Problem Set 4

What is a riboswitch? What is the function of the three domains?

- A riboswitch is a secondary structural element in mRNA (usually in the 5' untranslated region) that binds ligands to regulate mRNA translation. - The Aptamer Domain: binds the ligand - The Switching Sequence: changes secondary structure upon ligand binding and spans regions of both the aptamer domain and the expression platform domain - The Expression Platform: protein-encoding domain

What is a ribozyme? What are some common activities of ribozymes?

- A ribozyme is a ribonucleic acid chain, often with complex secondary structure, with the enzyme-like ability to catalyze reactions - The most common activities of ribozymes include cleavage and ligation of RNA and DNA, linkage of amino acids during protein synthesis, other RNA processing reactions such as splicing and tRNA biosynthesis

What are the primary domains in a helix-loop-helix transcription factor? Which helix binds DNA and how does it contact the DNA strand?

- As their name indicates, helix-loop-helix (HLH) transcription factors are composed of two alpha helices separated by a loop - The HLH monomers form dimers with one another, with the loops extended from each side like wings - When HLH transcription factors bind DNA, the H1 helices partially dissociate from one another and instead form a rigid structure that binds E-box response elements in the major groove on opposite sides of the DNA helix

Briefly describe the structure of DNA. What interactions stabilize this structure?

- B DNA is the standard conformation of the DNA double helix within the cell - In B DNA there are 10 nucleotide base pairs per turn, with a length of 3.4 Angstroms per pair and 34 Angstroms per turn - The nitrogenous bases are arranged inside the double helix (like stairs in a staircase), and because they are planar they stack closely together with hydrophobic interactions occurring between each base above and below - This hydrophobic stacking is a major stabilizing force of DNA structure - Hydrogen bonds between A and T (2 H bonds) and G and C nitrogenous bases (3 H bonds) link the two anti-parallel nucleic acid chains together, with the hydrophilic and negatively charged pentose-phosphate backbone (like the stair rail) exposed.

What is the function of Cro and lambda repressor?

- Cro functions as a switch along with lambda repressor to control the transition between lytic and lysogenic growth in phage - Genes encoding Cro and λ repressor are located on opposite sides of a region in the prophage called the operator (OR) region - If you transcribe right from the OR, Cro is made and lytic growth is initiated/ If you transcribe left, λ repressor is made and lysogenic growth continues - Both bind to the operator region (OR), with Cro binding to the left side and lambda repressor binding to the right. - If Cro is present, it blocks the λ repressor promoter, and if λ repressor is present, it blocks the Cro promoter. - RNA polymerase transcribes from the OR region towards the gene that is not blocked. - So, both Cro and lambda repressor proteins positively regulate transcription of their own gene and block transcription of the other

Describe the structure of Cro and by what mechanism Cro binds DNA

- Cro is a helix-turn-helix transcription factor that dimerizes so that there are two DNA binding regions 34 angstroms apart - The alpha 2 and alpha 3 helices of each of these monomers contact successive major grooves of DNA, with the alpha 3 helix, also known as the recognition helix sitting directly in the groove

What are the two primary domains in Leucine zipper (bZIP) transcription factors and how do they function? What parts of the DNA strand do leucine zipper transcription factors contact?

- DNA binding or basic region: binds DNA - The zipper region: mediates dimerization with a second leucine zipper monomer - Together the two halves of the leucine zipper dimer grip the DNA like chopsticks, binding to successive major grooves on opposite sides of the DNA helix

What are 3 common non-enzymatic reactions that can occur to nucleotides in a nucleic acid chain?

- Deamination: most commonly occurs to cytosine, converting it to uracil, but can also occur to adenine or guanine. Results in altered base pairing and can cause a permanent mutation in the DNA code if not repaired - Depurinations: loss of purine bases through hydrolysis (can also occur with pyrimidines, but much more slowly) - results in an abasic site in which the coding information is lost and can further destabilize the backbone because the furanose can open up to the linear aldehyde form of the pentose sugar - Thymine dimers: occur when UV light catalyzes formation of a cyclobutyl ring between adjacent pyrimidines

List the RNA functions that are facilitated by secondary structure

- Enables translation through formation of tRNAs - Controls whether non-standard amino acids are incorporated into a protein (e.g. 3' SECIS and selenocysteine incorporation) - Contributes to gene silencing through recognition and maturation of precursor miRNAs by DICER1 - Regulates whether an mRNA transcript is translated (i.e. riboswitches) - Generates conformations that enable catalytic activity (i.e. ribozymes) - Regulates pre-mRNA processing (i.e. intron secondary structure can bring splice sites together)

For Guanine, describe the structure of the "sensor" molecules that senses this metabolite, what the molecular function of the sensor is, and how the binding of guanine alters sensor function to adjust production of regulated enzymes

- Guanine is sensed by a riboswitch, which is an mRNA that binds ligands using an aptamer domain and then changes secondary structure of the switching domain to regulate whether the expression platform is accessible - The guanine riboswitch is present in the 5' end of mRNAs encoding purine biosynthetic enzymes. - When purine levels in the cell are high, then guanine is present to bind the aptamer domain of the riboswitch, this results in a secondary structural change that makes the expression platform unable to interact with the translational machinery - By regulating the purines biosynthetic enzyme levels at the translational level, bacterial cells can ensure that they aren't making purines when there are already enough present in the cell - However, if purine levels drop, the disassociation of guanine from the riboswitch leaves transcripts immediately accessible for translation to quickly address the need to make more

What are two common secondary structural motifs that occur in nucleic acid chains? How are they formed?

- Hairpins: occurs when a self-complementary palindromic repeat base pairs with itself to form a stem with a loop at the end - Cruciform: occurs when there are two complementary strands of a nucleic acid chain that form hairpins opposite one another to make a cross-shaped secondary structure

What structural family do homeobox transcription factors belong to? What part of the DNA is bound by homeobox transcription factors? What DNA residue motif is bound with very high affinity by homeobox transcription factors?

- Homeobox transcription factors are in the helix-turn-helix family - The alpha 3 (recognition) helix binds to the major groove of DNA, and arginines in the amino terminus tail bind to the minor groove - Stabilization occurs through several salt bridges and hydrogen bonds between the DNA backbone and HOX protein side chains - The alpha 3 helix binds to the sequence 5'-ATTA-3' with very high affinity

For Lactose, describe the structure of the "sensor" molecules that senses this metabolite, what the molecular function of the sensor is, and how the binding of guanine alters sensor function to adjust production of regulated enzymes

- Lactose is also sensed by a DNA-binding repressor protein - This protein occurs as two homodimers that associated with one another to form a tetramer. At the top of each dimer are two helix-turn helix domains that bind DNA through association with two successive major grooves in the promoter of the Lac operon. There are two additional helices, the hinge helices, which contact the minor groove. - The center of each Lac repressor monomer is a lactose-binding domain, and the tail of each monomer contains a helix that interacts with the helices from other monomers to form the tetramer. - Together, the two homodimers of the lac repressor bind to two separate regions of the lac operon promoter - The DNA in between these two binding sites becomes deformed into a hairpin that prevents association of the transcriptional machinery, and prevents the operon from being transcribed - The lac operon encodes genes that break down lactose to use as an energy source, and so it is beneficial to express these genes when lactose is present - To facilitate this, the lac repressor senses lactose through binding to the lactose binding domain. - When lactose is present and binds to this domain, there is an allosteric change that causes the helix-turn- helix binding domains to no longer be positioned to properly contact the DNA. - This results in the lac repressor falling off of the DNA and the hairpin structure reverts to normal B structure, allowing the transcriptional machinery to transcribe the genes encoding enzymes to break down lactose

For Tryptophan, describe the structure of the "sensor" molecules that senses this metabolite, what the molecular function of the sensor is, and how the binding of guanine alters sensor function to adjust production of regulated enzymes

- Like purines, tryptophan is metabolically expensive to make, and so bacteria don't make it unless they absolutely need to - Tryptophan is sensed by a DNA-binding protein repressor (the Trp repressor) that binds tryptophan - The Trp repressor is a dimer containing two helix-turn-helix domains that bind to DNA through association of the fifth alpha helix from each monomer with two successive major grooves of DNA located in the promoter for an operon encoding tryptophan biosynthetic enzymes. - The Trp repressor by itself is unable to bind DNA, because the two DNA binding domains are oriented at the wrong angle and too close to one another to span the two major grooves of DNA. This means that when there is no tryptophan present, Trp repressor can't bind to the operon encoding tryptophan biosynthetic genes, and so these genes are automatically transcribed so that the cell can make tryptophan - However, when there is tryptophan present, it binds to the Trp repressor and causes an allosteric change in structure. This change results in the two helix-turn-helix domains being oriented at just the right angle and distance to bind to the two major grooves of the Trp operon. Binding of Trp repressor to the promoter keeps these genes from being transcribed, thereby preventing the cell from making enzymes to synthesize tryptophan when it's not necessary

Mutations in certain arginine residues of p53 promote tumor formation. What are the functions of these arginine residues in p53? Why does mutating them promote cancers?

- Mutations associated with cancer occur in arginine residues of the p53 DNA binding domain, including Loops 2 and 3 and the SLH domain. - Arginine residues of p53 are involved in sequence-specific binding of nucleotides in the DNA major groove, binding sugar residues in backbone the minor groove, and stabilization of loop structure through coordination of the zinc ion - Depending on their location, mutations of Arg residues can cause an overall loss of stability of loop structure in the DNA binding region and compromised binding to the sugar residues or nucleotide bases, thereby disrupting p53's function in suppressing tumors

What is a zinc finger? What are the functions of the two zinc fingers in the glucocorticoid receptor? Briefly describe how the glucocorticoid receptor contacts DNA

- One zinc is bound by four Cys residues to stabilize the loop structure that properly positions the DNA binding alpha helix (recognition helix) of the glucocorticoid receptor. A second zinc stabilizes the structure of the dimerization loop which enables homodimerization of glucocorticoid receptors for DNA binding - The recognition helices of the two monomers bind to the glucocorticoid response element, interacting with successive major grooves on the same side of the DNA helix

RNA is less stable than DNA. Briefly describe the difference in chemistry underlying this instability.

- RNA is less stable than DNA because the hydroxyl group on the C2' position of the ribose acts as a nucleophile in a displacement reaction with an adjacent phosphate group and hydrolyzes the RNA backbone to yield a cyclic nucleotide and a shortened RNA chain - Because deoxyribose in DNA is missing this nucleophilic hydroxyl group, it is a more stable structure

What enzyme edits mRNA to convert adenosine bases to inosine? Briefly explain how incorporation of inosine into the coding region of an mRNA can change the acid sequence of the protein it is translated into

- The ADAR (Adenosine deaminase acting on RNA) enzyme deaminate adenosine to make inosine - Inosine can base pair with uracil, adenosine or cytosine, so when it is edited into RNA, a site that could previously only base-pair with uracil, can now base-pair with the other two bases as well - In the coding region of a transcript, this changes the codon at that site, so there is more than one tRNA that is complementary. - For instance, a change of AAC (encoding asparagine) converted to AIC could also be read to encode isoleucine or serine

What is the structure of the DNA binding domain of the TATA box binding protein and how does it interact with DNA? What molecular events does binding of the TATA box binding protein promote?

- The DNA binding domain of TATA box binding protein (TBP) is a 10 stranded beta sheet that forms a saddle shape. - Binding of TBP to the TATA box causes a deformation of DNA across the saddle shaped surface and acts as a scaffold for the recruitment of transcriptional machinery to initiate transcription of the upstream gene

Why is an alpha helix so commonly the structural motif in transcription factors that physically interacts with DNA?

- The diameter of the alpha helix is about 12 Angstroms - This width is just the right size to fit snugly into the groove of the major helix of DNA - Sidechains of bases on the side of the helix interacting with the DNA major groove are able to make hydrogen bonds with specific nucleotide bases