What is the alphabet code for protein molecules? Each of the 20 amino acids are represented by a single letter. What are the groups of amino acids based on their chemical properties and how do amino acids get these on the basis of the R groups? Of the 20 AAs, there are 4 groups: non-polar (hydrophobic), polar (hydrophilic), basic, acidic. What are the parameters that govern protein folding in 3D? Properties of R-groups, bond angles about alpha carbon. What are the primary, secondary, tertiary and quaternary structures of protein? Primary: the sequence of AAs; secondary: fundamental 3D structures such as a helix, b sheets; tertiary: folding into 3D structure into domains; quaternary: 3D arrangement of subunits (i.e. hemoglobin). Properties of a helix right-handed helix with 3.6 AAs per turn; backbone stabilized by H-bonding; further stabilization occurs with H-bonding between 1st and 4th amino acids; many are amphipathic (hydrophilic on one face, hydrophobic on the other). Properties of b sheets pleated sheets formed by two or more amino acid sequences within the same protein that are arranged adjacently and in parallel, but with alternating orientation such that hydrogen bonds can form between the two strands. What role does a turn in the protein structure do? Reverses the direction of the chain. Differentiate a motif, domain and subunit. A motif is a short pattern found in tertiary structures, such as helix-turn-helix. A domain is a basic unit of tertiary structure of a single polypeptide, which may include one or more motifs, and can fold and function independently. A sub-unit is a basic unit of quaternary structure which is a domain interacting with other polypeptides (domains) in the quaternary structure. What are the forces that operate in stabilizing a protein molecule in 3D? Hydrophobic/philic interactions with water; intramolecular H-bonding; intramolecular disulfide bonding (cysteine); interactions with inorganics such as Zn and Fe (cysteine, histidine); resistance of steric hindrance (proline) What are disulfide bonds and how are they different than the other forces in protein folding? S-S bonds formed between cysteine residues. They are covalent bonds, which differ from other forces which are weak bonds. What types of forces operate in the different levels of protein structures. Primary: intermolecular peptide bonds only. Secondary: intramolecular H-bonding; intramolecular disulfide bonding (cysteine); resistance of steric hindrance (proline). Tertiary: Hydrophobic/philic interactions with water; interactions with inorganics such as Zn and Fe (cysteine, histidine); intramolecular disulfide bonding (cysteine); Quaternary: interprotein interactions; interactions with inorganics (hemoglobin). How can phosphorylation of proteins regulate their activity and what residues participate in protein phosphorylation? Addition of phosphate groups can inhibit or stimulate the activities of different enzymes. Serine, Threonine, and Tyrosine are the only AAs that participate. Define enzymes and give their salient properties that make them catalysts. Enzymes are proteins that function as biological catalysts. Enzymes, like all catalysts, are not consumed in a reaction, do not change the ÆG of a reaction, and do lower the activation energy of a reaction. Enzymes are able to bind substrates at their active site such that orientation of the reactants is optimized to produce the product. Enzymes may also change the conformation of itself or substrates to facilitate production. What do you understand by the terms active site, substrate-binding site, catalytic site and allosteric site on an enzyme? A substrate-binding site on an enzyme is the location where the enzyme and substrate bond non-covalently. The active site is the location where the catalytic activity occurs. An allosteric site is a regulatory location that alters the enzymes activity when bound by an effector. What are feedback regulation and allosteric regulation? Feedback regulation occurs when the product of a reaction serves as an inhibitor of further production, typically by being able to bind to an enzyme early in its metabolic pathway. Allosteric regulation occurs when an effector (which is not a product) binds to an enzyme and changes its activity. What is the velocity of the enzyme catalysis reaction and what is the formula for it? v = dB/dt = k1 * [A] What is the Michaelis-Menton constant? What does it measure and what does the number associated with Km tell about how the enzyme will function? The M-M constant, Km, is the substrate concentration at 1/2 Vmax. Km measures the specificity of the enzyme. How are Vmax and Km affected by concentrations of the substrate and the enzyme. Vmax and Km are not affected by [E]. Vmax and Km increase as [S] increases. What is the primary step at which gene expression is regulated? Initiation of transcription. What are the unique features of prokaryotic gene expression? Coupled (simultaneous) transcription and translation; Genes can be organized in operons; A single RNA polymerase which does not require transcription factors. How does the strategy of using an operon help the prokaryotic cell in switching on a pathway? A single operator can control whether mRNAs for entire groups of related proteins are transcribed. What is a promoter? A region of DNA preceding a gene which can bind to the s subunit of RNA Pol and initiate transcription. What are the consensus sequences at the promoters called? Prokaryotes have consensus sequences at positions Ð10 (TATAAT) and Ð35 (TTGACA) relative to the start of the gene. How is a strong promoter different than a weak promoter? A strong promoter has sequences close to the consensus sequences. Strong promoters bind better to the holoenzyme. Strong promoters initiate more often, and have greater expression. Compare repressors and activators. Both are transcription factors, but repressors block transcription, while activators allow transcription to proceed. Explain induction and repression of the lac operon. A repressor enzyme will bind the operator of the lac operon when lactose is not present, blocking transcription of the lac operon. When lactose is present, it binds the repressor, which causes the repressor to change its conformation such that it can no longer bind the operator, which in turn allows for induction of transcription of the lac operon. When glucose is low, CAP and cAMP bind and activate the lac operon, which induces transcription. How is the lac operon a classical example of negative control of transcription regulation? The lac operon can be repressed to block its transcription, so the lac operon is an example of negative control. How is the trp operon regulation different than lac even though it still uses a repressor that exerts negative control of regulation? The trp operon is active by default and repressed (negatively controlled) when trp is abundant, whereas lac is repressed by default (negatively controlled) and active when lac is scarce. What is an apo-repressor and how does it get activated? It is a repressor that needs to be modified by another molecule before it will repress a gene. How does the lac operon serve as an example of both negative and positive regulation? By repressing when lactose is abundant, it is negatively controlled. When glucose is low and CAP and cAMP serve as an activator, it is positively controlled. What is catabolite repression? Explain its mechanism so that it explains when cells are in glucose containing media there is minimal use of other sugars as sources of carbon and energy. This allows the cell to optimize metabolism by choosing a better source of energy, such as glucose in preference to lactose. When glucose is plentiful, the cell will catabolize glucose. When glucose is scarce, it can choose to try to use any available sugar (i.e. lactose). When glucose is scarce, CAP and cAMP bind and activate the lac operon, so that lactose can be catabolized, but when glucose is available, cAMP is not formed, so lac is not activated and the cell uses only glucose. How do DNA binding proteins interact with DNA? Typically one portion of the protein binds non-covalently to DNA, while the remaining portions serve to stabilize the protein and/or DNA complex. What are the different types of motifs used in these interactions? Helix-turn-helix, helix-loop-helix, zinc fingers, leucine zippers, Beta-sheets, and Beta-hairpins are examples. What are enhancers and how can they work when they are far away from the gene that they are regulating? Enhancers are DNA sequences distinct from the promoter that can bind other proteins which then assist in the transcription activation process. Enhancers can be upstream or downstream of the transcription start site, and can be separated by long distances because they can use DNA looping to get the bound transcription factor near the transcription site. Compare rho-dependent and rho-independent termination of transcription. Rho-dependent termination is found only in prokaryotes, which uses the rho protein to bind past the end of the completed mRNA and cause RNA Pol to dissociate. Rho-independent termination relies upon the formation of hairpin loop structures via complementary base-pairing after the end of the gene, which causes the polymerase to dissociate. What is attenuation? Can you give an example of an operon that undergoes regulation through attenuation? Attenuation is conditional transcription termination. If an amino acid gene contains codons for itself, it will cause a coupled translating ribosome to stall if not enough of the amino acid is already present to form its tRNA. When this stall occurs, the remaining mRNA can form a conformation such that a rho-independent terminator is masked and the transcription and translation continue. If the amino acid is abundant, the ribosome will not stall and an alternate conformation of the mRNA forms which contains a rho-independent terminator, causing RNA Pol to dissociate prematurely and cease wasteful transcription and translation. Trp is an example operon. What are the different RNA polymerases in eukaryotes and how do they share the synthesis of different types of RNAs amongst themselves. Eukaryotes have RNA Pol I, II, and III . RNA Pol I is responsible for most rRNA transcription and does so in the nucleolus, but RNA Pol III transcribes the 5S subunit in a separate area of the nucleoplasm, which is then assembled with the other subunits outside the nucleus. What roles do RNA polymerase I and III do in the process of transcription? Pol I is responsible for most rRNA transcription, while Pol III is responsible for the small RNAs (snRNAs, scRNAs, etc.), tRNAs, and one subunit of rRNA. What is transcription initiation complex? The complex of RNA Polymerase, transcription factors, and mediator. What do you understand by basal transcription factors and tissue specific factors? Basal transcription factors are the minimal set of factors required for transcription to proceed. In the case of mRNA transcripts, these are TFIID (TBP, TAFs), TFIIB, TFIIF, RNA Pol II, TFIIE, and TFIIH. As for tissue-specific factorsÉ? What are the two distinct domains in euk transcription factors and what are their functions? DNA binding domain is responsible for recognizing the correct sequence of DNA and attaching to it. Activation domains are responsible for binding to the RNA Polymerase or other factors so that transcription can proceed. Describe in brief what you understand by helix-turn-helix, zinc fingers, leucine zipper and helix-loop-helix motifs that interact with DNA. They are common motifs that occur in DNA binding proteins, whose physical conformation enables different types of binding strategy. Describe in brief pol I and pol III mediated transcription of tRNA and small RNA molecules. Both are similar to Pol III in that a pre-initiation complex is required with the polymerase, but the complex is simpler and differs in the types of factors present. For Pol I, SL1 (Selectivity Factor 1) is a combination of TBP and TAFs is used, but is different from TFIID in that a TATA box is not required. Pol I also uses Upstream Control Elements to recruit Upstream Binding Factors that loop the DNA back towards the promoter and bind the loop to the complex. Pol III uses TBP, along with its own set of transcription factors (TFIIIA, B, C). What are the different types of modifications and processing events RNA molecules go through? Pre-mRNAs undergo capping and polyadenylation. RNAs are also filled with introns which must be edited out before translation; snRNPs are responsible for this activity. Finally, alternative and differential splicing means that finished RNAs may have their exons spliced together in different combinations to produce different RNAs and thus different end products. What is the process of capping? A 7-methylguanosine cap is added to the beginning of the mRNA, which is important since ribosomes will not recognize an mRNA without it. How do eukaryotic mRNAs get their appropriate 3Õ ends? The pre-mRNA is also polyadenylated, which adds a large number of adenosines to the tail, which stabilizes the pre-mRNA and increases its half-life by providing additional protection from exonucleases. What is splicing? Splicing is the removal of introns (RNA that isnÕt used for formation of the product) and ligating the exons (RNA that is used) back together to form a finished RNA. It is essentially post-transcriptional editing of the RNA to remove junk RNA and properly order useable RNA. How are the 5Õ and 3Õ ends of the exons brought together? The spliceosome is able to hold on to two exons at once, and after it removes an intervening intron, it can splice the two exons together. Describe in detail the formation of the spliceosome. Formed by complex of snRNPs. U1 snRNP binds to 5Õ starting splice site (5Õ-GUÉ-3Õ) at start of intron. U2 binds to branch point (5Õ-ÉAÉ-3Õ) within intron. U4/U5/U6 associate to form complex. Splicing commences, and a lariat loop is formed by binding the starting site to the branch point. The complex then recognizes the 3Õ ending splice site (5Õ-ÉAG-3Õ) at the end of the intron and excises the intron, and ligates the two intervening exons. What is differential and alternate splicing? Multiple different proteins can be produced from a single mRNA transcript by splicing the exons together in different patterns. What are the half-lives of prokaryotic and eukaryotic RNA molecules? Prok mRNAs have a half-life from 2 to 3 minutes. Euk mRNAs have a half-life from 30 minutes to 20 hours. How does RNA degradation affect gene expression? By degrading mRNAs faster, gene expression is reduced since the mRNA wonÕt be around as long for translation. What is the code for proteins? It is a 3-letter code, where each 3-letter sequence (codon) codes for one of the 20 amino acids. What is degeneracy of the code? Since there are 4^3 = 64 permutations for codons and only 20 amino acids to code for, the fact that multiple codons code for an amino acid is considered to be a degenerate code. What are stop codons? These codons (UGA, UAA, UAG) do not have corresponding tRNAs and serve as a signal to the translating ribosome that it should stop translation. Briefly describe Peptidyl tRNA, aminoacyl tRNA, peptidyl transferase. Peptidyl tRNA: a tRNA in the P site with the growing polypeptide chain attached to it. Aminoacyl tRNA: a ÒchargedÓ tRNA with the amino acid it codes for attached to the 3Õ CCA attachment site. Peptidyl Transferase: The portion of the ribosome responsible for enzymatically forming the peptide bond between the amino acid in the A site to the polypeptide chain in the P site. What are the roles of the tRNA molecules in the process of translation? tRNAs are responsible for bringing amino acids coded by the mRNA to the ribosome for addition to the polypeptide chain. What are the E, P, A sites on the ribosome? The A (aminoacyl) site is where a charged tRNA is bound by the ribosome. The amino acid from the charged tRNA is then transferred to the polypeptide chain by the ribosome. The P (peptidyl) site is the location where the growing polypeptide chain is kept. The E (exit) site is where the last used tRNA with the polypeptide chain moves to upon sensing a stop codon. The chain is then terminated and the finished polypeptide is released from the ribosome. Briefly review the structure of the tRNA molecule. A tRNA has a secondary structure similar to a cloverleaf, whose loops are formed by complementary base-pairing. At the bottom loop, a 3-letter anti-codon is present which specifies which amino acid to bind at the top, whose 3Õ end has the sequence 5Õ-CCA-3Õ appended to it. This sequence is able to bind to the amino acid. What are the two features of the tRNA molecules that determine the accuracy of the protein synthesis? The bottom loopÕs anti-codon sequence, and the 3Õ end which binds to the amino acid. Charging the tRNA with the correct amino acid, and proper pairing of the correct tRNAÕs anti-codon on the mRNA codon. What is amino acylation of tRNA molecules? ÒChargingÓ a tRNA so it binds to its respective amino acid. What is the enzyme that brings about this process? Is there any specificity to this? Recall how a tRNA molecule is loaded w/a acid. Each amino acid has its own tRNA which has its own aminoacyl synthetase. What is the wobble hypothesis? That the third position of a tRNAÕs anti-codon does not necessarily adhere to strict Watson & Crick base-pairing, and as a result can serve as a decoder for multiple codons. The third position may be a non-W&C base such as inosine which can base-pair with several bases (A, C, U), or it can show relaxed W&C rules (G-U) by having G and U pair with extra bases (G-C, G-U; U-A, U-G). How does wobble pairing protect from chaining the amino acid sequence in a protein from minor errors? It can still match up the correct amino acid if there is an error in the 3rd position. How does wobble pairing help the charging of tRNAs with Inosine residues in the anticodon loop? Since inosine can pair with 3 bases (A, C, U), the tRNA does not need to be precise and still end up with the correct amino acid bound. What are the unique features of prokaryotic translation? It can be coupled with transcription. Proks have the Shine-Dalgarno sequence in their mRNAs, which help align the ribosome in preparation for translation. Entire operons can be translated at once, since mRNAs are polycistronic. Proks do not require a 5Õ cap to begin translation. Ribosomes are slightly smaller than their euk counterparts. The methionine resulting from the start codon is formylated. What is SD sequence and how does that permit the translation of polycistronic mRNA molecules? The Shine-Dalgarno sequence in the UTR region of mRNAs helps align the ribosome in preparation for translation. It allows internal initiation Ð no 5Õ cap is required. With multiple translations possible, a SD sequence allows a ribosome to position itself next to a true AUG codon and not start translating from a random 5Õ-AUG-3Õ sequence occurring in the transcript. In euks, a 5Õ cap is required, so polycistronic mRNAs are not possible. How is prokaryotic translation initiated? The ribosome binds to the SD sequence with complementary base pairing, which allows it to find the AUG codon nearby. What is the role of the 5Õ cap structure in eukaryotic translation? In euks, the 5Õ 7-methylguanosine cap is required for the ribosome to recognize the mRNA.