2023_L13.pdf

1 Lecture 13 Transcription and Post-transcriptional Processing Translation and Post-translational Protein Processing 2 3 Enzymology of RNA Synthesis: RNA Polymerase DNA replication involves rapid chain growth at few intracellular sites, and transcri...

1 Lecture 13 Transcription and Post-transcriptional Processing Translation and Post-translational Protein Processing 2 3 Enzymology of RNA Synthesis: RNA Polymerase DNA replication involves rapid chain growth at few intracellular sites, and transcription involves slower growth at many sites. More RNA accumulates than DNA. The functions of the various RNA polymerase subunits can be determined by reconstitution 4 Enzymology of RNA Synthesis: RNA Polymerase RNA polymerase subunit structures in the three domains of life: The subunits are arranged by function rather than size. Homologous subunits are color coded. Subunits marked with an asterisk are conserved among the three eukaryotic RNA polymerases. These are core enzyme structures, not showing bound proteins that aid in template recognition. 5 Enzymology of RNA Synthesis: RNA Polymerase Crystal structures of RNA polymerases: a)Taq RNA polymerase complexed with DNA. a)Yeast RNA polymerase II. 6 Mechanism of Transcription Initiation and elongation steps of transcription by bacterial RNA polymerase: Transcription begins with sequence- specific interaction between RNA polymerase and a promoter site, where duplex unwinding and template strand selection occur.  Mechanism of Transcription 7 DNA bending in an open-promoter complex: This image is taken from the crystal structure of Taq RNA polymerase. Parts of have been removed to show interior structure; outline of is shown as a cyan line. The template DNA strand is shown in green, and the nontemplate strand is in yellow. Note the sharp bend in the template strand. The first two ribonucleotides to be incorporated (i and i + 1) are shown in orange and pink, respectively, close to a catalytically essential Mg2+. 8 MECHANISM OF TRANSCRIPTION  Mechanism of Transcription 9 The transcription bubble: The lengths of unwound DNA and DNA–RNA hybrid were originally estimated from reactivities of transcription complexes with reagents such as KMnO4, which oxidizes bases in single-strand nucleic acids. The length of DNA in contact with the enzyme is determined by footprinting. Six or seven nucleotides of RNA behind the DNA hybrid are protected from ribonuclease attack by binding to the enzyme.  Mechanism of Transcription 10 Cutaway view of the RNA polymerase II elongation complex: Direction of polymerase motion is left to right as shown. DNA entering the enzyme is gripped by protein “jaws” (upper jaw not shown in this cutaway model). The 3’ end of growing RNA is adjacent to one of the catalytically essential Mg2+ ions. The wall forces the DNA to turn. rNTPs probably enter the active site, as shown, through a funnel structure and pore. The 5’ end of the growing RNA chain is diverted from the DNA template by a protein loop called the rudder, which limits the length of RNA hybridized to template DNA. The rudder and lid, which guide the exit of RNA, emanate from a large clamp that swings from back to front, as shown, over the catalytic site and contribute to the binding of nucleic acids, and hence, to the high processivity of transcription.  Mechanism of Transcription 11 The nucleotide addition cycle in T. thermophilus RNA polymerase: Opening of the pincer permits translocation of the DNA, with just one template nucleotide available for base-pairing.  Mechanism of Transcription 12 Backtracking in an elongation complex: Above, the 3’ terminus of the transcript is in the active site (denoted by Mg2+). Below, the enzyme has slipped backward, leaving the transcript terminus at the end of a nonbase-paired RNA tail, some five nucleotides long. Transcription can resume either by forward sliding of polymerase back to the structure showed above or, more likely, by cleavage of the non-base-paired part of the transcript, creating a new base-paired 3’ terminus. 13 Mechanism of Transcription The frequency with which a gene is transcribed in E. coli genes is largely determined by the similarity between the gene’s promoter sequence and the consensus sequence. Conserved sequences in promoters recognized by E. coli RNA polymerase: Lengths of spacer sequences are also shown. Red arrows indicate transcription start site. 14 Mechanism of Transcription DNA sequences that promote factor- independent termination include a run of four to eight A residues and a GC-rich region that forms a stem–loop. In factor-dependent termination, protein acts as an RNA–DNA helicase, unwinding the template–transcript duplex and facilitating release of the transcript. 15 Mechanism of Transcription A model for factor-independent termination of transcription: a)An A-rich segment of the template has just been transcribed into a U-rich mRNA segment. a)RNA–RNA duplex, stabilized by G-C base pairs, eliminates some of the base pairing between template and transcript. a)The unstable A–U bonds linking transcript to template hybrid dissociate, releasing the transcript.  Mechanism of Transcription 16 r (rho) factor–dependent termination: r binds to a site on the nascent transcript and unwinds the RNA–DNA duplex. Once r reaches RNA polymerase, interaction with bound NusA protein (not shown) leads to termination. Rho's key function is its helicase activity Rho factor acts as an ATP-dependent unwinding enzyme, moving along the newly forming RNA molecule towards its 3′ end and unwinding it from the DNA template as it proceeds. 17 Mechanism of Transcription Crystal structure of E. coli r: a)RNA is shown in the central channel in orange. a)Schematic diagram of the helicase reaction. 18 Transcription and its Control in Eukaryotic Cells Eukaryotes have three kinds of nuclear RNA polymerases, each requiring additional protein factors to initiate transcription. o Pol I transcribes the major ribosomal RNA genes. o Pol III transcribes small RNA genes. o Pol II transcribes protein-encoding genes and a few small RNA genes. 19 Transcription and its Control in Eukaryotic Cells Transcription and processing of the major ribosomal RNAs in eukaryotes: The genes exist in tandem copies, separated by nontranscribed spacers. 20 Transcription and its Control in Eukaryotic Cells Preparation of a 5S rRNA gene for transcription: At least the three protein factors shown, plus RNA polymerase III, must assemble on the gene before transcription can occur. TFIIIA must bind to the gene before factors TFIIIC and TFIIIB can bind. Once the stable complex II has been formed, it will recycle with pol III to produce many RNA copies. An excess of 5S rRNA will form a complex with TFIIIA, inhibiting further transcription. 21 Transcription and its Control in Eukaryotic Cells Zinc fingers: a)The transcription factor TFIIIA binds to the 5S RNA gene via zinc fingers inserted into the major groove. The two major recognition regions, A block and C block, are contacted by fingers 7–9 and 1–3, respectively. a)Structure of a zinc finger. The structure shown is for a synthetic polypeptide with sequences found in a zinc finger protein.  Transcription and its Control in Eukaryotic Cells 22 Structures of four common types of DNA-binding motifs from eukaryotic transcription factors: (a) The zinc finger motif. (b) A helix–turn–helix motif. (c) A leucine zipper protein. (d) A helix–loop–helix motif. 23 Transcription and its Control in Eukaryotic Cells 24 Transcription and its Control in Eukaryotic Cells Structures of a few typical eukaryotic promoters: 25 Transcription and its Control in Eukaryotic Cells Transcription can be modified by binding of trans-acting factors, either in the promoter or in distant enhancers. 26 Transcription and its Control in Eukaryotic Cells A model for formation of a minimal preinitiation complex (PIC) for pol II on a TATA promoter: In the simplest situation, binding of TATA-binding protein (TBP) initiates the sequence. Alternatively, in vivo TFIID, which includes both TBP and associated factors (TAFs), is used. This will also result in binding of TFIIA. The series of dots indicates phosphorylation of the C-terminal domain (CTD) of Rpb1, the largest subunit of pol II. Phosphorylation is necessary for release of the enzyme from the initiation site.  Transcription and its Control in Eukaryotic Cells 27 Model of the TFIIA–TBP–TFIIB–promoter complex based on crystal structures of TBP–TFIIB–TATA and TFIIA–TBP–ATA complexes: 28 Transcription and its Control in Eukaryotic Cells A schematic representation of how DNA looping (perhaps mediated by nucleosomes) can bring enhancer-bound activator (or repressor) proteins into contact with TAFs associated with the core complex:  Transcription and its Control in Eukaryotic Cells 29 Acetylation of core histones: The general structure of each of the four core histones involves a helical “histone fold” domain plus an unstructured, highly basic N-terminal domain. Acetylation in nuclei occurs exclusively in the N-terminal domains, at the highly conserved sites indicate in red. 30 Transcription and its Control in Eukaryotic Cells Termination of transcription in eukaryotes: addition of poly(A) tails: There is an AATAAA sequence near the end of most eukaryotic genes. When this is transcribed to AAUAAA, it provides a signal for endonuclease cleavage and poly(A) tail addition. 31 Post-transcriptional Processing Bacterial transcripts undergo posttranscriptional processing, involving both endonucleolytic and exonucleolytic cleavage. 32 Post-transcriptional Processing Posttranscriptional processing in bacteria involves: Cleavage of the primary transcript. Modification of bases (in tRNA synthesis). Nontranscriptive nucleotide addition. Intron splicing (in a few cases).  Post-transcriptional Processing 33 Structure of a processed mRNA 5’ end: Details of the 5’ cap region are shown. Methyl groups that are added are in red.  Post-transcriptional Processing 34 mRNA capping l Regulation of nuclear export l Prevention of degradation by exonucleases l Promotion of translation l Promotion of 5′ proximal intron excision  Post-transcriptional Processing 35 Polyadenylation l multiple adenosine monophosphates at 3’ l protects the mRNA molecule from enzymatic degradation in the cytoplasm l aids in transcription termination and export of the mRNA from the nucleus  Post-transcriptional Processing 36 A schematic view of the mechanism of mRNA splicing: a) The overall process. b) The first transesterification reaction.  Post-transcriptional Processing 37 The overall process of splicing: The pre-mRNA plus assorted snRNPs (small nuclear ribonucleoproteins) assemble and disassemble a spliceosome, which carries out the splicing reaction. The snRNPs are designated U1, U2, and so on. In step 1 U1 is bound, which together with U2 binding (step 2) leads to a looped structure. Factors U4/6 and U5 then bind (step 3) and cleavage and transfer then occur (steps 4, 5). The spliceosome disassembles, releasing the ligated product (6) and the looped intron (7). This is degraded into small oligonucleotides (step 8). 38 Post-transcriptional Processing Kinetic analysis of spliceosome assembly: Each step in the assembly pathway was shown to be reversible, with rate constants in the forward direction as shown. Reversibility has not yet been detected in the activation step shown nor in mRNA release. SF3b is a protein splicing factor and NTC is a multiprotein complex called Prp19. 39 Post-transcriptional Processing Alternative splicing allows one gene to specify several proteins. Tropomyosin gene organization (rat) and seven alternative splicing pathways: Experimentally documented splicing pathways (solid lines) and others (dotted lines) inferred from nuclease protection mapping are shown. The smooth (SM) and striated (STR) exons encoding amino acid residues 39–80 are mutually exclusive, and there are alternative 3’–terminal exons as well. UT signifies untranslated regions. 40 Post-transcriptional Processing Possible mechanisms for alternative splicing by splice-site selection: 41 TRANSLATION 42 NUCLEAR-CYTOPLASMIC TRANSPORT 43 AN OVERVIEW OF TRANSLATION 44 AN OVERVIEW OF TRANSLATION Translation of an RNA message into a protein: As the ribosome moves along the message, it accepts specific aminoacyl tRNAs in succession, selecting them by matching the trinucleotide anticodon on the tRNA to the trinucleotide codon on the RNA message (step 1). The amino acid (in this example, the second one of the chain, Val) accepts the growing polypeptide chain (in this example the previously bound fMet) (step 2), and the ribosome moves on to the next codon to repeat the process, while releasing the deacylated transfer RNA that held the growing peptide in the previous cycle (the tRNA for fMet, step 3). The preceding steps are repeated, adding more amino acids to the chain, until a stop signal is read (step 4), whereupon a protein release factor causes both the polypeptide and the mRNA to be released.  AN OVERVIEW OF TRANSLATION 45 Transfer RNAs are the adaptor molecules that match amino acid to codon. Activation of amino acids for incorporation into proteins: A specific enzyme, aminoacyl-tRNA synthetase, recognizes both a particular amino acid and a tRNA carrying the corresponding anticodon. This synthetase catalyzes the formation of an aminoacyl tRNA, with accompanying hydrolysis of one ATP to AMP. Messenger RNA is read 5’ à 3’. Polypeptide synthesis begins at the N-terminus.  THE GENETIC CODE 46 Three concepts of genetic codes: Early research on the nature of the code quickly showed that a nonoverlapping, unpunctuated code (c) fit all experimental observations.  THE GENETIC CODE 47 The genetic code (as written in RNA): We show here the genetic code as used in most organisms. When AUG is used as a start codon, it codes for N-formylmethionine (fMet) in prokaryotes or methionine (Met) in eukaryotes The code is redundant (most amino acids are specified by more than one mRNA codon) but not ambiguous (more than one amino acid inserted at a given codon). Several codons may correspond to a single amino acid, sometimes via wobble in the 5’ anticodon position. 48 THE GENETIC CODE The wobble hypothesis: Pairing between two nucleotides in RNA molecules, that does not follow Watson-Crick base pair rules Due to the shape of the loop of tRNA one of the nucleotides will always be loosed (usually third one). 49 THE GENETIC CODE The wobble hypothesis: As an example, we show how the anticodon base G can pair with either C or U in a codon. Movement (“wobble”) of the base in the 5’ anticodon position is necessary for this capability (see arrow). 50 THE GENETIC CODE 51 THE GENETIC CODE Prokaryotic messengers contain translational start and stop signals, as well as a sequence that aligns the mRNA on the ribosome. All proteins start with N-fMet or Met, at least when they are first synthesized.  THE MAJOR PARTICIPANTS IN TRANSLATION: 52 MRNA, TRNA, AND RIBOSOMES The Shine-Dalgarno (SD) sequence is a ribosomal binding site in prokaryotic mRNA, generally located around 8 bases upstream of the start codon AUG. The RNA sequence helps recruit the ribosome to the mRNA to initiate protein synthesis by aligning the ribosome with the start codon.  THE MAJOR PARTICIPANTS IN TRANSLATION: 53 MRNA, TRNA, AND RIBOSOMES All tRNAs share a general common structure that includes an anticodon loop, which pair with codons, and an acceptor stem, to which the amino acid is attached. Structure of tRNAs: a) Generalized tRNA structure. b) A leucine tRNA from E. coli. c) A human mitochondrial tRNA for lysine.  THE MAJOR PARTICIPANTS IN TRANSLATION: 54 MRNA, TRNA, AND RIBOSOMES A sampling of the modified and unusual bases found in tRNAs:  THE MAJOR PARTICIPANTS IN TRANSLATION: 55 MRNA, TRNA, AND RIBOSOMES Unusual base pairings in tRNA: (a, b) Some unusual pair matches. (c, d) Some examples of triple interactions. R represents the ribosyl residue of the RNA chain. The bases prefixed by m are methylated at the carbon atom corresponding to the superscript. Numbers following the letters designating bases show the position in the sequence.  THE MAJOR PARTICIPANTS IN TRANSLATION: 56 MRNA, TRNA, AND RIBOSOMES Formation of aminoacyl tRNAs by aminoacyl tRNA synthetase: In step 1 the amino acid is accepted by the synthetase and is adenylylated, with the aminoacyl adenylate remaining bound to the enzyme. In step 2 the proper tRNA is accepted by the synthetase, and the amino acid residue is transferred to the 3’ OH of the 3’-terminal residue of the tRNA (class II enzymes) or to the 2’ hydroxyl, followed by isomerization to the 3’ aminoacyl-tRNA (class I enzymes). For class I enzymes the 2’ hydroxyl of the 3’- terminal AMP residue is the nucleophile for reaction 2.  THE MAJOR PARTICIPANTS IN TRANSLATION: 57 MRNA, TRNA, AND RIBOSOMES Major “identity elements” in some tRNAs: Red circles represent the positions that have been shown to identify the tRNA to its cognate synthetase. Shown also is a synthetic polynucleotide containing the G-U alanine identity element (in red), which is a good substrate for alanyl-tRNA synthetase.  THE MAJOR PARTICIPANTS IN TRANSLATION: 58 MRNA, TRNA, AND RIBOSOMES Major “identity elements” in tRNAs:  THE MAJOR PARTICIPANTS IN TRANSLATION: 59 MRNA, TRNA, AND RIBOSOMES A model of the E. coli glutaminyl tRNA synthetase coupled with its tRNA and ATP: The tRNA is represented by a detailed atomic model, the protein by its solvent-accessible surface (blue). The ATP (green) and the 3’ acceptor stem of the tRNA fit into a deep cleft in the synthetase. This cleft will also accommodate the amino acid. This is a monomeric class I synthetase.  THE MAJOR PARTICIPANTS IN TRANSLATION: 60 MRNA, TRNA, AND RIBOSOMES Components of bacterial and eukaryotic ribosomes: Bacterial (to the left) and eukaryotic (to the right) ribosomes are assembled along the same structural plan, with eukaryotic ribosomes being somewhat larger and more complex. The shapes of the ribosomal subunits were determined by electron microscopy.  THE MAJOR PARTICIPANTS IN TRANSLATION: 61 MRNA, TRNA, AND RIBOSOMES Assembly map for the 30S subunit: a)Assembly pathway in vitro, as determined by Traub and Nomura. Arrows indicate the obligatory nature of some protein-binding events. For example, S7 must bind before S9, S13, or S19, but once S7 is bound, any of these three proteins can be added. The earliest protein-binding events occur near the 5’ end of the 16S rRNA, and an intermediate to which 5’ - and central-domain proteins are bound must be formed before addition of 3’ domain proteins. b)In vivo assembly map, as determined by Williamson and colleagues. Parallel pathways begin, with proteins added at either the 5’ domain or the 3’ domain of 16S rRNA.  THE MAJOR PARTICIPANTS IN TRANSLATION: 62 MRNA, TRNA, AND RIBOSOMES A model of the 70S ribosome based upon early structural data: This model shows all three tRNA-binding sites occupied simultaneously, which does not normally occur. This view has the 30S subunit in front and the 50S subunit to the rear.  THE MAJOR PARTICIPANTS IN TRANSLATION: 63 MRNA, TRNA, AND RIBOSOMES A high-resolution model of the 50S ribosomal subunit: This view shows the two stalks and central protuberance (CP), seen also in the early electron micrographs. In this image RNA is in gray, and proteins are in gold. The peptidyltransferase site, ingreen, is identified from the binding of an inhibitor.  THE MAJOR PARTICIPANTS IN TRANSLATION: 64 MRNA, TRNA, AND RIBOSOMES A model of the 70 ribosome, with mRNA and tRNA bound: The 30S subunit is in light blue- green (RNA) and blue (protein), and the 50S subunit is in orange (RNA) and brown (protein). Two bound tRNAs can be seen.  MECHANISM OF TRANSLATION 65 SUMMARY Translation involves three steps: o Initiation o Elongation o Termination Each aided by soluble protein factors. 66 MECHANISM OF TRANSLATION Initiation of protein biosynthesis in prokaryotes: The ribosome contains three tRNA binding sites, shown here as E, P, and A; these are called the exit, peptidyltRNA, and aminoacyl-tRNA binding sites, respectively. The initiator AUG codon is positioned so that fMet-tRNA binds in the P site.  MECHANISM OF TRANSLATION 67  MECHANISM OF TRANSLATION 68 69 MECHANISM OF TRANSLATION In elongation, the growing peptide chain at the P site is transferred to the newly arrived aminoacyl tRNA in the A site. Translocation then moves this tRNA to the P site and the previous tRNA to the E site.  MECHANISM OF TRANSLATION 70 A schematic view of ribosome subunit rotational motions, based on crystal structures of ribosomes in intermediate states: (a) View from the bottom. 30S subunit (blue) is shown in starting conformation after termination (outlined in red) to a fully rotated conformation seen during elongation (black outline). (b) Side view. During transition to the fully rotated state, tRNAs shift from binding in A/A and P/P sites (30S/50S) to occupying hybrid sites A/P and P/E. (c) Rotation in another plane can move the head domain of the 30S subunit as much as toward the E site. 71 MECHANISM OF TRANSLATION Termination of translation in prokaryotes:  INHIBITION OF TRANSLATION BY 72 ANTIBIOTICS Some antibiotics that act by interfering with protein biosynthesis:  INHIBITION OF TRANSLATION BY 73 ANTIBIOTICS  INHIBITION OF TRANSLATION BY 74 ANTIBIOTICS 75 TRANSLATION IN EUKARYOTES A number of the common inhibitors of prokaryotic translation are also effective in eukaryotic cells. They include pactamycin, tetracycline, and puromycin. There are also inhibitors that are effective only in eukaryotes. Two important ones are cycloheximide and diphtheria toxin. o Cycloheximide inhibits the translocation activity in the eukaryotic ribosome and is often used in biochemical studies when processes must be studied in the absence of protein synthesis. o Diphtheria toxin is an enzyme, coded by a bacteriophage that is lysogenic in the bacterium Corynebacterium diphtheriae. 76 TRANSLATION IN EUKARYOTES ADP-ribosylated diphthamide derivative of histidine in eEF2: Synthesis of this derivative of a modified histidine in eEF2 using NAD+ is catalyzed by diphtheria toxin. eEF2 is inactivated, and protein synthesis is therefore blocked. 77 RATES AND ENERGETICS OF TRANSLATION Polyribosomes: Schematic picture of a polyribosome Each ribosome is to be imagined as moving from left to right. 78 RATES AND ENERGETICS OF TRANSLATION The energy cost for this process is high. If we examine the individual steps in protein synthesis described earlier, we can make the following estimate of the total energy budget for synthesizing a protein of N residues: Translation is fast but energy-expensive. About four ATP equivalents are needed for each amino acid added.  THE FINAL STAGES IN PROTEIN SYNTHESIS: 79 FOLDING AND COVALENT MODIFICATION Translation is immediately followed by various kinds of protein processing, including: o Chain folding. o Covalent modification. o Directed transport.  THE FINAL STAGES IN PROTEIN SYNTHESIS: 80 FOLDING AND COVALENT MODIFICATION A current model for protein secretion by prokaryotes: The new polypeptide chain (the pro- protein) complexes with SecB, which prevents complete folding during transport to the membrane. At the membrane an ATPase, SecA, drives translocation through the membrane with the aid of SecYEG, which forms a membrane pore. The leader sequence is then cleaved off the secreted protein by a membrane peptidase. 81 PROTEIN TARGETING IN EUKARYOTES Proteins destined for the cytoplasm, nuclei, mitochondria, and chloroplasts are synthesized in the cytoplasm. Those destined for organelles have specific targeting sequences.