nuclear proteins These proteins maintain the hnRNA in an untangled and accessible conformation The substrate for splicing, that is, intron excision and exon ligation, is the capped primary transcript emerging from the RNA polymerase II transcriptional apparatus Splicing occurs exclusively in the nucleus Consensus sequences define the exon/intron junctions in eukaryotic mRNA precursors Splicing of Pre-mRNA Capped, polyadenylated RNA, in the form of an RNP complex, is the substrate for splicing In "splicing," the introns are excised, and the exons are joined together to form mature mRNA The 5'-end of an intron in higher eukaryotes is always GU and the 3'-end is always AG All introns have a "branch site" 18 to 40 nucleotides upstream from 3'-splice site The branch site is essential to splicing Figure 29.41 Consensus Sequences at the Splice Sites in Vertebrate Genes The Splicing Reaction Proceeds Via Formation of a Lariat Intermediate Figure 29.42 shows the splicing mechanism The branch site is usually YNYRAY, where Y = pyrimidine, R = purine, and N is anything The lariat, a covalently closed loop of RNA, is formed by attachment of the 5'-P of the intron's invariant 5'-G to the 2'-OH at the branch A site The exons then join, excising the lariat. The lariat is unstable; the 2'-5' phosphodiester is quickly cleaved, and the intron is degraded in the nucleus. The Splicing Reaction Proceeds Via Formation of a Lariat Intermediate Figure 29.42 Splicing of mRNA precursors. A representative precursor mRNA is depicted. Exon 1 and Exon 2 indicate two exons separated by an intervening sequence (an intron) with consensus 5', 3', and branch sites. Splicing Depends on snRNPs Splicing depends on a unique set of small nuclear ribonucleoprotein particles - snRNPs, pronounced "snurps" A snRNP consists of a small RNA (100-200 bases long) and about 10 different proteins Some of the 10 proteins are general, some are specific (see Table 29.6) Major snRNP species are abundant, with more than 100,000 copies per nucleus Splicing Depends on snRNPs snRNPs and pre-mRNA form the spliceosome The spliceosome is the size of a ribosome, and its assembly requires ATP snRNPs Found in Spliceosomes snRNPs Form the Spliceosome Splicing occurs when the various snRNPs come together with the pre-mRNA to form the spliceosome snRNPs U1 and U5 bind at the 5'- and 3'- splice sites, and U2 snRNP binds at the branch site Interaction between the snRNPs brings 5'- and 3'splice sites together so the lariat can form, and exon ligation can occur Spliceosome assembly requires ATP-dependent RNA rearrangements catalyzed by spliceosomal DEAD-box ATPases/helicases Mammalian U1 snRNA Figure 29.44 The mammalian U1 snRNA can be arranged in a secondary structure where its 5'-end is singlestranded and can base-pair with the consensus 5'-splice site of the intron. Events in Spliceosome Assembly Figure 29.45 Events in spliceosome assembly. U1 snRNP binds at the 5'-splice site, followed by the association 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 branchpoint sequence with the 5'splice site, whereupon U4 snRNP is released. Alternative RNA Splicing Creates Protein Isoforms Many eukaryotic genes can give rise to multiple forms of mature RNA transcripts This may occur by: o Use of different promoters o Selection of different polyadenylylation sites o Alternative splicing of the primary transcript o A combination of these three mechanisms Alternative RNA Splicing Creates Protein Isoforms In constitutive splicing, every intron is removed, and every exon is incorporated into the mature RNA This produces a single form of mature mRNA from the primary transcript In alternative splicing, all the introns are removed, and selected exons are incorporated into the mature RNA Alternative RNA Splicing Creates Protein Isoforms Different transcripts from a single gene make possible a set of related polypeptides, termed protein isoforms, each with a slightly altered function The isoforms of fast skeletal muscle troponin T are an example of alternative splicing This gene consists of 18 exons, 11 of which are found in all mature mRNAs and are constitutive Five of the exons (4 through 8) are combinatorial, in that they may be included or excluded Alternative RNA Splicing Creates Protein Isoforms Two (16 and 17) are mutually exclusive – one is always present but never both 64 different mature mRNA can be formed from this gene by alternative splicing RNA Editing: Another Way To Increase the Diversity of Genetic Information RNA editing is a process that changes one or more nucleotides in an RNA transcript by deaminating a base, either A→I or C→U These changes alter the coding possibilities in a transcript, because I will pair with C (not U as A does) and U will pair with A (not G as C does) RNA editing can increase protein diversity by (1) Altering amino-acid coding possibilities (2) Introducing premature stop codons (3) Changing a splice site in a transcript Outline 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 gene expression be regulated once the transcript has been synthesized? (SKIP) 29.7 Can we propose a unified theory of gene expression? Outline 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 gene expression be regulated once the transcript has been synthesized? 29.7 Can we propose a unified theory of gene expression? (SKIP) Do You Understand… 1. RNA? 2. RNA polymerase? 3. Regulation of transcription? 4. DNA binding motifs? 5. Post-transcriptional processing?