BMSC 320: Nucleic Acids, From Central Dogma to Human Disease Lecture Notes PDF
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Dr. Anderson
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This document is lecture notes from BMSC 320 focusing on nucleic acids, from central dogma to human disease. It covers prokaryotic and eukaryotic gene structures, mechanisms of transcription, and the roles of specialized polymerases (RNA Pol I, II, and III).
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MSC 320: Nucleic Acids, From Central B Dogma to Human Disease WF 9:30 - 10:20 M Lecturer: Dr. Anderson Nucleic Acids Drawing Genes: Prokaryotic gene: - T he signal of termination of transcription is not from a Stop codon, there were structures that t...
MSC 320: Nucleic Acids, From Central B Dogma to Human Disease WF 9:30 - 10:20 M Lecturer: Dr. Anderson Nucleic Acids Drawing Genes: Prokaryotic gene: - T he signal of termination of transcription is not from a Stop codon, there were structures that terminated that transcription found on the transcript itself (ex. rho dependent, terminator structure) - There is the presence of3’ untranslated regionsafterthe termination sequence. - There can be multiple open reading frames (ORF), and the closest ORF to the promoter is typically replicated the most. Eukaryotic gene: - The mRNA has introns and exons, whereintrons, beingnon-econding, areremoved. - There are regulation sequences 800 to 1000 bp downstream the promoter before the transcription sequence. - Regulation can also be upstream to the open reading frame. - There can also be overlap between reading frames. - There can also be multiple promoters in a gene that can make different mRNAs. Eukaryotic Genes: 3 Specialized Polymerases P ol I- products areribosomal RNAs. Cells need them consistently and at high levels. They have their own specific promoters. P ol II- producesmRNAthat encodes for proteins. Also produces microRNAsand some non-coding RNAs. Pol III- small high quantitynon coding RNAsthatare needed in a cell(ex. tRNAs, 5S rRNA, 7SL RNA). ○ There are 64 codons (61 codons for AA + 3 stop codons) ○ An organism doesn't really produce all 61 tRNAs due to the wobbling effect of the 3rd letter of the codons that allow for redundancies in AA. RNA Pol I Promoter - roducesribosomal RNA (rRNA)which are the most abundantRNA in a cell. P - Itsconsensus sequence consistsof: ○ Upstream control element (UCE)- found at around -150to -100 ○ Core Promoter Element (CPE)- found around the startsite (+1). - Within a species, there isvery little variationofrRNA promoters but there are a lot of copies of their genes, especially in eukaryotic chromosomes. - Their genes are organized intandem arrays. (50 copiesin a row in one tandem array) - These arrays have the same promoter that are “ON” all the time. Initiation of transcription: - Initiation occurs whenUBF (upstream binding factor)binds to the UCE and Core sequence (consensus sequence). - SL1(s electivity factor 1- which contains TBP - TATA binding proteins) associates with UBF making the complex ready for the Pol I to bind. TATA binding proteins can be bound to all 3 Polymerases. - There is rapid recycling from one array to another as Pol I reaches the end of one array. 80%-90% of RNA in our cells are rRNAs, that’s why we need to make lots of them. In some genes, there are just about 100 bps between 2 arrays, which easily allows for rapid recycling of Pol I, but humans have around 1000 bps, this is solved by tightly compacting these regions (heterochromatin) so rapid recycling can occur. - The SL1-UBF complex remains at the promoter to recruit another RNA pol I. RNA Pol III promoter - orediverseandcomplexthan RNA pol I promoters.Its production can vary as there M may be RNAs that are present or absent depending on gene regulation. - RNA pol III specializes insmall and high-quantitynon-coding RNAs: tRNA, 5S rRNA, 7SL RNA, U6 snRNP (spliceosome RNA) - niquely, the promoters aredownstreamfrom the start site, resulting in an RNA that U hasa sequence of its promoter, which is importantbecause if the RNA somehow gets reverse transcribed to DNA and inserted back into the genome, it will still be able to reform a functional gene. - Unlike if an mRNA is reverse transcribed into a DNA, it will be useless and will not be expressed as it does not have a promoter. - Alu elementsare RNA pol III transcript so when theyare transcribed back into the genome, it has its own promoter. So they can be replicated and expressed. - Promoters vary by which conserved elements (boxes) they contain. ○ Different conserved elements will affect recruitment of RNA pol III and levels of those RNA molecules which means that there is some control of gene expression in this level. - There are also factors that are needed to recruit the polymerase: ○ TFIII - C (transcription factor 3 - C) recruits TFIII-B ○ TFIII - B recruits Pol III The TFIII -B remains bound on the DNA so it can quickly reinitiate transcription. TFIII-C is removed when transcription occurs. RNA Pol II promoters - as themost diverse and complexcontrol mechanismof the 3 RNA polymerases. H - This complexity is due to the variability in expression of different genes. ○ Some need to be up and downregulated across the 10,000-fold range in response to stimuli. ○ Some genes are expressed at high levels while others are kept at a constant baseline level. ○ Some are only expressed for a fraction of a percent of the organism’s lifetime and then never again. - Eukaryotes have ~10-100x more protein coding genes than bacteria. - Humans have ~20,000 genes. Structure: - RNA pol II core enzyme has 12 subunits with 5 some being homologous to bacterial subunits. - One of the structures that allow for control of the polymerase activation is its tail that can be phosphorylated or dephosphorylated. ○ CTD (carboxy-terminal domain)- the tail of the RNA pol II, results from many repeats of the 7-AA domain at the C-terminus that regulates the polymerase’s enzymatic activity. ○ Serine residues are where we can phosphorylate ○ 5 2 copies of CTD in vertebrates - allows to regulate whether the pol is at the start, the middle or the end of the transcription - Initiation of transcription at any genes is an issue of probability. - The probability depends on thestrength of the corepromoterin: recruiting the general transcription factors Recruiting additional or unique assisting TFs DNA sequence (histones/chromatins) accessibility. Core Pol II promoters: RE = TFII - B Recognition Element B TATA = TATA box INR = Initiator (found around +1) DPE = Downstream Promoter Element - ot all promoters have all of these boxes, but having all of them will yield the highest N degree of transcription Eukaryotic Transcription Cycle Overall process: 1. Recruitment/Assembly a. PIC formation b. Transcription bubble formation 2. Initiation a. Elongation complex b. Phosphorylation of CTD during initiation 3. Elongation a. Termination Complex 4. Termination a. CTD dephosphorylation 5. Recycling General transcription factors: - Are universal proteins required to recruit and assemble a working RNA pol II. - They assemble in order of D-A-B-F-E-H 1. TFIID- main “promoter finding” complex; similar to sigma factor in prokaryotes. 2. TFIIA- stabilizes D and B; helps binding of D and B together . 3 FIIB- helps to orient the laid pol II to directionally. T 4. TFIIF- stabilizes B and pol II; brings in pol II 5. TFIIE- stabilizes and regulate H 6. TFIIH- helicase and kinase to regulate pol II; phosphorylates the CTD of pol II to activate it. Formation of Preinitiation Complex (PIC) - TFIID binds to a DNA (on the euchromatin), it contains TBP that binds to TATA box. ○ Not all promoters have a TATA box, only ~¼ of promoters have one. - TBP binding in the minor groove causes the DNA to partially unwind and bend 80 degrees. ○ Major groove - has more information sequence ○ Minor groove - has a palindromic TATA sequence that confuses proteins that bind to it of the directionality of the gene. The different grooves arise due to the angle created by the bond between the sugar and the bases. The major groove is wider because the angle is wider, while the minor groove has a narrower bond. The difference in information between the grooves is the result of the exposed groups within that groove and the information that they convey regarding what bases are present. Major vs Minor Groove: ○ In a major groove, the order of the exposed groups between an A=T pair and a T=A pair and a G=C pair to a C=G pair are different, so proteins are able to distinguish one from the other and interact accordingly. ○ On the other hand, proteins are unable to distinguish between A=T and T=A or G=C and C=G due to their similar exposed groove. ○ Proteins with sequence specificity will bind on the major groove. Importance of TFIIB in directionality: ○ The TATA binding protein (TBP) works with a robust number of promoters, so it will bind on the minor groove which has a limited directionality, this is whyTFIIB is needed. ○ TFIID (contains TBP) binds to the promoter and the TFIIB binds to the BRE (upstream) and stabilizes and orients the TFIID to the right direction. ○ Once the TFIIB is bound, the RNA pol II can be recruited to the promoter with other general TFs. Specialized Transcription Factors in recruiting the RNA Poll II: The Role of Mediator ○ Various proteins (transcription factors) and DNA sequences (enhancers) turn ON genes when needed. Enhancer sequence can affect a gene that is a million bps away as long as it is cis (on the same strand as the enhancer) T he enhancers bind withactivating proteinsand they cannot interact directly with TFIID or TFIIB. ○ TheMediator Complexintegrates the ‘signals’ fromthe enhancer and activating proteins to bring the RNA pol II to the gene when it is needed. Protein + DNA binding can increase or decrease transcriptional efficiency ○ There are also sequences calledsilencersthat bindtorepressor proteinsthat block the mediator complex and ultimately block transcription. Role of Insulators ○ There are DNA sequences that bind to insulating proteins (CTCF) that form loops by bridging with another insulating protein bound to another insulator sequence. ○ This allows for enhancer regulation because they essentially limit the enhancer’s effects to only the genes within the loop.
