Lecture 9: Transcription I

Transcription Side notes: Information flow in the cell Transcription is the copying of one strand of DNA (the coding strand) into an RNA molecule (a transcript) RNA polymerase transcribes DNA into RNA RNA polymerase copies the coding strand of DNA by using the complementary strand (the template or non-coding strand) as a template RNA polymerase separates the DNA strands, and allows ribonucleoside triphosphates to base-pair with the template strand RNA polymerase is the key enzyme in transcription Steps in transcription: recognition & initiation Transcription starts when RNA polymerase binds to the DNA sequence just preceding the gene -- the promoter The transcription start site (TSS) is the first base to be transcribed RNA is transcribed in the 5' to 3' direction (so the template is read 3' to 5’) During initiation, the RNA polymerase separates the DNA strands to make a transcription bubble, and the first few ribonucleoside triphosphates are added while the RNA polymerase is at the promoter When the RNA is a su icient size, the RNA polymerase moves past the promoter and changes conformation to be more stably associated with the DNA (promoter clearance) Steps in transcription: elongation & termination During elongation, the RNA polymerase moves along the DNA, adding ribonucleotides and elongating the RNA transcript The RNA polymerase unwinds the DNA ahead of it, and the DNA re-pairs behind the enzyme, maintaining the transcription bubble along the DNA In the bubble, a short region of the transcript is paired with the DNA. The rest of the RNA is extruded from the polymerase Elongation continues until the polymerase meets a DNA sequence called a terminator that signals RNA synthesis to cease The RNA polymerase then dissociates from the DNA Types of RNA polymerase RNA polymerases are large multi-subunit enzymes Bacteria and archaea have a single RNA polymerase Eukaryotes usually have three RNA polymerases: RNA polymerase 1 (Pol I) transcribes large ribosomal RNA (rRNA) genes RNA polymerase 2 (Pol II) transcribes messenger RNA (mRNA) genes RNA polymerase 3 (Pol III) transcribes a variety of RNAs including transfer RNA (tRNA) and 5S ribosomal RNA Plants have a fourth RNA polymerase that transcribes regulatory RNAs Shared features of RNA polymerases across life All RNA polymerases have a core enzyme that catalyzes RNA synthesis, but this cannot act alone and relies on extra proteins. The same basic core structure is conserved between archaea, bacteria, and eukaryotes RNA polymerases are made up of subunits Bacterial RNA polymerase has 5 subunits -- 2 alpha, beta, beta prime, omega These subunit assemble into a complex with jaw-like lobes - the jaws are formed by the beta and beta prime subunits, with the two alpha subunits Transcription Side notes: Top hat l How many types of polymerase in: Bacteria: 1 Archaea: 1 Eukaryotes: 3+ and the omega subunit at the base. The base of the cle is the enzyme active site The alpha subunit has a N-terminal domain and C-terminal domain joined by a flexible linker RNA polymerases all have five central subunits that are similar in amino acid sequence, especially at the active site. Additional subunits in archaeal and eukaryotic RNA polymerases are arranged around the five central subunits Promoter recognition in bacteria: sigma factors RNA core polymerases can synthesize RNA, but cannot recognize promoters The core bacterial enzyme requires an extra subunit, the sigma factor, which directly contacts the promoter. Core enzyme + polymerase = holoenzyme Bacteria have di erent sigma factors that recognize di erent promoter sequences and promote transcription under di erent conditions Sigma factors bind sequences that define bacterial promoters Bacterial promoters generally have two elements: the -35 element and the 10 element. These are roughly 35 and 10 bases upstream of the transcription start site The three sigma domains are positioned to recognize specific promoter elements: Domain 2 binds to the -10 region and helps separate duplex DNA (promoter melting) Domain 3 recognizes the two bases of the extended -10 region Domain 4 recognizes the -35 element - it is a ached to a flexible part of the core enzyme which may let it accommodate di erent -35 to -10 spacing Top hat 2 How do alternate sigma factors activate different genes? 1. They bring RNA pol to different DNA sequences 2. They stop RNA pol from transcribing unless a signal is present 3. They degrade RNA pol in the presence of a signal Sigma factors can be regulated by signals or conditions Some sigma factors are regulated In response to environmental Or developmental conditions Sigmas can be regulated at the transcriptional or translational level, or by altering its mRNA or protein stability One way to regulate sigma factors is by use of pro-sigma factors. These have inhibitory domains that must be cleaved before the sigma can associate with the core enzyme Anti-sigma factors are proteins that bind to sigmas and inhibit their function Anti-sigma factors are involved in regulation of flagellum assembly in S. Typhimurium While the proteins that form the flagellum base are being synthesized, the anti-sigma FlgM binds to sigma factor to prevent it binding to the holoenzyme Sigma F promotes transcription of genes that are needed for completion of flagellum assembly In the late stages of flagellum protein synthesis, FlgM is exported from the cell, so sigma F can promote assembly gene transcription, allowing the flagellum to be put together General TFs guide RNA pol II to promoters in eukaryotes Eukaryotic and archaeal promoters also need extra proteins to target the Transcription Side notes: core enzyme to promoters: general transcription factors TFII proteins assemble at promoters with the core polymerase to form the pre-initiation complex GTFs can target specific promoter features Eukaryotic polymerases need the TATA binding protein (TBP) to initiate transcription (this is part of TFIID) Eukaryotic promoters generally have characteristic elements, though these are varied and hard to predict Promoters for pol II o en have a TATA box (TATAA consensus sequence) ~25-30 bp upstream of the transcription start Some other elements that may be present include the TFIIB recognition element (BRE), initiator element (INR), and downstream promoter element (DP; found downstream of the transcription start) Many promoters do not have any identifiable elements Top hat 3 Why is there a delay in lethality after ingesting alphaAmanitin? 1. RNA pol I and pol III can replace RNA pol II for a short time 2. It takes 2-3 days for digestion to work 3. RNA molecules created before ingestion can sustain protein production until they are degraded TFIID DNA binding is the First step in assembling the PIC The first step in assembling the pre-initiation complex (PIC) is binding of TFIID to the TATA box TFIID binds to the TATA box via TBP, which binds to the minor groove of DNA, inducing strong distortions in the DNA and causing local DNA unwinding Some other components of TFIID, called TBP associated factors (TAFs), mediate recognition of other promoter elements like INR and DPE A er TFIID and TFIIB have bound, TFIIA binds, and stabilizes TBP - DNA interactions, then TFIIE and TFIIH bind (TFIIH catalyzes ATP-powered unwinding of DNA) Assembling the pre-initiation complex The pre-initiation complex components are: RNA polymerase II core enzyme General transcription factors The pre-initiation complex is competent to initiate transcription in vitro. In vivo transcription requires additional protein complexes Another large complex, mediator (which has more than 20 subunits), is needed to activate many pol II transcribed genes RNA pol I and RNA pol III have distinct promoter targeting Core subunits are similar in eukaryotic pol I, pol II and pol III, and all three use TBP to initiate transcription, but TBP is part of di erent complexes in each polymerase Pol I, pol II, and pol III use di erent proteins to initiate transcription, recognize di erent promoters and have di erent general transcription factors RNA pol I (transcribes rRNA) binds to a promoter with a core element (recognized by TBP) and an upstream control element RNA pol III (transcribes 5S RNA and tRNA) recognizes key promoter elements that are downstream of the transcription start, as well as other upstream elements