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MCB 110
| Term | Definition |
|---|---|
| RNAP Composition | 5 polypeptide subunits make up core enzyme (holoenzyme) – with a separate subunit for promoter recognition |
| RNAP α subunit | 2x present – binds regulatory proteins/sequences |
| RNAP β subunit | Involved in catalysis – forms phosphodiester bonds |
| RNAP β' subunit | Bind the DNA template |
| RNAP ω subunit | Assembles the polymerase from subunits |
| RNAP σ subunit | Recognizes the promoter and initiates transcription – separates from core once a few bonds are formed. |
| σ70 and σ32 subunits | σ70 is used for most of E. coli's genes – σ32 promotes heat shock-induced genes |
| Promoters | DNA sequences -10 to -35 upstream from gene of interest –– These are where machinery binds and initiates transcription |
| Consensus Sequences | Prokaryotes mainly use two – Involve the most frequent nucleotides at certain locations. Determined statistically, not experiementally |
| Stages of Prokaryotic Transcription | Main steps are initiation, elongation, and termination |
| Prokaryotic Transcription Initiation (process) | RNAP slides on DNA scanning for the promoter, where σ factor binds and closes. DNA is then unwound in the -10 to -35 range – transcription begins, and σ dissociates after a few rNTPs are added |
| Prokaryotic Transcription Elongation (process) | Continues to add NTPs, releasing PPi from each non-initial nt (drives equilibrium forward.) Requires topoisomerases to undo DNA supercoiling |
| Rifampicin | Inhibits transcription elongation by binding to DNA strand and blocking movement of RNAP |
| Prokaryotic Transcription Termination (basic) | RNA strand dissociates, followed by RNAP – can be Rho-dependent or Rho-independent |
| Rho-Dependent Transcription Termination | Rho protein (ATPase) binds to Rho-loading site (Cys-rich) and translocates along RNA 5'-3', unwinding the RNA-DNA hybrid and causing RNA/RNAP to dissociate. |
| Rho-Independent Transcription Termination | RNAP is stalled at a CG-rich hairpin. If an AT-rich region is present after the hairpin, it is a "real terminator" and U-A interactions will cause RNA to dissociate, followed by RNAP. |
| Bicyclomicin | Antibiotic – Blocks catalytic water molecule needed for hydrolysis in termination, therefore blocks Rho activity |
| Transcription Regulatory Operons | Lac (lactose), Trp (tryptophan), PhoR/PhoB (phosphate, E. coli), GlnA (glutamine, prokaryotes) |
| Lac operon (makeup) | 3 separate genes making one polycistronic mRNA (LacZ [β-galactosidase], LacY [galactoside permease], LacA) and one separate repressor gene (LacI) |
| Mechanism of Lac operon | In the presence of lactose or synthetic IPTG, LacI produces a repressor which binds the primary operator and either of two secondary signal-propagating operators – this prevents RNAP binding |
| Secondary regulation of Lac operon | Catabolite Repression: In the absence of glucose (indep. of lactose) cAMP is produced and binds to CAP (catabolite activator protein). CAP-cAMP complex binds to Lac promoter and activates the Lac operon (assuming it's not being repressed) |
| Trp operon (makeup) | 5 genes (Trp A-E) involved in making Trp from other amino acids and constitutively expressed genes for a leader transcript (TrpL) and a repressor gene (TrpR) |
| Mechanism of Trp operon | Trp, when present, is a co-repressor – Complex with repressor (from TrpR) will bind to the operator and prevent RNAP binding |
| Secondary regulation of Trp operon | Attenuation: Involves an attenuator (hairpin region) and 4 similar regions following. |
| Mechanism of Attenuation | When Trp is low, regions 2 and 3 will form a hairpin and stall RNAP without ending transcription. When Trp is high, regions 3 and 4 will form a hairpin and its following U-rich region will cause RNA and RNAP to dissociate |
| PhoR/PhoB system (basic) | Responds to low concentrations of phosphate in E. coli |
| Mechanism of PhoR/PhoB system | At low concentrations of phosphate, a Pi leaves PhoR (sensor) and is transferred to PhoB (response regulator). This causes the cell to make proteins associated with low phosphate (PhoA, PhoS, PhoE, ugpB) |
| GlnA gene (mechanism) | Encodes glutamine synthetase, which makes glutamine from glutamate and ammonia. When Gln is low, NtrB phosphorylates NtrC dimer, which unwinds bound σ54 so it can co-transcribe GlnA with RNAP. |
| General Transcription Factors | Oct3/4, Sox2, Kif4, and c-Myc (Just Oct4 in MCB 130A). Can be used to create iPS (induced pluripotent stem) cells |
| Eukaryotic Type I Polymerase | Located in nucleolus – Transcribes 1 gene at ~200 copies, useful for bulk transcription |
| Eukaryotic Type II Polymerase | Located in nucleoplasm – Transcribes most human genes and has different α (binding) subunits than Types I and III, as well as a CTD tail |
| Eukaryotic Type III Polymerase | Located in nucleoplasm – transcribes 30-50 genes at variable numbers, useful for fast transcription. Very similar in structure to Type 1, only 2 more enzyme-specific subunits |
| CTD Tail | C-terminal domain on β' subunit of Type II polymerase – composed of 27-52 YSPTSPS repeats. Phosphorylation of residues on these repeats is crucial in transcription and RNA processing |
| α-aminitin | Potent toxin in Aminita phalloides (death cap mushroom) – Readily binds/inhibits Pol II and fucks shit up |
| Promoter Elements – Two Main Types | Promoter-proximal and enhancers. Enhancers can be separated from promoters by boundary/insulator elements – brought together by formation of loops in DNA. |
| Boundary/Insulator elements | Elements recognized by non-histone proteins – Separate enhancers from their promoters and prevent spreading of silenced heterochromatin |
| TATA Box and/or Initiator | Very common promoter-proximal elements found in highly transcribed genes – equivalent for purposes of this class, but generally both will not be found in the same gene |
| CpG Island Promoter elements | 20-50 nt CG-rich sequences found in 60-70% of genes involved in "housekeeping" (low, steady transcription). These rigid sequences are weakly bound by histones/nucleosomes and are more accessible to Pol II and general transcription factors |
| GC-Box | Promoter-proximal element with GGGCGG sequence; binding site for Sn1 activator. Often found ~110 bases upstream from the TATA box if one is present, may also be found in CpG promoter |
| Eukaryotic Transcription Initiation (Basic) | Revolves around formation of PIC (Pre-initiation complex) at core promoter |
| Eukaryotic Transcription Initiation Mechanism | Promoter is recognized by TFIID through its TBP unit. TFIID recruits TFIIA/B to stabilize it – TFIIB binds to the BRE region, forming DAB complex. TFIIF binds Pol II and recruits TFIIE and TFIIH. TFIIH unwinds DNA and releases elongation complex |
| TFIID | Recognizes promoter region (TATA/Inr) and binds it through TBP (TATA-binding protein) |
| DAB complex | TFIID recruits TFIIA/B to stabilize it; TFIIB (with its helper TFIIA) binds BRE (B recognition element) region, forming stable D-A-B complex. This complex is left at the promoter when Pol II moves to allow reinitiation |
| TFIIF | Also known as RAP30/74. Binds RNA Pol II and recruits TFIIH and its helper TFIIE; connects them to PIC somehow |
| TFIIH | Has both helicase and kinase activity. Uses ATP to unwind DNA at promoter region, then phosphorylates Ser5 of CTD tail (promoter clearance) |
| Pol II Pausing | After ~40 nts, negative elongation factors (NELF and DSIF) cause Pol II to pause. This generally occurs at key regulators of developmental signals and signal cascade transducers – allows for rapid induction. This is where the 5' cap is added to mRNA. |
| Post-capping (pause release) | p-TEFb phosphorylates NELF, DSIF, and Ser2 of CTD tail. Ser2 allows Pol II to continue, NELF dissociates, and DSIF becomes a positive elongation factor |
| Eukaryotic Transcription Elongation | Productively occurs after 60-70 nts, when TFIIE/H dissociate from Pol II. Activated by SEC – can be recruited to promoter by Tat and TAR RNA of HIV or to Hox genes by MLL/SEC fusion proteins in Mixed Lineage Leukemia. |
| SEC | Super Elongation Complex – Made up of positive elongation factors (P-TEFb and ELL2) and scaffold proteins (AFF1/4) |
| mRNA processing | Turns pre-mRNA into mRNA – Cotranscriptional process involving caping, splicing, and polyadenylation |
| Capping | Addition of a 7-methylguanosine cap to 5' end of mRNA. This cap prevents the 5' end from exonuclease activity, as well as stabilizing mRNA and promoting splicing, translation, export, etc. |
| Splicing | During transcription, introns are removed from pre-mRNA and the exons are bound together to form the mature mRNA |
| Splicing mechanism | Two transesterification reactions – always 2 phosphodiester bonds. Step 1: Cleavage of 5' splice site and joining of 5' end of intron to branch point. Step 2: Cleavage of 3' splice site and simultaneous ligation of exons – excision of lariat-like intron |
| Spliceosome | Made up of 5 snRNPs containing 5 snRNAs (U1, 2, 4, 5, 6). U4 unmasks U6 for catalytic activity; U1 and U2 find splice sites |
| Alternative Splicing | Removal of some exons while retaining others – allows many mRNAs to produced from one gene/pre-mRNA. We only covered one method – use of the cross-exon recognition complex |
| ESEs | Exonic Splicing Enhancers – bound by SR (Ser-Arg rich) proteins in alternative splicing, forming the cross-exon recognition complex |
| Cross-exon Recognition Complex | Involved in alternative splicing – SR proteins bound to ESEs. Promotes binding of U1 to 5' splice site, U2 to branch point, and U2AF (auxiliary factor) to 3' splice site. Exons containing this are always retained in splicing |
| Polyadenylation | 3' of mRNA is cleaved and a tail of multiple AMPs is added there – This step terminates transcription and adds stability to the mRNA, as well as protecting the 3' end from exonucleases and promoting translation and export. |
| CPSF | Cleavage and Polyadenylation Specificity Factor – recognizes the poly[A] signal |
| CStF | Cleavage Stimulatory Factor – Binds poly[A] signal in response to CPSF recognizing it |
| PAP | Polyadenylate Polymerase – Only adds adenosine bases, so it doesn't need a template. Binds prior to cleavage to make sure free reactive 3' OH end is immediately polyadenylated |
| CFI + CFII | Cleavage Factors I and II: Cleave RNA 10-35 nts away from poly[A] signal – only after PAP has been bound. |
| PABPII | Polyadenine Binding Protein II – After PAP has added ~12 bases, binds the complex at the tail and accelerates process until the tail is ~200 bases long. ALSO has a role in facilitating ribosome recapture by circularizing mRNAs during translation |
| Three Main DNA Binding Motifs | Zinc Finger Proteins, Basic helix-loop-helix (BHLH), and Leucine zippers (b-Zip) |
| Zn Finger Proteins | Consists of an antiparallel β sheet and an α-helix coordinated by Zn (Cys2His2). Inserts alpha helix into major groove of DNA for scanning – Generally binds to GC pairs. 3-4 zinc fingers are needed for sequence specificity. |
| Basic helix-loop-helix (bHLH) | Consists of 2 α-helices connected by a loop; usually binds DNA as a dimer. Larger helix in DNA-binding region – Many Arg and Lys residues |
| Leucine Zippers (b-Zip) | Leucine residue at every 7th position in the zipper domain. Usually binds DNA as a dimer; Many Arg and Lys residues in DNA-binding region |
| DNA binding dimerization | bHLH and b-Zips can form heterodimers or homodimers – provides variability and specificity, even allows them to form inhibitory heterodimers. Homodimers usually bind to DNA sequences with rotational (inverted) symmetry |
| Heterochromatin | Repressed DNA – Packed into histones and condensed in nucleosomes. Must be antirepressed (decondensed) before it can be activated |
| Euchromatin | Kept loosely in the cell – easily transcribed |
| Three ways of activating CHROMATIN for transcription | Covalently modify histone termini (post-translational modification) Move nucleosomes away from promoter using ATP (motor proteins) Use histone modifiers to recruit (co)activators (recruitment of accessory proteins) |
| Three ways NUCLEOSOMES can be affected, regulating transcription | Acetylation, Methylation, and Nucleosome Remodeling |
| Acetylation | Carried out by HATs and HDACs – neutralizes positive charge on histone tail lysines. DNA will be unable to bind as tightly, enabling access by transcription factors. |
| HAT | Histone Acetyl Transferase – Catalytic subunit is Gcn5 |
| HDAC | Histone Deacetylase Complex – Catalytic subunit is Rpd5 |
| Methylation | Occurs on specific residues – Can express or silence gene depending on which residue. Mutually exclusive with acetylation. |
| Nucleosome Remodeling | Nucleosomes covering the TATA box or enhancers can be "slid" off by helicase/ATPase to enable gene activation |
| Swi/Snf complex | Nucleosome remodeler in yeast |
| Three ways TRANSCRIPTIONAL REGULATORS can be controlled | cAMP signaling, NF–κB signaling, and Nuclear Receptor Family Transcription Factors |
| cAMP | Hunger signal in E. coli; secondary signal in eukaryotes. When a hormone/neurotransmitter binds to GPCR, adenyl cyclase is activated and produces cAMP from ATP |
| CREB | cAMP Response Element Binding Protein |
| CBP | CREB Binding Protein |
| NF-κB | "Master" immune system regulator – directly activates ~150 genes. Made up of p50 and p65 subunits; sequestered by I-κBα |
| NF-κB signaling process | TNF–α or IL-1 receptors are activated and activate TAK1 kinase in turn, which activates I-κB kinase. This phosphorylates I–κBα, triggering its ubiquitination. This "unmasking" of the NLS on p50 and p65 lets them be taken up into nucleus |
| cAMP signaling process | cAMP binds PKA's inhibitory subunit. PKA then phosphorylates CREB, which binds CRE and interacts with CBP ––> transcription activation |
| Nuclear Receptor Transcription Factors | A ligand (oft. steroid hormone) binds, competitively removing an inhibitor at the LBD (Ligand Binding Domain). TF can now enter nucleus and activate transcription |
| Glucocorticoid receptor | Given example of Nuclear Receptor TFs with Hsp90 inhibitor. Originally determined by immunofluorescence |
| Translation (basic) | Uses tRNAs (transfer RNAs) to turn mRNA codons into peptide sequences |
| tRNA charging | Aminoacyl tRNA synthetases attach a specific tRNA to its amino acid. Synthetase recognizes specific tRNA and aa – if incorrect, hydrolyzes bond and releases aa. Once the tRNA is charged, the ribosome will not dissociate. |
| Ribosome Sites | A – Entry site (except fMet). P – Attachment site. E – Exit site. |
| Prokaryotic Translation Initiation | Ribosome recognizes Shine-Delgarno Sequence and pairs 3' end of 16S rRNA with it – also requires an AUG start codon ~ 10 nts downstream. IFs (Initiation Factors) bind and begin translation |
| IF-1 | Associates with 30S subunit – Binds to A site of ribosome to prevent premature tRNA binding |
| IF-2 | Uses GTP to facilitate fMet–tRNA binding to 30S ribosomal subunit – this is the start of translation. |
| fMet | N-formylmethionine – Added to N-terminus at the start of translation in eukaryotes by transformylase. Ensures continuing residues are added to C-terminus |
| IF-3 | Binds to 30S subunit and allows its binding to mRNA. Prevents 50S from binding to 30S until removed |
| Prokaryotic Translation Elongation | GTP-bound Ef-Tu binds charged tRNAs and brings them to the A site, where they're connected by the peptidyl transferase ribozyme. Ribosome moves over and releases tRNA (translocation); repeat. |
| Prokaryotic Translation Termination | RFs (Response Factors) hydrolyze bonds in response to stop codon; all units are released |
| Eukaryotic Translation Initiation | 40S subunit of the ribosome recognizes the 5' cap on the mRNA, and the ribosome scans for the AUG codon and binds. |
| Circular Polyribosomes | PABPII and 5' cap interact to form circular mRNA – Allows translation by multiple ribosomes and facilitates ribosome recapture (may be the same) |
| miRNA | Micro RNAs – 21 nt sequence which can impair translation of complementary mRNA or induce its degradation. Closely complementary, but not exactly |
| siRNA | Small interfering RNA – 21 nt sequence which completely silences gene (perfectly complementary) and degrades relevant mRNA |
| Ferritin mRNA translation | When iron is low, IRP binds to IRE. When iron is present, it binds IRP, causing IRE to dissociate and allow translation of ferritin, which harmlessly sequesters iron in the cytoplasm of the cell |
| DNA Footprinting | Used to identify protein binding site. PCR the segment of interest, cleave with low conc. DNase and run it on a gel against the same sample with POI. "Protected" patch will not show up on gel. |
| DNA Affinity Chromatography | Used to purify DNA-binding proteins. Step 1: Purify cell lysate in matrix of varied DNA – Use low-salt wash to remove non-DNA-binders. Step 2: Purify DNA-binders in matrix of only DNA of interest; use medium-salt wash to remove others |
| Gel Shift/EMSA (Electrophoretic Mobility Shift Assay) | Used to test protein-DNA interactions. Mix DNA and protein with a labeled probe – Protein-DNA complex will move slower than free probe, protein, or DNA. |
| In vitro Transcription | Used to test transcriptional activity. In a dish/tube, mix purified GTFs, Pol II, DNA template, purified POI, and radiolabeled NTPs – transcription will be easily monitored |
| In vivo Transcription | Used to test transcriptional activity. Clone gene into a plasmid with a reporter tag; transform into cells, express, lyse, and analyze reporter tag |
| Promoter Bashing | Used to identify DNA elements affecting gene expression. Clone possible promoter region into plasmid with reporter tag and measure reporter transcription |
| Homology Search | Used to identify DNA elements affecting gene expression. Computationally determine likely DNA elements – these sequences are often highly conserved |
| DNA microarrays | Used to compare gene expression levels. Also known as a DNA chip – consists of "spots" of different DNAs (probes). Treatment with proteins with reporter tags will show bound hybrids |
| ChIP (Chromatin Immunoprecipitation) | Used to identify sites where proteins bind to DNA. Cross-link cells and DNA using formaldehyde and soniccate to break up DNA. Use bead-bound antibodies to pulldown POI-bound section, and use heat to reverse crosslink the protein and DNA |
| Analysis of ChIP | ChIP-PCR: Use (q)PCR to amplify pre-sequenced genes ChIP–Seq: Sequence everything pulled down (good for far proteins) ChIP-chip: Use microarray to identify all targets of an isolated protein |
Created by:
yazaria
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