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HMS Chemical Biology and Biochemistry > Oxygen Sensing > Flashcards

Flashcards in Oxygen Sensing Deck (49)
1

snRNA

small nuclear RNA

 

Involved in RNA splicing and processing

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snoRNA

small nucleolar RNA

 

Covalently modify RNA (tRNA, rRNA)

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lncRNA

Long non-coding RNA

 

Mostly unclear, but a few well-described lncRNAs, including Xist, which is involved in X-chromosome inactivation in females.

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Coding gene structure in DNA

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Organization of EPO gene

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nucleolus

where the expression of ribosomal RNA occurs and where ribosomes assemble

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Euchromatin vs Heterochromatin

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Nice Diagram of DNA structure

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DNA Supercoiling

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Histone octamer structure

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"Coactivators" and "Corepressors"

Co-activators and co-repressors bind to the regulatory transcription factors and activate or repress transcription through a variety of mechanisms.

 

DO NOT have a DNA binding domain

 

One example would be writers and erasers that are recruited by regulatory transcription factors.

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Extrusion of DNA and loop domains

The yellow rings here are referred to as "extrusion complexes."  They are made from cohesins.

 

The motifs upon which they land are CTCF motifs, which help direct which DNA should be extruded.

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Topologially Associated Domains

Neighboring cohesin-derived DNA loops that interact with one-another.  TADs contain actively transcribed chromatin, and tend to cluster with one another while inactive chromatin clusters with other inactive chromatin.

 

These acive TADs represent euchromatin, while inactive chromatin represents heterochromatin.

Among these differential clusters, chromatin originating from the same chromosome tends to cluster together.

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"Reader" protein

Protein which targets specific chromatin domains by recognizing histone tail modifications.

 

Display a diversity of structures and mechanisms for interacting with histones.  Each reader has an affinity for a specific type of modification.

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Five types of modification for histone tails

Acetylation

Methylation

Ubiquitination

Phosphorylation

Proline isomerization

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"Writer" proteins

Add histone modifications

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"Eraser" proteins

Remove histone modifications

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Components required for transcription

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Reactions Performed by Writers and Erasers

Notably, degree of methylation of lysine may also change.  Histone-associated lysines may be unmethylated, methylated, dimethylated, or trimethylated.

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How readers affect gene expression

An important point to emphasize is that regulatory transcription factors can also recruit writers and erasers. In this case, the writers or erasers are called co-activators or co-repressors, depending on whether they tend to activate or repress transcription

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Reader-Writer Epigenetic Propagation

When histone H3 lysine 9 is methylated, this recruits a reader called HP1, which can bind to itself and therefore promote a compact chromatin structure. HP1 can also bind the writer that makes the mark, a histone methyltransferase. Because the reader recruits its own writer, it helps maintain the mark, and can also lead to propagation of the mark down the chromatin. Specific sequences called boundary elements are necessary to prevent the propagation into active regions of chromatin.

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Pioneer Factors

Some transcription factors can bind to more compacted chromatin, and then initiate changes in chromatin structure. These are called pioneer factors because they can initiate the process of opening chromatin.

Other transcription factors, called non-pioneer factors, can only work on chromatin that has already been opened by pioneer factors.

 

Pioneer factors are particularly important in the initiation of cellular differentiation.

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Transcription Initiation

It is important to note that RNA polymerase cannot do this on its own—it needs a series of additional factors called general transcription factors (TFII proteins) to bind the promoter and initiate transcription.

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Transcription Elongation and Termination

The mechanism of termination differs for the various RNA polymerases. For RNA polymerase II, termination occurs when a specific sequence is reached that causes cleavage the mRNA and the addition of a polyA tail.

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Animal RNA Pol II

RNA polymerase II is composed of multiple subunits (12 in humans and yeast). Rbp1 is the largest subunit and contributes to the catalytic activity of the enzyme. Rbp1 also contains an extended C-terminal domain (CTD) that is composed of a repeating heptad (7 amino acids). These sequences become phosphorylated differentially during initiation and elongation. The bottom of the diagram shows an illustration of the relative size of the polymerase compared to the extended CTD. The CTD serves as a platform that allows other proteins to bind during the process of transcription and RNA processing (splicing). You can think of the CTD in a manner similar to a histone tail in that it gets modified by enzymes at various points during transcription (kinases act as ”writers”) and various proteins can then bind the phosphorylated CTD (“readers”). These readers can help promote processing of the newly synthesized mRNA as it emerges from the polymerase.

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general transcription factors

The general transcription factors help RNA polymerase where to bind the DNA, and therefore, where to start transcription. These proteins can be found at most/all genes that are transcribed. For this reason, they are called “general” transcription factors. There is a limited number of different general transcription factors. Some of these proteins bind directly to DNA whereas others do not.

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regulatory transcription factors

The regulatory transcription factors help decide how much of a transcript should be made. There are hundreds of different regulatory transcription factors, each of which has specificity for different DNA sequences. As we will discuss, these proteins do not work by themselves, but have to work in partnership with other proteins called co-regulators (co-activators or co-repressors, depending on how they affect transcription).

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TATA-box

There are a series of general transcription factors that recognize specific DNA sequences around the promoter. Some of these are located upstream of the promoter, like the TATA box, and others are located at the start site or downstream of the start site. (Note: in molecular biology, regions of evolutionarily conserved sequences are often called “boxes”. The TATA box is the best characterized sequence element, and is named because it is enriched in thymidine (T) and adenine (A) bases. It is recognized by a protein called TATA box-binding protein or TBP, which is part of a complex of proteins called TFIID (any time you see “TFII” this is a general transcription factor). Around the TATA box are additional sequence elements (BRE) that are recognized by a different general transcription factor called TFIIB. Furthermore, there are sequence elements at the transcription start site (Inr) and also downstream (DCE, MTE, DPE) that are also recognized by proteins in the TFIID complex.

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RNA Pol II and TATA-box interaction

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Establishing of preinitiation complex

See TATA-box interaction for first half.

 

Another general transcription factor called TFIIE then binds, which in turn helps recruit TFIIH. TFIIH has helicase activity and helps to unwind the DNA to allow transcription to begin. These proteins form what is called the preinitiation complex (PIC), which is located at the promoter but is not yet actively transcribing DNA.

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Transition from Preinitiation to Elongation

The C-terminal domain (CTD) of Pol II becomes phosphorylated by associated protein kinases once the complex moves into the elongation phase. The phosphorylated CTD can recruit other proteins that help with elongation or RNA processing, such as addition of the 5’ cap, splicing, or polyadenylation.

The diagram shows that the general transcription factors may be released, but it is now thought many of them remain associated with the promoter region for some time after Pol II moves into the gene body, creating a scaffold of factors that can help re-initiation of Pol II. Remember that an actively transcribed gene will be transcribed many times, so it is important to be able to reinitiate transcription quickly to support a high rate of mRNA synthesis.

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Response Elements

Control regions contain short DNA sequences called response elements. These specific sequences are recognized by regulatory transcription factors, which can “read” these sequences, as we will describe. The pink arrows indicate the position of the repeat elements that correspond to the binding site of the transcription factors. GRE=glucocorticoid response element; ERE=estrogen response element; VDRE=vitamin D3 response element; TRE=thyroid hormone response element; RARE= retinoic acid receptor response element.

These are just a few examples.

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Structure of a Regulatory TF

Regulatory transcription factors generally consists of a domain that binds to DNA response element and a domain that either activates or represses transcription. Transcriptional activation or repression is a consequence of the ability of the activation or repression domain to recruit additional proteins (co-activators or co-repressors) that affect the structure of the chromatin or activate the transcriptional machinery.

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Interaction of TFs with the major and minor grooves

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Helix-Turn-Heix motif

The recognition helix (red) is the helix that binds to the major groove. The amino acids in this helix are what interact with the exposed edges of each base in the major groove. The other helix is important for structural reasons.

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Helix-Loop-Helix motif

The helix-loop-helix contains two alpha helices connected by a series of unstructured amino acids (loop). This motif will bind to another protein with a helix-loop-helix motif, allowing both proteins to interact with the DNA through their recognition helices.

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Leucine Zipper motif

Regularly spaced leucines in an alpha helix in one protein interact with the regularly spaced leucines in an alpha helix of another protein. This interaction allows a conformation in which both proteins contact DNA on either side in the major groove. The lower part of each helix interacts with the major groove, using basic side chains.

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mediator complex

A large multisubunit complex which helps to activate RNA polymerase II.  The mediator can help recruit RNA polymerase to the promoter to form the pre-initiation complex (PIC), or it can help stimulate the transition from initiation to elongation.

 

An example of a co-activator recruited by regulatory transcription factors.  

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Helicase can be thought of as a

co-activator

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Activator Directed Histone Hyperacetylation

The regulatory transcription factor (shown as a dumbell structure with a DNA-Binding domain (blue, DBD) and an activation domain (green, AD) binds to a response element (RE) on the DNA. The activation domain binds to a protein complex that contains an enzyme that can acetylate histones (HAT or histone acetyl transferase). The HAT enzyme acetylates the tails of histones in the neighborhood of where the enzyme is bound. The increase in acetylation of the histones helps make the chromatin more accessible to the general transcription factors, such as the TATAbinding protein (TBP). In human cells there are multiple HAT enzymes including protein complexes called CBP and P300.

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Mediator Complex and Cyclin C-CDK8

Cyclin-C CDK8 functions to control how much and what type of transcription takes place within the cell.  CDK8 is one of the "transcriptional CDKs", which directly regulate gene transcription by modifying TFs.

If CDK8 is active, reinitiation will be strongly suppressed, and it will phosphorylate in an activatory or deactivatory manner many transcription factors to skew the transcriptional profile of the cell.  If not, reinitiation will occur frequently.  Thus, CDK8 activity inhibits cell growth.

CDK8 activity also activates genes involved in non-glucose sugar metabolism.

 

Think of it like a proofreading mechanism:  The mediator complex forms and makes the decision of whether or not to form the PIC and proceed with initiation.  Cyclin-C-CDK8 has a chance to read the TFs and say: "Yes, we should proceed with this change given the cell's current state" or "No, we should conserve our resources instead of upregulating these genes."  This way the cell saves the resources it would have spent on RNA that does not apply well to its situation.

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Chromatin remodeling protein complexes

In addition to post-translational modifications of histone tails, there are protein complexes that use ATP to remodel chromatin structure, and have homology to DNA helicases.

They have subunits that are ATPases (enzymes that hydrolyze ATP), and they are thought to use the energy from ATP hydrolysis to break histone/DNA contacts and allow movement of the histone octamer, although it is not yet clear exactly how this happens. They are recruited to the DNA by regulatory transcription factors. There are complexes that can lead to more accessible chromatin, and complexes that can generate chromatin that is repressive towards transcription. These remodeling proteins could also help move nucleosomes to promote transcription by RNA polymerase during the elongation phase.

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X chromosome and Barr bodies on the side of the nucleus

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Transcriptional Repression by Histone Modification and X-chromosome inactivation

This slide shows an example of the activity of the polycomb genes, which promote formation of condensed heterochromatin by modifying histone H3 on lysine 27. This leads to formation of a stable, repressed complex. In females, this type of mechanism is important for inactivating one copy of the X chromosome. The key principle here is that the post-translational modifications of histones enable other proteins to now bind the histones. In other words, the polycomb protein (PC) can only bind to the histone when the histone is methylated at a specific lysine residue. This allows the polycomb protein to bind, which in turn binds the PRC1 protein, forming heterochromatin that cannot be transcribed

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Summary of Transcriptional Regulation

General transcription factors define where RNA polymerase II will bind, while regulatory transcription factors, via the action of co-activators and the mediator influence, how much transcription takes place

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Developmental Control of Transcription

Patterns of gene expression that change as a cell develops from its less differentiated form to its final mature adult form

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Homeostatic Control of Transcription

In the mature cell, changes in gene expression that help the cell carry out its normal functions, possibly changing in response to the environment

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Transcriptional Regulation Cascade