BIOMG 3320 Group 4 (Lectures 8-10)

• DNA probes that are complementary to the sequence of RNAs are covalently bound to a glass slide. Probes for one specific RNA species are grouped at one specific spot on the glass slide; we can look at thousands of probes on one slide.
• Allow genome-wide measurements of levels of gene expression.
• Levels of mRNAs in the cell are measured simultaneously.
• Levels of non-coding RNAs can also be measured.
• Relative measurement of RNA abundance in one sample compared to the other sample.
• mRNA length cannot be measured.

• Isolate RNA of interest
• Make cDNA from RNA using reverse transcriptase. Incorporate a fluorescent label into cDNA. (ex. Red for egg cells, green for tadpole cells)
• Mix cDNA at equal ratios, flow over microarray. cDNA will hybridize to complementary probes.
• After washing away excess, image the microarray to measure the fluorescent signal at each spot.
• Relative color intensity at each spot allows you to compare the expression of that RNA species between your two samples. (ex. Red means more expressed in egg, yellow means equally expressed.)

• Low sensitivity makes it difficult to measure expression of lowly expressed genes.
• Cross-hybridization can occur between RNA species with overlapping or similar sequences.

• Transcription unit, from which RNA is produced. Contains exons and introns.
• The promoter: the general transcription factors and RNA polymerase accumulate before transcription.
• Enhancers: sequences of DNA that can be very far from the promoter and that serve to recruit regulatory transcription factors that control expression of the gene.

• Similar to prokaryotic RNA polymerases in their overall structure and catalytic activity.
• Has additional subunits that provide more layers of regulation and transcription, and are more specialized.
• Crab-claw shape that houses the active site where RNAis polymerized.
• Minor proofreading capability in the same catalytic site.
• Multiple protein subunits with comparable functions, BUT NO SIGMA FACTOR

• Pol 1: ribosomal RNA (which makes up nearly 50% of all RNAs in cell.
• Pol 2: messenger RNA (and a handful fo others)
• Pol 3: non-coding RNAs (tRNAs)

• Makes the rRNA precursor.
• The rRNA promoter has two components bound by two different factors required to recruit RNAP1:
• The core promoter: which overlaps with the transcription site. Bound by the SL1 factor, which are a complex of four monomers called the TATA binding protein
• The upstream control element (UCE: which sits 100bp upstream of the start site. Bound by the UCE binding factors.
• There are no distal enhancers associated with this promoter.

• Makes tRNAs and other non-coding RNAs in addition to one subunit of rRNA.
• Its promoters are downstream of the start site.
• TFIIIC recognizes and binds the Box A and Box B regions.
• It then recruits TFIIB, which again contains TBP.
• Together, they recruit Pol 3 and transcription begins.

• TBP, in complex with TFIID, binds to the minor group in the TATA box, bending the DNA nearly 90 degrees.
• TFIIA binds and assists with the stability of TBP-promoter and assists TFIIB-promoter interaction.
• TFIIB contacts the TFIIB recognition element in the promoter and TBP. TFIIB is recruited to the BRE (B recognition element) directly or through physical interaction with TBP.
• TFIIB helps to point the PIC in the right direction for transcription by binding to bent DNA.
• RNAP 2 and TFIIF together are recruited by TFIIB to the promoter. TFIIF prevents Pol2 from binding to non-specific DNA sequences.
• TFIIE binds the complex and recruits TFIIH, whose ATP-dependent activity contribute to DNA helix melting and phosphorylation events that lead to promoter escape.
• The double helix melts, forming the DNA bubble that characterizes the open complex.
• The open complex proceeds into initiation, which produces abortive transcripts as in prokaryotes.
• The transcription complex escape the promoter and proceeds into elongation, and ends transcription with termination.

• An average Pol 2 promoter contains an initiator sequence, where the transcription start site lies
• A TATA box around the -30 position, and various regulatory sequences that can be near or very far from the core promoter.
• There are a number of general transcription factors that are required for Pol2 recruitment and various initiation-related factors.

• The PIC is the step that is controlled to determine whether or not expression occurs, but for many genes in multicellular organisms, pausing is also a point of regulation.
• Pol 2 that has escaped the promoter can be bound by pausing factors DSIF and NELF (negative elongation factor), which temporarily halt elongation.
• P-TEFb (positive transcription elongation factor) is a kinase that, when recruited, phosphorylates DSIF and the Pol 2 CTD, which releases NELF and allows Pol 2 continue.

• 7-methylguanine cap forms at the 5’ end of the mRNA
• Promotes nuclear transport
• Stabilizes RNA (protects against degradation)
• Stimulates translation

• Regions of DNA that contain histone proteins are called chromatin
• A single unit of a histone core wrapped by DNA is called a nucleosome.
• The histone protein core is an octomer consisting of two H2A/H2B dimers and one H3/H4 tetramer. Each of these protein subunits within the octamer have N-terminal tails that project outward from the nucleosome.
• Euchromatin is the transcriptionally active form of chromatin that is more spaced apart.
• Heterochromatin is the transcriptionally inactive form that is more tightly packed.

The pattern of covalent chemical modifications made to the N-terminal tails of histones that determine in part whether chromatin is packaged as euchromatin or heterochromatin.

• Activators recruit histone acetyl-transferases (HATs) that acetylate lysines on the nearby histone tails.
• Acetylation contributes to histones repelling each other.

• The FACT complex (facilitates chromatin transcription) dismantles histone octamers.
• As the polymerase rolls through the region, the FACT complex reassembles histones in the past region.
• FACT pulls an H2A/H2B dimer out of the nucleosome ahead of Pol2, holding it nearby but out of the way. This loosens the histone-DNA contacts enough to allow passage.
• FACT chaperones the H2A/H2B dimer back into the nucleosome after Pol 2, restoring the stable chromatin structure.

• Binds TATA box and bends DNA nearly 90 degrees.
• Part of TFIID complex
• Binds TFIIB

• Binds TBP, recruits TFIIF and Pol2
• Binds to DNA minor groove bent by TBP, guiding PIC in the correct direction for transcription.

Complexed with Pol2, binds TFIIB. Prevents Pol2 binding outside promoters.

• ATPase dependent helicase activity
• TFIIE binds the complex and recruits TFIIH, whose ATP-dependent activity contribute to DNA helix melting and phosphorylation events that lead to promoter escape.

Pausing factors in initiation

Pause releasing factor

• A completely transcribed pre-mRNA is cleaved away from Pol 2 after Poly-A signal sequence emerges, but RNAP will continue transcribing it until it is terminated by one of the two models.
• Two models:
• Torpedo model: Relies on Rat1/hXrn2
• Poly-A signal sequence results in the cleavage and release of the nascent RNA, which has a 5’-capped. The exposed uncapped RNA trailing out of Pol II is quickly digested by Rat1/hXrn2.
• The ramming of Rat1 into Pol II knocks it off the DNA and terminated transcription, similar to rho-dependent termination in prokaryotes.
• Allosteric model:
• When factors associated with Pol II leave after pre-mrNA cleavage and polyadenylation, Pol 2 undergoes a conformation change that reduces its efficiency and leads to spontaneous jumping off from the DNA.

• Poly-A signal sequence results in the cleavage and release of the nascent RNA, which has a 5’-cap. The exposed uncapped RNA trailing out of Pol II is quickly digested by Rat1/hXrn2.
• The ramming of Rat1 into Pol II knocks it off the DNA and terminated transcription, similar to rho-dependent termination in prokaryotes.

When factors associated with Pol II leave after pre-mrNA cleavage and polyadenylation, Pol 2 undergoes a conformation change that reduces its efficiency and leads to spontaneous jumping off from the DNA.

• Occurs right before termination, resulting in indirect termination of transcription and a 3’ poly(A) tail nearly 200 nucleotides long.
• After the pol-A signal sequence (AAUAAA) is transcribed and emerges from Pol 2, CstF (cleavage stimulation factor) and CPSF (cleavage and polyadenylation specificity factor) jump off the CTD and onto the RNA
• CstF leaves after CPSF catalyzes cleavage
• CPSF recruits Poy-A-polymerase, which synthesizes poly(A) tail in the absence of a DNA template. As the tail is generated, it is bound by many Poly-A binding proteins, which contribute to the export and protection functions of the tail.
• After 200 adenisones are added, CPSF and PAP leave.

• Promote export
• Stabilize RNA
• Stimulate translation

• Human RNAP 2 has a tail at the C-terminus (CTD) made up of around 52 repeats fo a 7-amino acid sequence. The CTD tail resides near the channel for the nascent RNA.
• The CTD is phosphorylated in different patterns during each stage of transcription. These patterns attract the factors needed for the different RNA-processing events.
• The phosphorylation pattern put in place during promoter escape recruits 5’-capping enzymes, whereas the pattern in place near the end of elongation recruits the factors needed for pre-mRNA cleavage and polyadenylation.

Capping factors

Splicing factors

• A coactivator of transcription, it takes in signals by binding to activators/repressor to make the “yes or no” decision on whether to initiate transcription.
• Activators bind to enhancer sequences in the DNA and can encourage transcription by interacting with Mediator or by recruiting chromatin remodeling and modifying enzymes.
• Repressors prevent transcription by interacting with the mediator, competing with transcription activators on enhancer binding, and by inhibiting general transcription factors.
• High mobility group proteins can alter the overall architecture of chromatin.

Gene that researchers attach to a regulatory sequence. LacZ reporter turns cells to blue when expressed.

• GAL4 has a DNA binding domain and an activation domain, which binds with the pre-initiation complex to facilitate transcription.
• The reporter construct is a cassette of DNA engineered and placed in cells. It contains a GAL4 binding site upstream of lacZ, which causes cells to turn blue when expressed. When cells are given the GAL4 activator, it binds to the reporter and activates it.
• When the cells are given GAL4 DNA binding domain only, they bind to the reporter but don’t activate expression.
• When the reporter is modified to have a bacteria-derived LexA binding site and given Lex-A protein DNA-binding domains, they bind to the reporter but don’t induce expression.
• When the cells with a LexA site reporter are given a fusion protein that contains LexA BD and GAL4 AD, it binds to the reporter via the LexA BD and induces expression through GAL4 AD.

• Competition: A repressor binds the DNA overlapping with the activator binding site, preventing the activator from binding.
• Inhibition: The repressor binds the DNA apart from the activator, but it then binds with and inhibits the activation domain of the activator.
• Direct Repression: The repressor binds mediator and inputs a signal that prevents expression.
• Indirect repression: The repressor recruits chromatin histone modifying enzymes and/or modeling complexes that inactivate regions of DNA, usually by converting euchromatin to heterochromatin.
• Mig1 is a repressor that binds near GAL1 and recruits another repressor called Tup1, which inactivates expression through indirect and direct repression.

• A method of sequencing and measuring the quantity of thousands of mRNAs simultaneously.
• After extracting total RNA from sample, the mRNAs are isolated by binding their pol(A) tails to single-stranded DNA oligos of repeating Ts attached to beads.
• The mRNAs are fragmented and primed from reverse transcription with either poligo d(T) primers or random primers, producing cDNAs.
• The cDNAs are sequenced via Illumina sequencing and individual reads are mapped to the known genome. The number of reads for a gene tells you about the expression level of the gene.

• TATA box: where TBP binds to from pre-initiation complex
• Enhancers: where activator and repressor proteins bind
• Insulator elements: prevent enhancers from influencing wrong promoter.

Activator or repressor binds to mediator, sending signal to repress or enhance transcription.

• Shows you what DNA sequences a targeted protein is binding. A method to measure protein-DNA interaction.
• Genomic DNA is fragmented.
• We form reversible covalent attachments between proteins and DNA. Crosslinking proteins to the binding DNA prevents them from falling off during the experiment.
• Use antibody specific to protein of interest to purify binding DNA fragments.
• We then use Illumina sequencing to analyze the DNA fragments that were obtained.

Create separated domains of transcriptional regulation by blocking the interaction between an enhancer and promoters in directional fashion.

• Covalent modification: Covalent modifications to histone tails convert heterochromatin to euchromatin and vice versa.
• Structural modification: Activators recruit chromatin-remodeling complexes that loosen and rearrange the way DNA is wrapped around histones. Does not involve covalent changes to chromatin. Changes accessibility of binding sites in DNA to proteins.

• Writing (acetylases, methylases, phosphorylases)
• Erasing (deacetylases, demethylases, dephosphorylases)
• Reading (bromodomain, chromodomain, PHD finger, WD40 repeat)

• K9 methylation acts to condense chromatin by recruiting HP1 that further condense chromatin.
• However, K9 methylation and S10 phosphorylation together can dissociate HP1 and open chromatin.

• Different regions of the chromosome gave different histone marks associated with them.
• Histone code is not stable and it is reversible by erasing enzymes. However, it is still considered to be epigenetic modification.

• Rap1 binds and recruits Sir complex.
• Sir2 deacetylates proximal nucleosomes.
• Deacetylated nucleosomes bind more Sir complex, spreading the silenced domain.

• Polycomb complex:
• An example where the DNA sequence triggers the epigenetic changes.
• PRE (polycomb response elements), DNA sequence that triggers repression.
• PRE is recognized and bound by PHO-RC
• PHO-RC recruits PRC2, polycomb repressive complex 2.
• PRC2 contains histone methyltransferase

• Stable change (unchanged during cell division) in gene expression without changing the DNA code.
• In vertebrates, happens through DNA methylation.

• Happens in vertebrates. Epigenetic change.
• Majority of CpG is methylated (C is methylateD)
• Methylation represses transcription.
• Transposons are a good example of this - they’re highly methylated and not expressed.
• Proteins that bind methylated DNA can recruit histone deacetylase and chromatin remodeling complex for further repressing the gene, locking the DNA in a state of repression.
• Changes in methylation associated with cancer.
• Ageing generally results in loss of methylation.

• De novo methyltransferase: Establish methylation patterns in early development
• Maintenance methyltransferase: After replication, maintains methylation patterns during cell division, allowing for epigenetic regulation.

• Methylation marks are removed after each generation, except for imprinted genes.
• Imprinted genes: only kind of gene where methylation marks aren’t removed during fertilization.

Bisulphite sequencing.

• Cooperativity though protein-protein interactions
• Two factors have their own adjacent binding sites, but their interaction can enhance DNA binding and activation activity.
• Alternatively, they can be connected through a third intermediate.
• Indirect effects through chromatin remodeling
• The binding of one transcription factor can cause the chromatin to rearrange and open, either on its own or through a modifier, thus exposing the binding site for the second factor.

• 1. Nucleophilic attack of phosphodiester bond by 2′ OH (of the same sugar) leading to two RNA fragments, one with a 2′ 3′ cyclic phosphate and the other with a 5′ OH.
• 2. Reaction is initiated and stimulated by OH-.
• 3. RNA hydrolysis places a limitation on the size of RNA-based genomes – as the probability of a hydrolysis increases with the number of phosphopdiester bonds subject to cleavage (i.e., genome size), and such cleavage would be problematic for genetic material. Only relatively small RNA genomes (all viral) exist.