Main Ideas - DNA and RNA are similar, but have clearly distinct structural and functional features!- Single stranded RNA molecules can form complex secondary structures by intramolecular base pairing!- RNA is synthesized by RNA polymerases in transcriptional. Transcriptional activity of genes varies greatly.!- Transcription is different from DNA replication: faster, higher error rate, unidirectional, multiple copies per gene.!- Transcription: Initiation-->Elongation-->Termination Lecture Objectives 1) Understand multiple levels of control in gene expression 2) Explain the basic concepts of RNA structure and function 3) Describe the main steps and factors of DNA transcription 4) Compare prokaryotic and eukaryotic transcriptional initiation (including the involved complexes 5) Understand the basic concepts of how transcription by eukaryotic RNA polymerase II is regulated 6) Relevance for medical practice and disease!!!!!1) Understand multiple levels of control in gene expression 2) Explain the basic concepts of RNA structure and function - RNA= ribonucleic acid, DNA= deoxyribonucleic acid!- RNA single stranded, DNA double stranded!- RNA contains uracil instead of thymine, pairs with adenine!-->has this because deaminationn of C-->U, uracil glycosylase eliminates U from DNA-->cells can scan for deamination of DNA and know it's wrong. Call tell diff between RNA and DNA.!- RNA can fold into secondary structures! 3) Describe the main steps and factors of DNA transcription - Synthesizing a complementary RNA strand!- One strand of the DNA is used as the template, the growing RNA strand is complementary to the template strand!- sense strand: same seq. as the RNA transcript- anti-sense strand= template strand - DNA is transcribed by RNA polymerases!- RNA poly catalyze formation of phosphodiester bonds!- RNA strand always synthesized in the 5'->3' direction!- Many RNA transcripts can be made from 1 gene (unlike DNA replication)!- Transcriptional activity of a gene depends on a lot of things: cell/tissue, developmental stage, phase of cell cycle, health of the cell!- Genes can be transcribed many times in a row-->put a bunch of RNA polys on a DNA strand-->work in tandem to maximize transcription!- there are many diff. types of RNA, mRNA is only 3-5% of total RNA!!Transcription Process A. Initiation- gene promoter on the DNA is recognized by transcription machinery and the RNA poly binds and opens up the DNA double helix B. Elongation- RNA poly moves along DNA and syn. a new RNA transcript C. Termination- a signal on the DNA mediates termination of transcription 4) Compare prokaryotic and eukaryotic transcriptional initiation (including the involved complexes - Bacteria have 1 RNA poly, 1 helper factor= sigma. Sigma recognizes the promoter->RNA poly will start synthesizing and moving--> kicks of the sigma factor-->stop signal is med. by the RNA as a portion of the new RNA folds back on itself (hairpin)-->RNA poly falls off!- E. coli has 2 promoter regions (-35 sequence and -10 sequence ) that are upstreeam of the start site. It has a clear orientation that tells the RNA poly where to start and what direction to go. Bacteria don't splice things out.!- transcription orientation is determined by the promoter sequence (whether you go left or right essentially)!- Eukaryotes= much more complicated!!- Have 3 RNA polymerases: RNA pol I (most rRNA), II (all protein-coding genes), and III (tRNA, snRNA,)!- Need general transcription factors to initiate transcription (proks only need sigma)!- Need to access the DNA in the chromatin!- Each RNA poly recognizes a diff. promoter type--> thus they only transcribe the genes that they're supposed to be responsible for!- promoter= core promoter + upstream control elements (enhancers, silencers)!- RNA poly II needs several transcription factors that help to position it at the promoter, unwind the DNA helix, and release RNA pol II into elongation mode = promoter clearance - euks have many general transcription factors that all need to get together to help the RNA poly find it, sit down, and go into elongation mode!- DNA bends by the binding of the TATA binding protein-->bent DNA landmark to recruit other general transcription factors!- To synthesize RNA 1) Open up the DNA 2) Use ATP to get RNA poly into motion!- RNA pol II requires additional factors!- Core= RNA pol II + GTFs!- Mediator helps other transcription factors (which can be very far apart) talk to the RNA pol 2-->affecting the effeciency of transcription!- Need histone modifiers and chromatin remodeling complexes to open the DNA!- Transcription elongation is coupled to RNA processing!- add CAP to 5' end!- Remove introns by splicing!- add polyA tail to 3' end!- these affect termination 5) Understand the basic concepts of how transcription by eukaryotic RNA polymerase II is regulated - See above and below6) Relevance for medical practice and disease - HDAC inhibitors= make DNA accesible for transcriptional repressors, inhibit transcription factors. Even though normally acetlylation loosens the histone and DNA interactions making txn more favorable, but essentially the loosened interactions allow access to anything (incl. repressors) - DNA intercalators= block transcription elongation in proks and euks, chemotherapy! - New cancer treatment strategy= inhibit specific txn factors to target cancer -Rifampicin= binds only bacterial RNA polymerase, prevents bacterial growth. Used to treat bacterial infection incl. TB. Resistance= mutation in an RNA poly subunit.!- transcription inhibitors harmful= alpha-amanitin--> specifically inhibits RNA pol II. Common cause of fatal mushroom poisonin (deathcap).
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