Adv. Cell Chapter 7 Reading Guide

What is a cis-regulatory sequence 'logo'? Why are they depicted this way?

"logo" displays the range of sequences recognized by a particular transcription regulator. Since they recognize range of closely related sequences with the affinity of the protein for the DNA varying according to how closely the DNA matches the optimal sequence, depicted this way.

what is leucine zipper?

- 2 alpha helices one from each monomer joined together to form a short coiled-coil - two alpha helices separate from each other to form a Y-shaped structure which allows their side chains to contact major groove - bind to DNA as dimers

what is helix-loop-helix?

- consists of short alpha helix connected by a loop to a second larger alpha helix, flexibility allows one helix to fold back and park against other forming dimerization surface - two helix structure binds to both DNA and to the two-helix structure of a second protein to create either a homodimer or heterodimer - two alpha helices that extend from dimerization contact with major groove of DNA

what is helix-turn-helix?

- constructed from two alpha helices connected by a short extended chain of amino acids which creates the "turn" - C-terminal helix called recognition helix fits into the major groove of DNA, amino acid side chains play a role in recognizing the specific DNA sequence to which the protein binds - bind in dimers (pairs) - major groove of DNA

what is homeodomain?

- three alpha helices folded together and packed tightly together via hydrophobic interactions, helices 2 and 3 resemble helix-turn-helix - helix 3 makes contact with major groove, Asn of helix 3 contacts an adenine, flexible arm attached to helix 1 creates contact with nucleotide pairs in minor groove - bind solo

What are the steps in the pathway leading from DNA to protein?

1. Cell controlling when and how often a given gene is transcribed (transcriptional control) 2. Controlling the splicing and processing of RNA transcripts (RNA processing control) 3. Selecting which completed mRNAs are exported from the nucleus to the cytosol and determining where in the cytosol they are localized (RNA transport and localization control) 4. Selecting which mRNAs in the cytoplasm are translated by ribosomes (translational control) 5. Selectively destabilizing certain mRNA molecules in the cytoplasm (mRNA degradation control) 6. Selectively activating, inactivating, degrading or localizing specific protein molecules after they have been made (protein activity control)

The book indicates that while we do not have hard and fast rules about what differences exist between any one cell type and another we do know some generalities. Using the 4 points on page 371 - write out these differences in your own words.

1. Many cellular process in a cell is common to other cells. Two cells in an organism contain similar proteins, enzymes, and repair genes. 2. Specialized cells are abundant in RNA and protein and can't be detected in other cells. An example would be hemoglobin only found in RBCs. 3. In a one moment a human cell expresses 30-60% of its genes. The level of RNA expression between different human cells varies as well as the same gene found in all cells. There are many ways RNA can be regulated. Coding RNAs in specialized cell types are different

How long are the patterned stretches of DNA that are normally read by GRPs?

20 contacts with DNA to be able to bind

what is cis-regulatory sequences?

5-10 nucleotide pairs in length, on same chromosome (cis) to the genes they control - short stretches of double-helical DNA of defined sequence

How does a cell determine which of its thousands of genes to transcribe?

A cell uses a group of proteins - transcription regulators. These proteins recognize specific sequences of DNA (5-10 nucleotide pairs in length) that are often called cis-regulatory sequences due to having to be on the same chromosome to the genes they control. Transcription regulators bind to the sequences and this binding puts into motion a series of reactions that specify which genes are to be transcribed and at what rate.

Is this is a weak or strong interaction? How specific?

An individual contact is weak but 20 of the bonds would very strong and ensures interaction is highly specific and strong. DNA protein interactions includes some of the tightest and most specific molecular interactions known in biology.

Why did biologists originally suspect that genes were selectively lost after cell differentiation? Did this turn out to be the case?

Biologists thought that cell differentiation was irreversible, so that meant that genes might have been selectively lost when the cell differentiated. This is not the case since we now know that cell differentiation does not occur without changes in nucleotide sequence of a cell's genome.

How do cell types in a multicellular organism become different from on another?

Cell types in a multicellular organism become different because they synthesize and accumulate different sets of RNA and protein molecules.

What are the fundamental components of a regulatory region?

Cis regulatory sequence, short unique sequence that can be read and bound, same chromosome typically not on a separate chromosome, can be anywhere away or close to gene

Where do DNA-protein interactions rank as far as strength and specificity when compared to all molecular interactions in biology? Is this surprising?

DNA-protein interactions are the tightest and most specific molecular interactions. No.

What various types of regulatory regions exist?

Different sequences of Cis-regulatory sequences and located anywhere on same chromosome, 10% of protein-coding regions of most organisms are devoted to transcription regulators, intergenic regions (upstream from transcription start)

When and why is glucocorticoid released?

Glucocorticoid is released into the body during periods of starvation or intense exercise. Glucocorticoids signal the liver to increase the production of energy from amino acids and other small molecules.

What types of bonds and how many are formed between the DNA and the protein?

Hydrogen bonds, ionic bonds and hydrophobic interactions - about 20 bonds

What is the resulting signal to liver cells? (Include overall outcome and individual protein expression differences.)

Liver cells increase production of energy from amino acids and other small molecules signaled by glucocorticoids. The proteins produced are the enzyme, tyrosine aminotransferase, when hormone is no longer present the production of the proteins decrease down to their normal unstimulated liver cells.

what is a major helix?

Major groove is wider and displays more molecular features than a minor groove. Major groove is made up of nearly all transcription regulators.

Where on the DNA are the patterns different enough to be distinguished? Why?

Major groove is wider and displays more molecular features than the minor groove. Nearly all transcription regulators make majority of contacts with major groove.

what is a minor?

Minor groove is when backbones are close together

Do cells express different genes by altering the nucleotide sequence of their DNA as they differentiate? Describe the experiments used to test this proposal and show that the genomes of differentiated cells have all the information needed to form a new organism.

No, cells do not change the nucleotide sequence of DNA for differentiation. The experiment that explains differentiated cells is the frog experiment. Nucleus of fully differentiated frog cell was injected into a frog egg that had the nucleus removed. The injected nucleus cell can guide the egg to produce a normal tadpole. The cells that make up the tadpole contains differentiated cells that came from the injected nucleus DNA sequence.

Are there features of the gene expression pattern that don't change? Why would this be useful?

No, other features of gene expression pattern do not change and give each cell type its permanently distinctive character. This would be helpful in distinguishing certain genes.

What is nucleosome remodeling? Is it necessary for access to DNA? Why or why not?

Nucleosome remodeling can alter the structure of the nucleosome allowing transcription regulators to access the DNA. Without remodeling regulators can still gain limited access to DNA in a nucleosome because "breathing" occurs at end of nucleosome which exposes the DNA and allows regulators to bind.

How can cooperation among transcription regulators become greater? Why does this occur?

Nucleosome remodeling complexes can cause cooperation of transcription regulators to be greater. If one transcription regulator binds its cis regulatory sequence and attracts the chromatin remodeling complex the localize action of the remodeling complex can allow a second transcription regulator to efficiently bind near by.

Why do nucleosome promote cooperative but not obligatory binding?

Nucleosomes promote cooperative binding because a regulatory protein entering the DNA of nucleosome will prevent the DNA from tightly rewrapping around the nucleosome core. This increases affinity of a second transcription regulator for cis regulatory sequence. Also when two transcription regulators interact with each other that leads to a greater cooperative effect. This can also lead to the displacement of the histone core of nucleosome.

What does it mean that dimer structures form 'obligatorily'? What is the resulting binding curve? When do dimers bind DNA cooperatively instead of obligatorily? What is the resulting binding curve?

Obligatorily means the collection of non covalent bonds that holds the above dimers and heterodimers together. Its extensive because they structures form obligatory and never fall apart. The resulting binding curve for the transcription regulator, which is the fraction of DNA bound as a function of protein concentration, has a standard exponential shape. Dimers bind to DNA cooperatively due to predominately existing as monomers in solution and dimers/heterodimers are held together weakly. The resulting binding curve is sigmoidal in shape.

In order to act as a transcriptional regulator, a protein must bind to a specific point in the DNA. Where on the DNA do these proteins bind?

On the major groove - the double helix is studded with DNA sequence information that transcription regulators recognize.

What happens when the hormone is no longer present? Why do you think this is?

Once the hormone is no longer present, the production of these proteins drops to its normal, unstimulated level in liver cells. This occurs as a negative feedback loop, so once the body reaches the levels or energy it needs, so no longer starving or no intense exercise, the body tells the liver there is no need to produce energy any further.

What is cooperative binding?

Over a range of concentrations of the transcription regulator, binding is more of an all-or-none phenomenon than for noncooperative binding. So in terms of most protein concentrations, the cis-regulatory sequence is either nearly empty or nearly fully occupied and rarely is somewhere between.

Molecular recognition in biology generally relies on what?

Relies on the exact fit between the surfaces of two molecules

How do they "recognize" bases (at what point in the molecules)?

The edge of each base pair presents a distinctive pattern of hydrogen bond donors, acceptors and hydrophobic patches in both major and minor grooves.

What ultimately determines the time and place that each gene is transcribed?

The positions, identity and arrangement of cis-regulatory sequences embedded in genome - is what determines the time and place that a gene is transcribed.

What happens to liver cells exposed to glucocorticoid hormone?

The production of a set of proteins is dramatically increased as well as an enzyme, tyrosine aminotransferase.

What is the second, indirect mechanism for cooperative binding? Why does it arise?

The second, indirect mechanism for cooperative binding is one that arises from the nucleosome structure of eukaryotic chromosomes it occurs because the monomers have only a weak affinity for each other.

What kinds of structural motifs are usually involved in making contact with DNA? Is this surprising?

The structural motifs are a small set of DNA-binding structural motifs and they are made up of alpha helices and beta sheets that bind to major groove. No.

At what level is the control of gene expression able to be exercised?

There are many steps in the pathway for DNA to protein. Any step of this pathway can be regulated so the control of gene expression can be exercised at any level.

Which of these points is most important for the predominance of genes? Why

Transcriptional controls are the most important for the predominance of genes because only transcriptional control ensures that the cell will not synthesize superfluous intermediates.

What additional contributions to DNA-binding specificity must be present? Why?

Transcriptional regulators that form dimers with both monomers making nearly identical contacts with DNA. This doubles the length of the cis-regulatory sequence recognized greatly increases both the affinity and the specificity of transcription regulator binding.

What happens in fat cells exposed to glucocorticoid? What is this an example of?

When a fat cell is exposed to glucocorticoid it reduces the production of tyrosine aminotransferase. This is an example of a general feature of cell specialization, different cell types respond differently to the same extracellular signal.

Does having the DNA sequence of an organism enable us to reconstruct that organism? Why or why not?

Yes, this is due to cell differentiation. An example of using the DNA sequence of an organism to reconstruct that organism was done with a frog. Nucleus of fully differentiated frog cell was injected into a frog egg that had the nucleus removed. The injected nucleus cell can guide the egg to produce a normal tadpole. The cells that make up the tadpole contains differentiated cells that came from the injected nucleus DNA sequence.

What is a nucleosome? Do transcription regulators bind nucleosomes better or worse than naked DNA? Why?

a nucleosome a section of DNA that is wrapped around a core proteins. Transcription regulators bind to DNA in nucleosomes with lower affinity than they do to naked DNA. There are two reasons for this: 1) surface of cis-regulatory sequence recognized by the transcription regulator may be facing inward on the nucleosome, toward the histone core and not be readily available to regulatory protein. 2) if cis- regulatory sequence is exposed on outside of nucleosome, many transcription factors subtly alter their conformation of DNA when they bind and these changes are generally opposed by tight wrapping of DNA around histone core.

Summarize how nucleosomes effect the binding of transcription regulators as depicted in Fig 7-11:

a. When breathing occurs with a nucleosome the cis regulatory sequence opens up a little b. When cis regulatory sequence is located near end of nucleosome transcription regulator binds with 20x less affinity c. Transcription regulator will bind 200 fold less affinity to cis regulatory sequence if it is located in the middle of nucleosome d. One transcription regulator can facilitate binding of another regulator by destabilizing the nucleosome - more likely to get a polymerase

what is zinc finger?

contains one or more zinc atoms as structural component, zinc atom holds an alpha helix and a beta sheet together - found in cluster with the alpha helix of each finger contacting the major groove of DNA forms a continuous stretch of alpha helices along groove

what is heterodimerization?

different monomers formed by different transcription regulators - strength is that same transcriptional factor can be reused, different combinations

what are transcription regulators?

group of proteins - bind to DNA in nucleosomes with lower affinity than they do to naked DNA - proteins recognize specific sequences of DNA (usually 5-10 nucleotide pairs) that are often called cis-regulatory sequences because they are on same chromosome of the genes they control

what is combinatorial control?

mechanism by which relatively small numbers of transcription factors can control the expression of a much larger number of genes with finely tuned temporal and spatial patterns

what is cellular differentiation?

process where cell changes from one cell type to another - cell changes to a specialized type

What is gene expression?

process where instructions in our DNA are converted into a functional product like a protein

what are gene regulatory protein?

small molecules that can make a protein active or inactive by changing its ability to bind DNA - influences regions of DNA molecules

How versatile are these structural motifs in binding various cis-regulatory sequences? Why?

the amino acid side chains that extend from these protein motifs make the specific contacts with DNA. So a given structural motif can be used to recognize many different cis-regulatory sequences depending on specific side chains present.

what is homodimerization?

two of the same monomer - easier to regulate since it's the same protein - can quickly switch it on and off

what is a housekeeping protein?

utilized in genetic machinery of gene expression - proteins involved in basic functioning of a cell or set of cells ie: enzymes involved in synthesis of DNA, RNA, protein