:PROPERTIES: :ID: 4a34f08a-c003-4d88-968c-10e298ef2793 :END: #+title: Genetics: DNA Transcription #+filetags: :genetics:lecture_notes: #+STARTUP: latexpreview RNA is the vessel upon which transcription takes place. An intermediary molecule must be needed since DNA never leaves the nucleus but protein synthesis occurs in the cytoplasm. RNA is the molecule that brings the message from DNA to the ribosomes, which contruct the protens. Thus, RNA is a /messenger./ Two scientists, Volkin and Astrachan, used radioactive Phosphorus to: * Show a burst of RNA synthesis following bacteriophage infection. * Labeled RNA degraded quickly. Spiegelman saw that RNA could be made from a viral genome. This brings us to the *Central Dogma of Biology:* DNA |\ v/ transcription RNA |\ v/ translation Protein There are also many types of RNA, each with specific functions that will be touched on in other notes. * mRNA: Codes for proteins * rRNA: structural component for ribosomes and other complexes * tRNA: Responsible for facilitating the contruction of polypeptide chains * snRNA: Responsible for assisting in pre-mRNA splicing * miRNA: used to block the expression of mRNA * Transcription Overview - RNA is made first as *pre-mRNA* - Then, it is spliced/cut/etc. - Precursors for RNA synthesis are called *ribonucleoside triphosphates* or (RNTPs) - Only *one* strand of DNA serves as the template for the new RNA * RNA is complimentary to template only * This means RNA is *identical* to non-template strand (but with uracil instead of thymine) - RNA chains can be synthesized /de novo/ *they don't need a primer.* - Synthesis is in the 5' --> 3' direction. (For more info see[[id:3a5368b9-d120-40d0-907f-9b59cc53d653][Genetics: DNA Replication]]) - The current base in question on a strand is labeled as +1. bases ahead of it (downstream) are +2, +3, etc. Bases behind (upstream) are -1, -2, -3, etc. Transcription comes in these steps: 1. Initiation 2. Elongation 3. Termination 4. Splicing 5. RNA Editing * Initiation (From Perspective of Bacteria) - Binding of *RNA Polymerase* * Holoenzyme contains 5 subunits * $\alpha$ subunits are involved in assembly * $\beta$ subunits contain rNTP binding site * $\beta$' subunits contain template binding region * $\sigma$ subunits recognize *promoter sites* (specific sequences that function as the anchor site for RNAP) - Actual chain synthesis is not recognized to happen at least for another 5-9 base pairs after the promoter sequence. After 10 base pairs, much more stability is acheived. the length of the RNAP enzyme may also contribute to this. - $\sigma$ subunits prevents random transcriptional initiation. * -10 and -35 promoter sequence * -35: TTGACA (non-template) * -10: TATAAT (non-template) * After these promoter sequences, there is a /range/ over which RNAP begins to polymerize (Transcription Start Site). * /The actual RNA is usually begun with a purine, that is, an A or a G./ * So when there a lost of Cs and Ts...you can be confident that the polymerizing began at a conspicuous A or G. In reality it may be harder to visualize the start of an RNA molecule. - There are four types of sigma factors, which are activated based on the nature of the external environment: * $\sigma^{70}$: normal factor * $\sigma^{32}$: heat shock response * $\sigma^{54}$: nitrogen metabolism * $\sigma^{23}$: viral infection - Sigma is released after initiation. * Elongation - Unwinding and rewinding - 2,400 nucleotides/min in /E.coli/ - RNA:DNA hybrid is very short. - The single-stranded RNA is mostly "grown" off the DNA template strand, threaded through the RNAP. * Termination There is Rho-dependent and Rho-independent termination processes. *Rho-independent:* * Termination region in DNA * G:C - rich region followed by a stretch of of T's * For example: CGGCCCATTTTTTT (non-template) * A hairpin can form from the strong, tighly-knit interactions between C's and G's. The T's form a weaker bond with A's. * So essentially it is a strong hairpin region followed by a weak region. * RNA folds sharply in on itself due to C:G interactions. And what's left (all those U's) is weakly bound to the DNA. Thus, it can detach and terminate easily. *Rho-dewpendent*: * 50-90 bp long stretch of C's * *Rho* binds to the RNA and moves 5' --> 3' * when Rho "catches" RNAP, it literally pulls the chain free * Rho appears to chase the RNAP * However, in prokaryotes, translation happens concurrently with transcription. Eventually the ribosomes encounter their stop sequences and disengage. * This allows Rho to bind to it and engage the RNAP. * Transcription in Eukaryotes - There are multiple Polymerases * 10 or more subunits each * RNA polymerase I, II, and III * RNAP I: Nucleolus, rRNA, not 5S rRNA * RNAP II: Nucleus, nuclear pre-mRNA (does much of the process as the prokaryote RNAP discussesd above) * RNAP III: Nucleus, tRNA, snRNA, 5S rRNA - However, these RNAP's /cannot initiate by themselves./ * Uses transcription factors, TFIID, TFIIA, TFIIB, (Transcription Factor for polymerase II A, etc.) * *TFIID* binds first. It contains *TBP* which searches for the eukaryotic promoter region called the "Tata-box" (found at -30) * *TFIIA and TFIIB* then follow, helping TFIID bind more securely * *TFIIE and TFIIH* then bind. TFIIH can use ATP. * *TFIIB* defines the directionality of the transcription. If you think about this, this also mean it determines which strand is the template and which is the non-template. Sometimes, divergent transcription can happen. The purpose is not known, but it may actually serve to help the process. TFIIB also acts as a molecular "ruler" for RNAP II. - RNAP I promoters are typically bipartite. Both are G:C-rich. These are not "Tata-boxes" - RNAP III promoters are typically /downstream/ of the TSS. This means it binds to the DNA before its promoter sequence, meaning that sequence is actually included on the fabricated pre-mRNA strand. * mRNA Processing - 7-methyl guanosine cap * Initiation of translation * Protects growing mRNA strand - Poly(A) Tail * Protects mRNA from degradation] * Aids in transportation * Added /after/ transcription * Splicing - Removal of introns from pre-mRNA - The recognizable features of introns are given below 5' GT........UACUAAC.........AG 3' GT and AG can be thought of as "bookends" of the introns. - These introns don't have caps or tails, so they are digested. *Type 1 Splicing* * Shape driven * Requires endonuclease and ligase * Enzymes recognize shape and clip intron by location, not by base-pair. *Type 2 Splicing* * No protein activity * Requires guanosine with 3' OH end * Two phosphodiester bond transfers *Type 3 Splicing (Spliceosome)* * Involves spliceosomes, which are RNA/protein structures * U1, U2, U4, U5, U6. * *U1* binds to 5' splice site (GT) * *U5* binds to other end (AG) * *U2* recognizes the UACUAAC sequence on the intron * *U4* and *U6* do not make contact with the RNA but provide structure.