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Molecular Genetics
Exam 3
| Term | Definition |
|---|---|
| Transcription | production of an RNA polymer complementary to a DNA strand |
| RNA synthesized 5'- --> 3' | 3' OH attacks phosphate group and is catalyzed by magnesium ion |
| template strand | noncoding DNA stand that serves as a template for transciption |
| Magnesium in nucleotide polymerization | mg ions are bound to polymerase and with mg the oxygen can attack the phosphorus to polymerize |
| Stages of Transcription | Initiation, elongation, termination |
| transcription initiation | Initiates at promoter elements |
| transcription elongation | Creates a bubble that is about 12 bases that opened DNA slides along |
| transcription termination | different for prokaryotes and eukaryotes |
| prokaryotic RNA polymerase, RNAP core | 5 unit complex: alpha subunit, beta subunit, beta prime, omega subunit |
| alpha subunit | (2x), enzyme assembly and promoter recognition |
| beta subunit | catalytic center for RNA synthesis, where magnesium ions bind |
| beta prime | binds to the DNA template and ensures stability of the RNA-DNA hybrid |
| Omega subunit | Assists in enzyme assembly and stability, holds everything together |
| sigma factors | inititiation binds to RNA polymerase and then helps it find a promoter squence |
| sigma 70 | most common sigma factor that is used in typical growth conditions |
| GreA/B factors | elongation- helps RNAP overcome stalling - binds to secondary catalytic site which helps RNA polymerase be an endonuclease to overcome the stall |
| nus factors | elongation and termination- stabilizes elongation in gene body and/or promote proper termination at correct termination sequence |
| Nus E | associates with a ribosome, no nucleus and transcription and translation occur simultaneously |
| Nus G | associates with the polymerase, keeps ribosomes and polymerase close together during the paired up process |
| Nus A | stabilizes RNA-RNA polymerase combo |
| Nus B | helps load Nus R |
| Rho factor | termination- dissociates the RNA at the termination sequence |
| rut | bacterial transcripts with a sequence at the 3' end that is made into the RNA that utilizes Rho sequence |
| Rho steps | Rho binds to rut, charges down RNA molecule, slams into RNA polymerase, takes RNA with it when it leaves |
| transcriptional regulator elements | RNAP sits down on a promoter, +1, -10, -35 |
| +1 transcriptional regulator | fist transcribed base, usually a A or G |
| -10 transcriptional regulator (TATA box) | sequence recognized by sigma, this is upstream from transcription start site |
| -35 transcriptional regulator | sequence recognized by sigma |
| Nut site | N utilization sequence in the 5'UTR, this contains Box A sequence, while Nus B/E also bind there It helps stabilize RNAP + RNA complex |
| Rut site | Rho utilization sequence, can be in the 3' UTR or in the coding sequence, recruits Rho before it traverses RNA toward RNAP |
| RNAP stall at terminator hairpin | 1 Rho dependent termination- Rho catches up to it and dissociates the RNA 2 Rho independent termination- hairpins form in RNA causing Nus A to bind which dissociates the RNA |
| super coiling | negative supercoiling helps dissociate the double helix which positively affects transcription by priming it for melting by sigma |
| transcription bubble can cause supercoiling | positive- in front and is relaxed by topoisomerase 2 (gyrase_ negative- coils behind and is relaxed by topoisomerase 1 |
| Eukaryotic RNAPs | 3 RNAP enzyme complexes that exhibit division of labor mostly by gene type |
| transcription factors | modulate transcription while not actually preforming it themselves, they bind DNA and/or RNAP and/or other transcription factors, either help or hinder |
| general transcription factors | they are distinct loader complexes that recognize promoter sequences they first assemble at promoters and then recruit polymerase to join |
| RNA polymerase 1 | rRNA polymerase, synthasize most rRNAs, works in the nucleolus, and is the busiest |
| RNA polymerase 2 | synthesizes mRNA, snRNA, miRNA, piRNA, and IncRNA in the nucleoplasm |
| RNA polymerase 3 | synthesizes tRNA, snRNA, snoRNA, and some specific rRNAs in the nucleoplasm |
| Eukaryotic RNAP entourage overview | each has a unique set of GTF that help it recognize and bind promoters |
| TTA-binding protein (TBP) | common to all three GFT sets, causes a significant kink in the DNA at the TATA box, required to break the hydrogen bonds that hold the two strands together within the promoter |
| once RNAP is recruited the pre-initiation complex (PIC) forms | most GTFs get left at the promoter or dissociate from DNA once RNAP starts transcription (promoter clearance) |
| spt5 | GTF that travels with RNAP to help stabilize elongation and help terminate the nusG homolog |
| RNA polymerase 2- initiation | - TFIID- largest RNAPII GTF complex that binds promoters (TBP and TAFs) - RNAPII recruited and forms PIC they also interact with other TF-bound regulatory elements via the mediator complex |
| enhancers | can be upstream or downstream of the promoter they are affecting, inside the intron of the gene they regulate, or on a neighboring gene |
| enhancer-driven gene regulation | Involved with RNAPII, 1) enhancer bound by transcription factors 2) gets looped up the the promoter of a gene they are regulating 3) interact with the mediator of the complex 4) mediatory interactions with GTF and RNAP |
| regulatory element is an enhancer bound by factors that positively regulate transcription | transcription go (positively) |
| regulatory element is a repressor bound by factors that inhibit transcription | mediator complex will stop polymerase from assembling or initiating transcription |
| cohesion and CTCF | participate in looping process which helps bring regulatory DNA to promoters |
| GTF | essential for basal transcription of all genes, primarily located near the promoter region, primarily regulated by coregulators and chromatin modifications |
| other TF | regulate the expression of specific genes, can bind to various sites including enhancers and repressors, can be regulated by various factors including signaling pathways and environmental cues |
| TFIIH | helps unwind the DNA as it phosphorylates a serine on the C terminal domain (CTD) of Rbp1 in RNAPII |
| RNAPII- elongation | further phosphorylated in the CTD as the promoter is cleared. dephosphorylation as elongation nears completion, TFIIS, guanylyltransferase binds to the RNAPII Rbp1 CTD, intron removal via the spliceosome is coupled |
| TFIIS | catalyzes RNAP11 RNA clevage during a stall |
| Guanylytransferase | binds to the RNAPII Rbp1 CTD which adds the guanine that will become the 7-MeG cap during the early stages of elongation |
| RNA Polymerase II- Termination | RNAPII terminates transcription as a result of a poly A signal sequence (AAUAAA) in the RNA transcript which is recognized by the cleavage and polyadenylation specificity factor (CPSF), clevage, poly A, Xrn2 exonuclease |
| cleavage and polyadenylation specificity factor (CPSF) | 1)10-30 nucleotides downstream of the poly A signal by cleavage stimulation factor (CstF) 2) poly A polymerase (PAP) adds poly A tail to the free 3' end of the cleaved RNA transcript 3) RNAPII released from DNA template by Crn2 exonuclease |
| Pabn1 | helps regulate tail length and marks it for nuclear transport |
| Xrn2 | exonuclease that grabs onto the free 5' end of the RNA that RNAPII is still making and chews it up and dissociates the polymerase from the template |
| RNA polymerase I initiation | SL1 complex and upstream binding factor (UBF) bind to promoter, restricted to nucleolus where the chromosomal regions with the most rRNA genes stay |
| SL1 complex and upstream binding factor | help recruit RNAPI to promoter to make preinitiation complex |
| RNA polymerase I elongation | A12.2 cleavage of RNA in backtracked RNAP |
| RNA polymerase I termination | TTF-1 binds a terminator sequence (Sal Box) on the DNA which stalls RNAPI and RNAPI eventually falls off |
| RNA polymerase III initiation | TFIIIA, B, and C recruit RNAPIII |
| Variable promoters | many are contained completely in the transcribed region some use TATA box and others use sites inside of the gene |
| RNA polymerase III elongation | faster and pauses less than the others but it does proofread less than the others making it more error prone, mainly makes tRNAs |
| RNA polymerase III termination | intrinsic like meaning no blocking proteins and no protein that chew up RNA or rams off polymerase, instead it has a poly u sequence |
| poly u sequence | terminator in RNA, that causes weak association with template As and RNAPII disengages |
| Major RNA processing events | 5' caps, splicing, poly A, ribozymes, RNA editing, |
| 5' caps | add stability to RNA, 7- methylGuanosine, noncanonical caps (NCCs), |
| 7-methylGuanosine | eukaryotes only, 5' to 5' linkage with a shared triphosphate, methylated guanine nucleotide, guanylyltransferase enzyme rides along with the polymerase and sticks the cap on |
| non-canonical caps (NCCs) | all groups of life demonstrate some amount of 5' cap including NAD and FAD, may be found in prokaryotes and eukaryotes |
| nicotinamide adenine dinucleotide (NAD) | contributes to stability of transcript it is attached to |
| splicing | occurs during transcription, RNA is cut then put back together at the same time |
| Vast majority of RNA splicing is | intramolecular, two steps, and removal of a segment |
| basic splice anatomy | donor (5' site) is just after first exon that is getting joined, acceptor (3' site) just before the next exon getting joined, and branch point which is in the middle and usually adenosine |
| typical mechanism for intron excision | 2 step lariat formation two OH attacks that leave us with RNA thats bound to itself in a loop- 2'OH attacks 5' phosphate and 5'OH attacks the 3' phosphate |
| three main types of introns | spliceosomal/nuclear, group 1, and group 2 |
| spliceosomal/nuclear introns | eukaryotes only as they require the enzyme complex called spliceosome to be removed from RNA, no secondary structure, 3 types of intron splice sites (hyper conservered) |
| intron splice sites/recognition sequences | CU-AG, GC-AG, AU-AC |
| group 1 | autocatalytic- introns cut themselves out as a result of their sequence, prokaryotes and organelles, secondary structure twists RNA up to preform the two transesterification reactions |
| group 2 | autocatalytic, prokaryotes and organelles, spliceosomal intron mechanism resembles |
Created by:
kverkon