Lecture 9: Transcription - DNA-dependent synthesis of RNA PDF

Lecture 9 Transcription: DNA-dependent synthesis of RNA Additional material for this lecture may be found in: § Lehninger’s Biochemistry (8th ed), chapter 26: p. 960-972 RNA METABOLISM - DNA-Dependent Synthesis of RNA (this lecture) - Post-transcriptional processing of RNA (Lectures 1011a) - RNA-Dep...

Lecture 9 Transcription: DNA-dependent synthesis of RNA Additional material for this lecture may be found in: § Lehninger’s Biochemistry (8th ed), chapter 26: p. 960-972 RNA METABOLISM - DNA-Dependent Synthesis of RNA (this lecture) - Post-transcriptional processing of RNA (Lectures 1011a) - RNA-Dependent Synthesis of RNA and DNA (Lecture 11b) Learning objectives: – Transcription: DNA-Dependent Synthesis of RNA Transcription in E. Coli Transcription in Eukaryotes Overview of RNA Function Ribonucleic acids play three well-understood roles in living cells: – Messenger RNAs encode the amino acid sequences of all the polypeptides found in the cell – Transfer RNAs match specific amino acids to triplet codons in mRNA during protein synthesis – Ribosomal RNAs are the constituents of ribosomes and have structural and catalytic functions Ribonucleic acids play several less-understood functions in eukaryotic cells: – MicroRNA appears to regulate the expression of genes, possibly via binding to specific nucleotide sequences – Long noncoding RNAs, or lncRNAs, recently discovered, are transcribed RNA molecules longer than 200 nucleotides that do not code for proteins and appear to contribute to the control of cell differentiation and maintenance of cell identity. Ribonucleic acids act as genomic material in certain viruses (RNA viruses) PRINCIPAL TYPES OF RNAs OVERVIEW OF RNA METABOLISM Ribonucleic acids are synthesized in cells using DNA as a template in transcription – Transcription is tightly regulated in order to control the concentration of each protein Being mainly single-stranded, many RNA molecules can fold into compact 3D structures with specific functions – Some RNA molecules can act as catalysts (ribozymes), often using metal ions as cofactors Most eukaryotic ribonucleic acids are processed after synthesis – Elimination of introns; joining of exons – Poly-adenylation of the 3’ end – Capping the 5’ end TRANSCRIPTION IN E. COLI DNA-DEPENDENT SYNTHESIS OF RNA FEATURES OF TRANSCRIPTION IN E. COLI The nucleoside triphosphates add to the the 3’ end of the growing RNA strand The growing chain is complementary to the template strand in DNA The synthesis is catalyzed by RNA polymerase RNA polymerase covers about a 35 bp-long segment of DNA (see footprinting) TRANSCRIPTION BY RNA POLYMERASE IN E. COLI § The catalytic mechanism of RNA synthesis by RNA polymerase is essentially the same as that used by DNA polymerases. § The reaction involves two Mg2+ ions, coordinated to the phosphate groups of the incoming nucleoside triphosphates (NTPs) and to three Asp residues, which are highly conserved in the RNA polymerases of all species. § One Mg2+ ion facilitates attack by the 3’-hydroxyl group on the α phosphate of the NTP; the other Mg2+ ion facilitates displacement of the pyrophosphate, and both metal ions stabilize the pentacovalent transition state. FIRST STEPS IN TRANSCRIPTION RNA polymerase binds to sequence called promoter to begin transcription – Primer is not required Growing end of new RNA temporarily base-pairs with DNA template for ~8 bp DNA duplex unwinds, forming a “bubble” of ~17 bp RNA Polymerase generates positive supercoils ahead, later relieved by topoisomerases THE TRANSCRIPTION “BUBBLE” For synthesis of an RNA strand complementary to one of two DNA strands in a double helix, the DNA is transiently unwound. About 17 bp of DNA are unwound at any given time. RNA polymerase and the transcription bubble move from left to right along the DNA (in the 5’ to 3’ direction) as shown, facilitating RNA synthesis. The DNA is unwound ahead and rewound behind as RNA is transcribed. As the DNA is rewound, the RNA-DNA hybrid is displaced and the RNA strand is extruded. MOVEMENT OF RNA POLYMERASE ALONG THE DNA AND GENERATION OF POSITIVE SUPERCOILS Changes in the supercoiling of DNA brought about by transcription: Movement of an RNA polymerase along DNA tends to create positive supercoils (overwound DNA) ahead of the transcription bubble and negative supercoils (underwound DNA) behind it. In a cell, topoisomerases rapidly eliminate the positive supercoils and regulate the level of negative supercoiling The RNA polymerase is in close contact with the DNA ahead of the transcription bubble as well as with the separated DNA strands and the RNA within and immediately behind the bubble. A channel in the protein funnels new NTPs to the polymerase active site. The polymerase footprint encompasses about 35 bp of DNA during elongation. BE CAREFUL WITH TERMINOLOGY DNA Template Strand – serves as template for RNA polymerase DNA Coding Strand – the non-template strand; has the same sequence as the RNA transcript Template and non-template (coding) DNA strands. The two complementary strands of DNA are defined by their function in transcription. The RNA transcript is synthesized on the template strand and is identical in sequence (with U in place of T) to the non-template strand, or coding strand. ***Regulatory sequences are listed by the coding strand sequence. BOTH STRANDS MAY ENCODE FOR PROTEINS Coding information may be on either strand (thus the two stands “top” and “bottom” can encode for proteins. – Adenovirus (one of the causative agents of the common cold) for example has a linear genome in which: Each strand codes for a certain number of proteins. The direction of transcription is determined by the orientation of the promoter at the beginning of each gene. Organization of coding information in the genome of adenovirus (causes upper respiratory tract infections in some vertebrates): The genetic information of the adenovirus genome is encoded by a double-stranded DNA molecule of 36,000 bp, both strands of which encode proteins. The information for most proteins is encoded by (that is, identical to) the top strand by convention, the strand oriented 5′ to 3′ from left to right. The bottom strand acts as template for these transcripts. However, a few proteins are encoded by the bottom strand, which is transcribed in the opposite direction (and uses the top strand as template). Synthesis of mRNAs in adenovirus is actually much more complex than shown here. Many of the mRNAs shown for the upper strand are initially synthesized as a single, long transcript (25,000 nucleotides), which is then extensively processed to produce the separate mRNAs. RNA POLYMERASE IS A LARGE ENZYME WITH NO PROOFREADING CAPABILITY RNA polymerase holoenzyme has five core subunits of a2bb’w (Mr 390.000) plus a sixth called s or s70 (Mr 70.000) RNA Pol lacks 3’à 5’-exonuclease, so has high error rate of 1/104–1/105 RNA Pol binds to promoter regions to initiate transcription STRUCTURE OF BACTERIAL RNA POLYMERASE The several subunits give the enzyme the shape of a crab claw. The pincers are formed from the large b and b’ subunits The s70 subunit rests on top of the crab claw and threads through the RNA exit channel Two a subunits function in assembly and binding to UP (upstream promoter) elements The b subunit is the main catalytic subunit (polymerization activity) The b’ subunit is responsible for DNA binding (not shown) The s 70 subunit directs enzyme to the promoter The w appears to protect the polymerase from denaturation PROMOTERS IN E. COLI THAT BIND THE SAME RNA POLYMERASE HAVE COMMON FEATURES A-T−rich sequences (Upstream Promoter Elements or UP elements) between −40 and −60 binds the a subunit of polymerase and promote strand separation UP elements govern efficacy of RNA Pol binding and therefore affect gene expression level Two consensus sequences at −10 (TATAAT) and −35 (TTGACA) for s subunit binding – Called TATA sequences SOME E. COLI PROMOTERS RECOGNIZED BY AN RNA POLYMERASE HOLOENZYME CONTAINING σ70 § The non-template strand of the consensus sequence for E. coli promoters recognized by s70 is shown, read in the 5’→3’ direction, as is the convention for representations of this kind. § The sequences vary from one promoter to the next, but comparisons of many promoters reveal similarities, particularly in the –10 and –35 regions. § The sequence element UP, not present in all E. coli promoters, generally occurring in the region between –40 and –60 strongly stimulate transcription at the promoters that contain them. § The consensus sequence for E. coli promoters recognized by s70 is shown second from the top. Spacer regions contain slightly variable numbers of nucleotides (N). Only the first nucleotide coding the RNA transcript (at position +1) is shown. RNA POLYMERASE LEAVES ITS FOOTPRINT ON A PROMOTER Premise: DNA bound a by protein (RNA Pol or any other protein) will be protected from chemical or enzymatic cleavage at the binding site of the protein. 1) Isolate a DNA fragment thought to contain a binding site 2) Radiolabel the DNA 3) Bind protein to DNA in one test tube; keep another as a “naked DNA” control in another test tube 4) Treat both samples with chemical or enzymatic agent to cleave the DNA 5) Separate the fragments by gel electrophoresis and visualize bands on X-ray film or imager plate PROTEIN-DNA FOOTPRINTING Theory: Footprint analysis of the RNA polymerasebinding site on a DNA fragment. Separate experiments are carried out in the presence (+) and absence (–) of the polymerase. Experiment: Footprinting results of RNA polymerase binding to the lac promoter In this experiment, the 5’ end of the nontemplate strand was radioactively labeled. Lane C is a control in which the labeled DNA fragments were cleaved with a chemical reagent that produces a more uniform banding pattern. TRANSCRIPTION INITIATION AND ELONGATION HAVE SEVERAL STEPS RNA Pol binds to promoter in presence of s – Creates a closed complex (DNA is not unwound) Open complex forms – Region from ~−10 to ~+2 unwinds RNA Pol moves away from promoter – s is replaced by protein NusA TRANSCRIPTION INITIATION AND ELONGATION IN E. COLI § Initiation of transcription requires several steps generally divided into two phases, binding and initiation. In the binding phase, the initial interaction of the RNA polymerase with the promoter leads to formation of a closed complex, in which the promoter DNA is stably bound but not unwound. A 12 to 15 bp region of DNA—from within the –10 region to position +2 or +3—is then unwound to form an open complex. Additional intermediates (not shown) have been detected in the pathways leading to the closed and open complexes, along with several changes in protein conformation. The initiation phase encompasses transcription initiation and promoter clearance (steps 1 through 4). § Once elongation commences, the σ subunit is released and it is replaced by the protein NusA. The polymerase leaves the promoter and becomes committed to elongation of the RNA (step 5). § When transcription is complete, the RNA is released, the NusA protein dissociates, and the RNA polymerase dissociates from the DNA (step 6). Another σ subunit binds to the RNA polymerase and the process begins again. TRANSCRIPTION TERMINATION Two types of termination in E. coli 1) r-independent – Characterized by a self-complementary region (palindrome) followed by 3 or more Us near the 3’ end of the transcript – Self-complementary region in transcript mRNA form a hairpin 15−20 nucleotides before the 3’ end Makes the RNA Polymerase pause, then dissociate 2) r-dependent (less understood) – common CA-rich sequence called a rut site (Rho utilization element) is present in the termination region – r protein is a helicase, binds to rut site – r protein processes until termination site reached. Makes the RNA Polymerase pause, then dissociate TERMINATION OF TRANSCRIPTION IN E. COLI Rho independent termination. RNA polymerase pauses at a variety of DNA sequences, some of which are terminators. One of two outcomes is then possible: - either the polymerase bypasses the site and continues on its way, - or the complex undergoes a conformational change (isomerization). During isomerization, intramolecular pairing of complementary sequences in the newly formed RNA transcript may form a hairpin that disrupts the RNA-DNA hybrid, the interactions between RNA and the polymerase, or both. An RNA-DNA hybrid region at the 3’ end of the new transcript is relatively unstable, and the RNA dissociates from the complex completely, leading to termination. At nonterminating pause sites, the complex may escape after the isomerization step to continue RNA synthesis. Rho Dependent termination. RNAs that include a rut element recruit the Rho helicase. The Rho helicase migrates along the mRNA in the 5’à 3’ direction and separates it from the polymerase. RNA HAIRPIN FORMATION IN r-INDEPENDENT TERMINATION OF TRANSCRIPTION A r independent termination signal found at the 3‘end of an mRNA transcript (formation of a hairpin) r-DEPENDENT TERMINATION OF TRANSCRIPTION RNAs that include a rut site (purple) recruit the ρ helicase (ρ factor). The ρ helicase migrates along the mRNA in the 5’Ž3’ direction and separates it from the polymerase. unwinding of the DNA:RNA hybrid by helicase TRANSCRIPTION IS A MAJOR TARGET FOR REGULATION Transcription is energy-intensive so it is logical to regulate gene production at this level Regulation is achieved in many ways: – One way is to regulate the affinity of RNA polymerase for a promoter using: Different promoter sequences Activator proteins Repressor proteins SUMMARY ON RNA POLYMERASE OF E. COLI 3 Stages of RNA synthesis: Initiation; Elongation; Termination RNA Polymerase: Searches DNA for initiation sites (promoters) Unwinds a short stretch of dsDNA Selects the correct NTPs (W-C base-pairing rules) and catalyzes phosphodiester bond formation Does not require a primer to initiate RNA synthesis Elongation proceeds in 5’ to 3’ direction Is completely processive Detects termination signals Its activity can be modified by activator and repressor proteins TRANSCRIPTION IN EUKARYOTES DNA-DEPENDENT SYNTHESIS OF RNA EUKARYOTES CONTAIN SEVERAL DISTINCT RNA POLYMERASES RNA polymerase I synthesizes pre-ribosomal RNA (precursor for 28S, 18S, and 5.8 rRNAs) RNA polymerase II is responsible for synthesis of mRNA – Very fast (500–1000 nucleotides/sec) – Specifically inhibited by mushroom toxin a-amanitin – Can recognize thousands of promoters RNA polymerase III makes tRNAs and some small RNA products Plants appear to have RNA polymerase IV that is responsible for the synthesis of small interfering RNAs Mitochondria have their own RNA polymerase FEATURES OF SOME PROMOTERS RECOGNIZED BY EUKARYOTIC RNA POLYMERASE II The TATA box is the major assembly point for the proteins of the preinitiation complexes of Pol II. The DNA is unwound at the initiator sequence (Inr), and the transcription start site is usually within or very near this sequence. In the Inr consensus sequence shown here, N represents any nucleotide and Y represents a pyrimidine nucleotide. Additional sequences around the TATA box and downstream (to the right as shown here) of Inr may be recognized by one or more transcription factors. The sequence elements of Pol II promoters summarized here are much more variable and complex in comparison to E. coli promoters. Many of the sequences are located within a few hundred base pairs of the TATA box on the 5’ side; others may be thousands of base pairs away. EUKARYOTIC mRNA TRANSCRIPTION INVOLVES MANY PROTEINS Eukaryotic mRNA transcription relies on protein-protein contacts – Many highly conserved transcription factors RNA Polymerase II: – Large complex of 12 subunits Some subunits have some structural homology to bacterial RNA polymerase – Has a carboxy-terminal domain with highly conserved repeats INITIATION, ELONGATION AND TERMINATION OF EUKARYOTIC TRANSCRIPTION Initiation: Assembly of RNA Polymerase at promoter - Initiated by TATA box-Binding Protein (TBP) with the promoter - TBP is part of multi-subunit complex TFIID (other proteins include TFIIB, TFIIA, TFIIF, TFIIE and TFIIH) - Helicase activity in TFIIH unwinds DNA at the promoter - Kinase activity in TFIIH phosphorylates the polymerase at the CTD (carboxy-terminal domain) changing the conformation and enabling RNA Pol II to transcribe Elongation: Elongation factors bound to RNA Polymerase II enhance processivity and coordinate post-translational modifications Termination: RNA Polymerase II is dephosphorylated Regulation of transcription is complex (wide variety of proteins interacting with different proteins of the transcription machinery) TRANSCRIPTION AT RNA POLYMERASE II PROMOTERS TBP (often with TFIIA and sometimes with TFIID) and TFIIB bind sequentially to a promoter. TFIIF plus Pol II are then recruited to that complex. The further addition of TFIIE and TFIIH results in a closed complex. Within the complex, the DNA is unwound at the Inr region by the helicase activity of TFIIH and perhaps of TFIIE, creating an open complex that completes assembly. The carboxyl-terminal domain of the largest Pol II subunit is phosphorylated by TFIIH, and the polymerase then escapes the promoter and initiates transcription. Elongation is accompanied by the release of many transcription factors and is also enhanced by elongation factors After termination, Pol II is released, dephosphorylated, and recycled. FUNCTION OF TFIIH IN REPAIR Repair is linked to transcription in eukaryotes. Transcribed genes are more actively repaired than silent genes TFIIH also has a role in nucleotide-excision repair (NER) – Recruits the NER complex at a lesion Genetic repair diseases are associated with TFIIH defects – Xeroderma pigmentosum, etc. RNA POLYMERASES CAN BE SELECTIVELY INHIBITED Actinomycin D and Acridine – Intercalate in DNA and prevents transcription (works both in prokaryotes and eukaryotes) Rifampicin – Binds to b-subunit of bacterial RNA Pols (works only in prokaryotes) a-Amanitin from mushroom Amanita phalloides (works only in eukaryotes) – Blocks Pol II and Pol III of predators of the mushroom – But does not block Pol II of the mushroom itself ACTINOMYCIN D AND ACRIDINE, INHIBIT DNA TRANSCRIPTION BY INTERCALATING IN DNA, THEN BENDING IT (a) The shaded portion of actinomycin D is planar and intercalates between two successive G≡C base pairs in duplex DNA. The two cyclic peptide moieties of actinomycin D bind to the minor groove of the double helix. Sarcosine (Sar) is N-methylglycine; meVal is methylvaline. Acridine also acts by intercalation in DNA. (a) A complex of actinomycin D with DNA. The DNA backbone is shown in blue, the bases are grey, the intercalated part of actinomycin (shaded in (a)) is orange, and the remainder of the actinomycin is red. The DNA is bent as a result of the actinomycin binding.