Bio 329- Exam 2

What is the difference between genetic and epigenetic variation? Name one important form of epigenetic variation based on a histone modification.

. Genetic variation results from change in the DNA sequence of the genome. Epigenetic variation results from modification of chromatin state. One example of an epigenetic modification is methylation of lysine 9 on histone H3.

How do transcriptional activators and repressors differ in terms of their effect on gene expression?

Activators increase the transcription of the genes that they regulate, while repressors reduce transcription.

What are the four levels at which gene expression may be regulated in eukaryotic cells?

Gene expression can be regulated at the levels of transcription, mRNA processing, translation, and post-translational control.

How does the small ribosomal subunit know where to start initiation of translation in prokaryotes? How does this step differ in eukaryotes?

In prokaryotes, the small ribosomal subunit recognizes where to initiate translation by binding to the Shine-Dalgarno sequence, which is found upstream of the start codon. This binding is mediated by base pairing of the ribosomal RNA sequence with the Shine-Dalgarno sequence. In eukaryotic translation, the small subunit assembles as part of a 43S complex which associates with the 5' cap of the message. The complex then scans along the message until it finds a start codon in an appropriate sequence context (e.g. Kozak's sequence).

What are the general steps in the processing of a pre-mRNA into an mRNA? What is the role of the snRNAs and the spliceosome?

RNA polymerase II assembles a primary transcript that is complementary to the DNA of the entire transcription unit. Electron microscopic examination of transcriptionally active genes indicates that RNA transcripts become associated with proteins and larger particles while they are still in the process of being synthesized. These particles, which consist of proteins and ribonucleoproteins, include the agents responsible for converting the primary transcript into a mature messenger. This conversion process requires addition of a 5' cap and 3' poly(A) tail to the ends of the transcript, and removal of any intervening introns. Once processing is completed, the mRNP, which consists of mRNA and associated proteins, is ready for export from the nucleus. The 5' ends of all RNAs initially possess a triphosphate derived from the first nucleoside triphosphate incorporated at the site of initiation of RNA synthesis. Once the 5' end of an mRNA precursor has been synthesized, several enzyme activities act on this end of the molecule. In the first step, the last of the three phosphates is removed, converting the 5' terminus to a diphosphate. Then, a GMP is added in an inverted orientation so that the 5' end of the guanosine is facing the 5' end of the RNA chain. As a result, the first two nucleosides are joined by an unusual 5'-5' triphosphate bridge. Finally, the terminal, inverted guanosine is methylated at the 7-position on its guanine base, while the nucleotide on the internal side of the triphosphate bridge is methylated at the 2' position of the ribose. The 5' end of the RNA now contains a methylguanosine cap. The methylguanosine cap at the 5' end of an mRNA serves several functions: it prevents the 5' end of the mRNA from being digested by exonucleases, it aids in transport of the mRNA out of the nucleus, and it plays an important role in the initiation of mRNA translation. The 3' end of an mRNA contains a string of adenosine residues that forms a poly(A) tail. As a number of mRNAs were sequenced, it became evident that the poly(A) tail typically begins 10 to 30 nucleotides downstream from the sequence AAUAAA. This sequence in the primary transcript serves as a recognition site for the assembly of a large complex of proteins that carry out the processing reactions at the 3' end of the mRNA. The poly(A) processing complex is also physically associated with the phosphorylated CTD of RNA polymerase II as it synthesizes the primary transcript. Included among the proteins of the processing complex is an endonuclease that cleaves the pre-mRNA downstream from the 6 recognition site. Following cleavage by the nuclease, an enzyme called poly(A) polymerase adds 250 or so adenosines without the need of a template. The poly(A) tail together with an associated protein protects the mRNA from premature degradation by exonucleases. In addition to formation of the 5' cap and poly(A) tail, those parts of a primary transcript that correspond to the intervening DNA sequences (the introns) must be removed by a complex process known as RNA splicing. To splice an RNA molecule, breaks in the strand must be introduced at the 5' and 3' ends (the splice sites) of each intron, and the exons situated on either side of the splice sites must be covalently joined (ligated). It is imperative that the splicing process occur with absolute precision, because the addition or loss of a single nucleotide at any of the splice junctions would cause the resulting mRNA to be mistranslated. The pre-mRNA is not able to splice itself; it requires a host of small nuclear RNAs (snRNAs) and their associated proteins. As each large hnRNA molecule is transcribed, it becomes associated with a variety of proteins to form an hnRNP (heterogeneous nuclear ribonucleoprotein), which represents the substrate for the processing reactions that follow. Processing occurs as each intron of the pre-mRNA (hnRNA) becomes associated with a dynamic macromolecular machine called a spliceosome. Each spliceosome consists of a variety of proteins and a number of distinct ribonucleoprotein particles, called snRNPs because they are composed of snRNAs bound to specific proteins. Spliceosomes are not present within the nucleus in a prefabricated state, but rather they are assembled as their component snRNPs bind to the pre-mRNA. Once the spliceosome machinery is assembled, the snRNPs carry out the reactions that cut the introns out of the transcript and paste the ends of the exons together. The excised introns, which constitute roughly 90 percent of the average mammalian pre-mRNA, are simply degraded within the nucleus. Taken together, removal of an intron requires several snRNP particles: the U1 snRNP, U2 snRNP, U5 snRNP, and the U4/U6 snRNP, which contains the U4 and U6 snRNAs bound together. In addition to its snRNA, each snRNP contains a dozen or more proteins.

Which initiation system, prokaryotic or eukaryotic, involves scanning?

Scanning is a feature of eukaryotic translation. In prokaryotes, the small ribosomal subunit binds directly to the Shine-Dalgarno sequence and does not need to scan.

Describe the nature of the interaction between tRNAs and aminoacyl-tRNA synthetases. Describe the nature of the interaction between tRNAs and mRNAs. What is the wobble hypothesis?

) It is critically important during polypeptide synthesis that each transfer RNA molecule be attached to the cognate amino acid. Amino acids are covalently linked to the 3' ends of their cognate tRNA(s) by an enzyme called an aminoacyl-tRNA synthetase (aaRS). Although there are many exceptions, organisms typically contain 20 different aminoacyl-tRNA synthetases, one for each of the 20 amino acids incorporated into proteins. Each of the synthetases is capable of "charging" all of the tRNAs that are appropriate for that amino acid (i.e., any tRNA whose anticodon recognizes one of the various codons specifying that amino acid). Aminoacyl-tRNA synthetases provide an excellent example of the specificity of protein-nucleic acid interactions. Certain common features must exist among all tRNA species specifying a given amino acid to allow a single aminoacyl-tRNA synthetase to recognize all of these tRNAs while, at the same time, discriminating against all of the tRNAs for other amino acids. Information concerning the structural features of tRNAs that cause them to be selected or rejected as substrates has come primarily from two sources: I. Determination of the three-dimensional structure of these enzymes by X-ray crystallography, which allows investigators to identify which sites on the tRNA make direct contact with the protein. The two ends of the tRNA (the acceptor stem and anticodon)) are particularly important for recognition by most of these enzymes. II. Determination of the changes in a tRNA that cause the molecule to be aminoacylated by a non-cognate synthetase. It is found, for example, that a specific base pair in a tRNAAla (the G-U base pair involving the third G from the 5' end of the molecule) is the primary determinant of its interaction with the alanyl-tRNA synthetase. Insertion of this specific base pair into the acceptor stem of a tRNAPhe or a tRNACys is sufficient to cause these tRNAs to be recognized by alanyl-tRNA synthetase and to be aminoacylated with alanine. Aminoacyl-tRNA synthetases carry out the following two-step reaction: In the first step, the energy of ATP activates the amino acid by formation of an adenylated amino acid, which is bound to the enzyme. This is the primary energy-requiring step in the chemical reactions leading to polypeptide synthesis. Subsequent events, such as the transfer of the amino acid to the tRNA molecule in step 2, and eventually to the growing polypeptide chain, are thermodynamically favorable. The PPi produced in the first reaction is subsequently hydrolyzed to Pi, further driving the overall reaction toward the formation of products. In the second step, the enzyme transfers its bound amino acid to the 3' end of a cognate tRNA. Among the 20 amino acids incorporated into proteins, some are quite similar structurally to others. The synthetases that catalyze reactions dealing with these "similar-looking" amino acids typically possess a second editing site in addition to the catalytic site. If the synthetase reaction happens to place an inappropriate amino acid on a tRNA in the catalytic site, a proofreading mechanism of the enzyme is activated and the bond between the amino acid and tRNA is severed in the editing site. Given the fact that 61 different codons can specify an amino acid, a cell might be expected to have at least 61 different tRNAs, each with a different anticodon complementary to one of the codons. But the greatest similarities among codons that specify the same amino acid occur in the first two nucleotides of the triplet, whereas the greatest variability in these same codons occurs in the third nucleotide of the triplet. Consider the 16 codons ending in U. In every case, if the U is changed to a C, the same amino acid is specified. Similarly, in most cases, a switch between an A and a G at the third site is also without effect on amino acid determination. The interchangeability of the base of the third position led Francis Crick to propose that the same transfer RNA may be able to recognize more than one codon. Crick's proposal that the same transfer RNA may be able to recognize more than one codon was termed the wobble hypothesis and suggested that the steric requirement between the anticodon of the tRNA and the codon of the mRNA is very strict for the first two positions but is more flexible at the third position. As a result, two codons that specify the same amino acid and differ only at the third position should use the same tRNA in protein synthesis. Once again, a Crick hypothesis proved to be correct. The rules governing the wobble at the third position of the codon are as follows: U of the anticodon can pair with A or G of the mRNA; G of the anticodon can pair with U or C of the mRNA; and I (inosine, which is derived from guanine in the original tRNA molecule) of the anticodon can pair with U, C, or A of the mRNA. As a result of the wobble, the six codons for leucine, for example, require only three tRNAs.

Explain what is meant by stating that the genetic code is triplet and non-overlapping? What did the finding that DNA base compositions varied greatly among different organisms suggest about the genetic code? How was the identity of the UUU codon established?

) One of the first models of the genetic code was presented by the physicist George Gamow, who proposed that each amino acid in a polypeptide was encoded by three sequential nucleotides. In other words, the code words, or codons, for amino acids were nucleotide triplets. He reasoned that it would require at least three nucleotides for each amino acid to have its own unique codon. If one considers the number of words that can be spelled using an alphabet containing four different letters corresponding to the four possible bases that can be present at a particular site in DNA (or mRNA), there are 4 possible one-letter words, 16 (42) possible two-letter words, and 64 (43) possible three-letter words. Because there are 20 different amino acids (words) that have to be specified, codons must contain at least 3 successive nucleotides (letters). In a non-overlapping code, each nucleotide along the mRNA would be part of one, and only one, codon. Crick summarized his ideas about the genetic code in a paper entitled "On Protein Synthesis" and presented it at University College London in September 1957. The paper, according to the historian Horace Judson, "permanently altered the logic of biology." In it Crick proposed the "sequence hypothesis," which held that genetic information was encoded in the sequence of the bases in DNA. Francis Crick predicted that the genetic code was degenerate on theoretical grounds when he considered the great range in base composition among the DNAs of various bacteria. A degenerate genetic code is one in which at least some of the amino acids would be specified by more than one codon. He came to this conclusion because it had been found that the G + C content of the DNA of various organisms could range from 20 percent to 74 percent of the genome, whereas the amino acid composition of the proteins from these organisms showed little overall variation. This suggested that the same amino acids were being encoded by different base sequences, which would make the code degenerate. Given a triplet code that can specify 64 different amino acids and the reality that there are only 20 amino acids to be specified, the question arises as to the function of the extra 44 triplets. If any or all of these 44 triplets code for amino acids, then the code would be degenerate. As it turns out, the code is highly degenerate, as nearly all of the 64 possible codons specify amino acids. Those that do not (3 of the 64) have a special "punctuation" function— they are recognized by the ribosome as termination codons and cause reading of the message to stop. By 1961, the general properties of the code were known, but not one of the coding assignments of the specific triplets had been discovered. At the time, most geneticists thought it would take many years to decipher the entire code. But a breakthrough was made by Marshall Nirenberg and Heinrich Matthaei who used an enzyme polynucleotide phosphorylase to synthesize their own artificial genetic messages and then determine what kind of protein they encoded. The first message they tested was a polyribonucleotide consisting exclusively of uridine; the message was called poly(U). When poly(U) was added to a test tube containing a bacterial extract with all 20 amino acids and the materials necessary for protein synthesis (ribosomes and various soluble factors), the system followed the artificial messenger's instructions and manufactured a polypeptide. The assembled polypeptide was analyzed and found to be polyphenylalanine - a polymer of the amino acid phenylalanine. Nirenberg and Matthaei had thus shown that the codon UUU specifies phenylalanine.

What does it mean that replication is semiconservative? How was this feature of replication demonstrated in bacterial cells? In eukaryotic cells?

The Watson-Crick proposal made certain predictions concerning the behavior of DNA during replication. According to the proposal, each of the daughter duplexes should consist of one complete strand inherited from the parental duplex and one complete strand that has been newly synthesized. Replication of this type is said to be semiconservative because each daughter duplex contains one strand from the parent structure. Two other possibilities were considered: conservative and dispersive. In conservative replication, the two original strands would remain together (after serving as templates), as would the two newly synthesized strands. As a result, one of the daughter duplexes would contain only parental DNA, while the other daughter duplex would contain only newly synthesized DNA. In dispersive replication, the parental strands would be broken into fragments, and the new strands would be synthesized in short segments. Then the old fragments and new segments would be joined together to form a complete strand. As a result, the daughter duplexes would contain strands that were composites of old and new DNA. At first glance, dispersive replication might seem like an unlikely solution, but it appeared at the time to be the only way to avoid the seemingly impossible task of unwinding two intertwined strands of a DNA duplex as it replicated. To decide among these three possibilities, it was necessary to distinguish newly synthesized DNA strands from the original DNA strands that served as templates. This was accomplished in studies on bacteria in 1957 by Matthew Meselson and Franklin Stahl who used heavy (15N) and light (14N) isotopes of nitrogen to distinguish between parental and newly synthesized DNA strands. These researchers grew bacteria in medium containing 15N-ammonium chloride as the sole nitrogen source. Consequently, the nitrogen-containing bases of the DNA of these cells contained only the heavy nitrogen isotope. Cultures of "heavy" bacteria were washed free of the old medium and incubated in fresh medium with light, 14N-containing compounds, and samples were removed at increasing intervals over a period of several generations. DNA was extracted from the samples of bacteria and subjected to equilibrium density-gradient centrifugation. In this procedure, the DNA is mixed with a concentrated solution of cesium chloride and centrifuged until the double-stranded DNA molecules reach equilibrium according to their density. In the Meselson-Stahl experiment, the density of a DNA molecule is directly proportional to the percentage of 15N or 14N atoms it contains. If replication is semiconservative, one would expect that the density of DNA molecules would decrease during culture in the 14N-containing medium. After one generation, all DNA molecules would be 15N-14N hybrids, and their buoyant density would be halfway between that expected for totally heavy and totally light DNA. As replication continued beyond the first generation, the newly synthesized strands would continue to contain only light isotopes, and two types of duplexes would appear in the gradients: those containing 15N-14N hybrids and those containing only 14N. As the time of growth in the light medium continued, a greater and greater percentage of the DNA molecules present would be light. However, as long as replication continued semiconservatively, the original heavy parental strands would remain intact and present in hybrid DNA molecules that occupied a smaller and smaller percentage of the total DNA. The results of the density-gradient experiments obtained by Meselson and Stahl demonstrate unequivocally that replication occurs semiconservatively. By 1960, replication had been demonstrated to occur semiconservatively in eukaryotes as well. The original experiments were carried out by J. Herbert Taylor. Cultured mammalian cells were allowed to undergo replication in bromodeoxyuridine (BrdU), a compound that is incorporated into DNA in place of thymidine. Following replication, a chromosome is made up of two chromatids. After one round of replication in BrdU, both chromatids of each chromosome contained BrdU. After two rounds of replication in BrdU, one chromatid of each chromosome was composed of two BrdU-containing strands, whereas the other chromatid was a hybrid consisting of a BrdU-containing strand and a thymidine-containing strand. The thymidine-containing strand had been part of the original parental DNA molecule prior to addition of BrdU to the culture.

What is meant by the term RNA world? What type of evidence argues for its existence?

The discovery that RNAs are capable of catalyzing chemical reactions has had an enormous impact on our view of biological evolution. Ever since the discovery of DNA as the genetic material, biologists have wondered which came first, protein or DNA. The dilemma arose from the seemingly non-overlapping functions of these two types of macromolecules. Nucleic acids store information, whereas proteins catalyze reactions. With the discovery of ribozymes in the early 1980s, it became apparent that one type of molecule - RNA - could do both. These findings have fueled the belief that both DNA and protein were absent at an early stage in the evolution of life. During this period, RNA molecules performed double duty: they served as genetic material, and they catalyzed chemical reactions, including those required for RNA replication. Life at this stage is described as an "RNA world." Only at a later stage in evolution were the functions of catalysis and information storage taken over by protein and DNA, respectively, leaving RNA to function primarily as a go-between in the flow of genetic information. Many researchers also believe that splicing provides an example of a legacy from an ancient RNA world. The best argument for an RNA world is that the two requirements for a living organism (the ability to carry a code and the ability to catalyze a reaction) are wrapped up in one molecule, RNA. For example, RNA is responsible for catalyzing the formation of a peptide bond. DNA is better at carrying a code because of its stability and proteins are more versatile as enzymes, but RNA still serves as the go-between between the two other macromolecules. The transition from the RNA world to the present version of the world may have occurred in the following way. It is speculated that amino acids may have been used initially as adjuncts (cofactors) to enhance catalytic reactions carried out by ribozymes. Over time, ribozymes presumably evolved that were able to string specific amino acids together to form small proteins, which were more versatile catalysts than their RNA predecessors. Ribosomes - the ribonucleoprotein machines responsible for protein synthesis - are essentially ribozymes at heart, which provides strong support for this evolutionary scenario. As proteins took over a greater share of the workload in the primitive cell, the RNA world was gradually transformed into an "RNA- protein world." At a later point in time, RNA was presumably replaced by DNA as the genetic material, which propelled life forms into the present "DNA-RNA- protein world." The evolution of DNA may have required only two types of enzymes: a ribonucleotide reductase to convert ribonucleotides into deoxyribonucleotides and a reverse transcriptase to transcribe RNA into DNA. The fact that RNA catalysts do not appear to be involved in either DNA synthesis or transcription supports the idea that DNA was the last member of the DNA-RNA-protein triad to appear on the scene. Somewhere along the line of evolutionary progress, a code had to evolve that would allow the genetic material to specify the sequence of amino acids to be incorporated into a given protein.

How is the specificity of nucleotide incorporation determined?

The specificity of nucleotide incorporation is determined by the sequence of the template strand of DNA. As the polymerase moves along the DNA template, it incorporates complementary nucleotides into the growing RNA chain. A nucleotide is incorporated into the RNA strand if it is able to form a proper (Watson-Crick) base pair with the nucleotide in the DNA strand being transcribed. For example, an incoming adenosine 5'-triphosphate pairs with a thyminecontaining nucleotide of the template and an incoming uracil 5'-triphosphate pairs with an adenine containing nucleotide of the template

Distinguish between the two-dimensional and three-dimensional structure of RNAs.

Two-dimensional renderings of RNA structure show the extensive base-pairing between different regions of the single strand of RNA. Stems are the regions where base-pairing is occurring and loop regions are areas where base-pairing is not occurring. Three-dimensional renderings are usually based on X-ray diffraction data and do not emphasize base-pairing; they illustrate the arrangement in space of the various segments of an RNA relative to the other segments (see: tRNA).

Describe the mechanism of action of DNA polymerases operating on the two template strands and the effect this has on the synthesis of the lagging versus the leading strand.

As Watson and Crick first discovered, the two strands of a DNA helix have an antiparallel orientation. The diagram of DNA replication first presented by Watson and Crick depicted events as they would be expected to occur at the replication fork. The diagram suggested that one of the newly synthesized strands is polymerized in a 5' → 3' direction, while the other strand is polymerized in a 3' → 5' direction. Investigators began to wonder if some other enzyme was responsible for the construction of the 3' → 5' strand and if the enzyme worked differently in the cell than under in vitro conditions. The lack of polymerization activity in the 3' → 5' direction has a straightforward explanation: DNA strands cannot be synthesized in that direction. Rather, both newly synthesized strands are assembled in a 5' → 3' direction. During the polymerization reaction, the OH group at the 3' end of the primer carries out a nucleophilic attack on the 5'-phosphate of the incoming nucleoside triphosphate. The polymerase molecules responsible for construction of the two new strands of DNA both move in a 3'-to-5' direction along the template, and both construct a chain that grows from its 5'-P terminus. Consequently, one of the newly synthesized strands grows toward the replication fork where the parental DNA strands are being separated, while the other strand grows away from the fork. Although this solves the problem concerning an enzyme that synthesizes a strand in only one direction, it creates an even more complicated dilemma. It is apparent that the strand that grows toward the fork can be constructed by the continuous addition of nucleotides to its 3' end. But evidence was soon gathered to indicate that the strand that grows away from the replication fork is synthesized discontinuously, that is, as fragments. Before the synthesis of a fragment can be initiated, a suitable stretch of template must be exposed by movement of the replication fork. Once initiated, each fragment grows away from the replication fork toward the 5' end of a previously synthesized fragment to which it is subsequently linked. Thus, the two newly synthesized strands of the daughter duplexes are synthesized by very different processes. The strand that is synthesized continuously is called the leading strand because its synthesis continues as the replication fork advances. The strand that is synthesized discontinuously is called the lagging strand because initiation of each fragment must wait for the parental strands to separate and expose additional template. Both strands are probably synthesized simultaneously, so that the terms leading and lagging may not be as appropriate as thought when they were first coined. Because one strand is synthesized continuously and the other discontinuously, replication is said to be semi-discontinuous. The discovery that the lagging strand is synthesized in pieces raised a new set of perplexing questions about the initiation of DNA synthesis. One wonders how the synthesis of each of these fragments begin when none of the DNA polymerases are capable of strand initiation. Further studies revealed that initiation is not accomplished by a DNA polymerase but, rather, by a distinct type of RNA polymerase, called primase, that constructs a short primer composed of RNA, not DNA. The leading strand, whose synthesis begins at the origin of replication, is also initiated by a primase molecule. The short RNAs synthesized by the primase at the 5' end of the leading strand and the 5' end of each Okazaki fragment serve as the required primer for the synthesis of DNA by a DNA polymerase. The RNA primers are subsequently removed, and the resulting gaps in the strand are filled with DNA and then sealed by DNA ligase. The formation of transient RNA primers during the process of DNA replication is a curious activity. It is thought that the likelihood of mistakes is greater during initiation than during elongation, and the use of a short removable segment of RNA avoids the inclusion of mismatched bases.

Describe the steps during initiation of transcription in bacteria.

Bacteria, such as E. coli, contain a single type of RNA polymerase composed of five subunits that are tightly associated to form a core enzyme. If the core enzyme is purified from bacterial cells and added to a solution of bacterial DNA molecules and ribonucleoside triphosphates, the enzyme binds to the DNA and synthesizes RNA. The RNA molecules produced by a purified polymerase, however, are not the same as those found within cells because the core enzyme has attached to random sites in the DNA, sites that it would normally have ignored in vivo. If, 4 however, a purified accessory polypeptide called sigma factor (s) added to the RNA polymerase before it attaches to DNA, transcription begins at selected locations. Attachment of the s factor to the core enzyme increases the enzyme's affinity for promoter sites in DNA and decreases its affinity for DNA in general. As a result, the complete enzyme is thought to slide freely along the DNA until it recognizes and binds to a suitable promoter region. Bacterial cells possess a variety of different s factors that recognize different versions of the promoter sequence.

Why is it important in mismatch repair that the cell distinguish the parental strands from the newly synthesized strands?

Cells can remove mismatched bases that are incorporated by the DNA polymerase and escape the enzyme's proofreading exonuclease. This process is called mismatch repair (MMR). A mismatched base pair causes a distortion in the geometry of the double helix that can be recognized by a repair enzyme. But the enzyme must "recognize" which member of the mismatched pair is the incorrect nucleotide. If it were to remove one of the nucleotides at random, it would make the wrong choice 50 percent of the time, creating a permanent mutation at that site. Thus, for a mismatch to be repaired after the DNA polymerase has moved past a site, it is important that the repair system distinguish the newly synthesized strand, which contains the incorrect nucleotide, from the parental strand, which contains the correct nucleotide. In E. coli, the two strands are distinguished by the presence of methylated adenosine residues on the parental strand. DNA methylation does not appear to be utilized by the MMR system in eukaryotes, and the mechanism of identification of the newly synthesized strand remains unclear.

How to characterization of proteins by polyacrylamide gel electrophoresis and Western blotting?

Electrophoresis is a powerful technique widely used to separate proteins and it depends on the ability of charged molecules to migrate when placed in an electric field. Protein separation by electrophoresis is usually accomplished using native polyacrylamide gel electrophoresis (PAGE). The proteins are driven by an applied current through a gelated matrix composed of polymers of a small organic molecule (acrylamide) that is cross-linked to form a molecular sieve. A polyacrylamide gel may be formed as a thin slab between 2 glass plates or as a cylinder formed within a glass tube. Once the gel has polymerized, the slab (or tube) is suspended between 2 compartments containing buffer in which opposing electrodes are immersed. The protein sample is prepared in a solution of glycerol or sucrose whose density prevents mixing with the buffer in the upper compartment. A voltage is then applied between the buffer compartments and current flows across the slab, causing the proteins to move toward the oppositely charged electrode. Separations are typically carried out using alkaline buffers, which make the proteins negatively charged and cause them to move toward the positively charged electrode, the anode, at the gel's opposite end. After electrophoresis, the slab is removed from the glass plates (or tube) and stained. The relative protein movement through a polyacrylamide gel depends on the charge density (charge per unit of mass) of the molecules. The greater the charge density, the more forcefully the protein is driven through the gel, and thus the more rapid is its rate of migration, but charge density is only one important factor in PAGE fractionation; size and shape also play a role. The polyacrylamide forms a cross-linked molecular sieve that entangles proteins passing through the gel. The larger the protein is the more it becomes entangled in the sieve and the more slowly it migrates. Shape is also a factor because compact globular proteins move more rapidly than elongated fibrous proteins of comparable molecular mass. The acrylamide and cross-linking agent concentrations used in making the gel are also important factors affecting movement. If acrylamide is less concentrated, there is less cross-linking and the more rapidly a given protein migrates. A gel containing 5% acrylamide might be useful for separating proteins of 60 - 250 kDa, while a gel of 15% acrylamide might be useful for separating proteins of 10 - 50 kDa. One can follow the progress of electrophoresis by watching the migration of a charged tracking dye that moves just ahead of the fastest proteins. When the tracking dye has moved to the desired location, the current is turned off and the gel is removed from its container. Gels are typically stained with Coomassie Blue or silver stain to reveal the locations of the proteins. In a Western blot, the proteins on the membrane are identified by their interaction with specific antibodies.

What is RNA editing, and how can it increase the number of proteins that can be formed from a single pre-mRNA transcript?

Gene expression can be regulated at the posttranscriptional level is by RNA editing, in which specific nucleotides are converted to other nucleotides after the RNA has been transcribed. RNA editing can create new splice sites, generate stop codons, or lead to amino acid substitutions. Although not nearly as widespread as alternative splicing, RNA editing is particularly important in the nervous system, where a significant number of messages appear to have one or more adenines (A) converted to inosines (I). This modification involves the enzymatic removal of an amino group from the nucleotide. Inosine is subsequently read as a G by the translational machinery.

Identify the structure of all 5 bases found in DNA and RNA

Two-dimensional renderings of RNA structure show the extensive base-pairing between different regions of the single strand of RNA. Stems are the regions where base-pairing is occurring and loop regions are areas where base-pairing is not occurring. Three-dimensional renderings are usually based on X-ray diffraction data and do not emphasize base-pairing; they illustrate the arrangement in space of the various segments of an RNA relative to the other segments (see: tRNA)

Describe some of the ways that the initiation step of translation differs from the elongation steps of translation.

) Once it attaches to an mRNA, a ribosome always moves along the mRNA from one codon to the next, that is, in consecutive blocks of three nucleotides. To ensure that the proper triplets are read, the ribosome attaches to the mRNA at a precise site, termed the initiation codon, which is specified as AUG. Binding to this codon automatically puts the ribosome in the proper reading frame so that it correctly reads the entire message from that point on. During initiation, an mRNA does not bind to an intact ribosome, but to the small and large subunits in separate stages; this is unlike elongation where the full complex has already been assembled during initiation. The first major step of initiation is the binding of the small ribosomal subunit to the first AUG sequence (or one of the first) in the message, which serves as the initiation codon. Several of the steps in initiation require the help of soluble proteins, called initiation factors (designated as IFs in bacteria and eIFs in eukaryotes). There are other factors, called elongation factors, which are needed for elongation. Cells possess two distinct methionyl-tRNAs: one used to initiate protein synthesis and a different one to incorporate methionyl residues into the interior of a polypeptide. The initiator aa-tRNA is positioned within the P site of the ribosome, where the anticodon loop of the tRNA binds to the AUG codon of the mRNA. During elongation, all of the aa-tRNAs are positioned initially in the A site of the ribosome rather than in the P site. During initiation, the small ribosomal subunit moves to the initiator codon before any tRNAs enter the ribosome at the P site and bind to the small ribosomal subunit and the mRNA. During elongation, the full ribosome moves three nucleotides toward the 3' end of the mRNA (translocates) after the peptide bond has been formed.

How does methylation of DNA affect gene expression? How is it related to histone acetylation or histone methylation? What is meant by the term 'genomic imprinting'?

. One of the key factors in silencing a region of the genome involves a phenomenon known as DNA methylation. Examination of the DNA of mammals and other vertebrates indicates that as many as 1 out of 100 nucleotides bears an added methyl group, which is always attached to carbon 5 of a cytosine. Methyl groups are added to the DNA by a family of enzymes called DNA methyltransferases encoded in humans by DNMT genes. This simple chemical modification is thought to serve as an epigenetic mark or "tag" that allows certain regions of the DNA to be identified and utilized differently from other regions. In mammals, the methylcytosine residues are part of a 5'-CpG-3' dinucleotide within a symmetrical sequence. As a true epigenetic mark, the pattern of DNA methylation must be maintained through repeated cell divisions. This is accomplished by an enzyme, Dnmt1, which travels with the replication fork and methylates the daughter DNA strands by copying the methylation pattern of the parental strands. The majority of methylcytosine residues in mammalian DNA are located within noncoding, repeated sequences, and primarily transposable elements. Methylation is thought to maintain these elements in an inactive state. In addition to the general suppression of transposable elements, DNA methylation has long been implicated in the repression of transcription of specific genes. In recent years, techniques have been developed to determine the specific cytosine residues that are methylated in any given population of cells across the entire genome. These studies confirm that (1) the promoter regions of inactive genes tend to be more heavily methylated than the promoter regions of active genes and (2) DNA methylation patterns vary from one cell type to another, reflecting the differential activity of genes among various tissues. Most evidence suggests that methylation of a gene's promoter serves more to maintain that gene in an inactive state than as a mechanism for initial inactivation. As an example, inactivation of the genes on one of the X chromosomes of female mammals occurs prior to a wave of methylation of gene promoters that is thought to convert the DNA into a more permanently repressed condition. DNA methylation has been closely linked with another repressive epigenetic mark—histone methylation. It may be that gene inactivation begins with the establishment of a transcriptionally repressive pattern of histone modifications in the core histones of promoter regions and that these modified histone tails then recruit the DNA methylation machinery to those nucleosomes. Once the DNA in these regions is methylated, the methylated cytosine residues can serve as binding sites to recruit additional histone modifying enzymes that further repress and compact the chromatin of that promoter. Histone acetylation has an effect opposite from histone methylation; it can lead to transcriptional activation of the gene in question. It had been assumed until the mid-1980s that the set of chromosomes inherited from a male parent was functionally equivalent to the corresponding set of chromosomes inherited from the female parent. But, as with many other long-standing assumptions, this proved not to be the case. Instead, certain genes are either active or inactive during early mammalian development depending solely on whether they were brought into the zygote by the sperm or the egg. For example, the gene that encodes a fetal growth factor called IGF2 is only active on the chromosome transmitted from the male parent. In contrast, the gene that encodes a specific potassium channel (KVLQT1) is only active on the chromosome transmitted from the female parent. Genes of this type are said to be imprinted according to their parental origin. Imprinting can be considered an epigenetic phenomenon, because the differences between alleles are inherited from one's parents but are not based on differences in DNA sequence. It is estimated, based largely on the study of mutant mice, that the mammalian genome contains at least 80 imprinted genes located primarily in several distinct chromosomal clusters. Genes are thought to become imprinted as the result of selective DNA methylation of certain regions that control the expression of either the male or female alleles. As a result, the maternal and paternal versions of imprinted genes differ consistently in their degree of methylation. Furthermore, mice that lack a key DNA methyltransferase (Dnmt1) are unable to maintain the imprinted state of the genes they inherit. The methylation state of imprinted genes is not affected by the waves of demethylation and re-methylation that sweep through the early embryo. Consequently, the same alleles that are inactive due to imprinting in the fertilized egg will be inactive in the cells of the fetus and most adult tissues. The major exception occurs in the germ cells, where the imprints inherited from the parents are erased during early development and then reestablished when that individual begins to produce his or her own gametes.

What is the role of cyclic AMP in the synthesis of β-galactosidase?

. Repressors, such as those of the lac and trp operons, exert their influence by negative control, as the interaction of the DNA with this protein inhibits gene expression. The lac operon is also under positive control, as was discovered during an early investigation of a phenomenon called the glucose effect. If bacterial cells are supplied with glucose as well as a variety of other substrates, such as lactose or galactose, the cells catabolize the glucose first and ignore the other sugars. The glucose in the medium acts to repress the production of various catabolic enzymes, such as -galactosidase, that would allow utilization of the other sugars. In 1965, a surprising finding was made: cyclic AMP (cAMP), previously thought to be involved only in eukaryotic metabolism, was detected in cells of E. coli. It was found that the concentration of cAMP in the cells was related to the presence of glucose in the medium; the higher the glucose concentration, the lower the cAMP concentration. Furthermore, when cAMP was added to the medium in the presence of glucose, the catabolic enzymes that were normally absent were suddenly synthesized by the cells. Although the exact means by which glucose lowers the concentration of cAMP has still not been elucidated, the mechanism by which cAMP overcomes the effect of glucose is well understood. cAMP acts in prokaryotic cells by binding to a protein, the cAMP receptor protein (CRP). By itself, CRP is unable to bind to DNA. However, the cAMP-CRP complex recognizes and binds to a specific site in the lac control region. The presence of the bound cAMP-CRP causes a change in the conformation of the DNA, which makes it possible for RNA polymerase to transcribe the lac operon. The binding site in the promoter of the lac operon for RNA polymerase is not a high-affinity binding site, so initiation of transcription is extremely inefficient except in the presence of the cAMP-CRP complex. Even when lactose is present and the lac repressor is inactivated, RNA polymerase cannot transcribe the lac operon unless the levels of cAMP-CRP are high. Because of the inverse relationship between cAMP levels and glucose levels, transcription of the lac operon is thus regulated by glucose levels. As long as glucose is abundant, cAMP concentrations remain below that required to promote transcription of the operon.

What types of regulatory sequences are found in the regulatory regions of the DNA upstream from a gene, such as that which encodes for PEPCK? What is the role of these various sequences in controlling the expression of the nearby gene(s)?

. The ligand-bound receptor binds to a specific DNA sequence, called a glucocorticoid response element (GRE). For the PEPCK gene, this site is located upstream from the core promoter, and binding activates transcription of the gene. Identical or similar GRE sequences are located upstream from a number of other genes on different chromosomes. If the site is the exact preferred binding site, the gene will be highly responsive to elevated glucocorticoid levels, whereas genes with imperfect binding sites will respond in accordance with the change in binding affinity for the glucocorticoid-receptor complex. Consequently, a single stimulus (elevated glucocorticoid concentration) can simultaneously activate a number of genes, each at its own precise level, allowing a comprehensive, finely tuned response that meets the needs of the cell. The preferred glucocorticoid response element consists of the following sequence, where n can be any nucleotide. 3'-TCTTGTnnnACAAGA-5' 5'-AGAACAnnnTGTTCT-3' A symmetrical sequence of this type is called a palindrome because the two strands have the same 5' to 3' sequence. The GRE is seen to consist of two defined stretches of nucleotides separated by three undefined nucleotides. The twofold nature of a GRE is important because pairs of GR polypeptides bind to the DNA as dimers in which each subunit of the dimer binds to one-half of the DNA sequence indicated above. The importance of the GRE in mediating a hormone response is most clearly demonstrated by introducing one of these sequences into the upstream region of a gene that normally does not respond to glucocorticoids. When cells containing DNA that has been engineered in this way are exposed to glucocorticoids, transcription of the gene downstream from the transplanted GRE is initiated. The GRE situated upstream from the PEPCK gene, and the other response elements are referred to as distal promoter elements to distinguish them from the proximal promoter elements situated closer to the gene or the core promoter, which dictates the site of initiation. The expression of most genes is also regulated by even more distant DNA elements called enhancers. An enhancer typically contains multiple binding sites for sequence-specific transcriptional activators. Enhancers are often distinguished from promoter elements by a unique property: they can be situated either upstream or downstream of the start site and they can even be inverted (rotated 180°), without affecting the ability of a bound transcription factor to stimulate transcription. Deletion of an enhancer can decrease the level of transcription by 100-fold or more. A typical mammalian gene may have a number of enhancers scattered within the DNA in the vicinity of the gene. Different enhancers typically bind different sets of transcription factors and respond independently to different stimuli. Some enhancers are located thousands or even tens of thousands of base pairs upstream or downstream from the gene whose transcription they stimulate. Even though enhancers and promoters may be separated by large numbers of nucleotides, enhancers are thought to stimulate transcription by influencing events that occur at the core promoter. Enhancers and core promoters can be brought into proximity because the intervening DNA can form a loop through the interactions of bound proteins. If enhancers can interact with promoters over such long distances, what is to prevent an enhancer from binding to an inappropriate promoter located even farther downstream on the DNA molecule? A promoter and its enhancers are, in essence, "cordoned off" from other promoter/enhancer elements by specialized boundary sequences called insulators. One of the most active areas of molecular biology in the past decade or so has focused on the mechanism by which a transcriptional activator bound to an enhancer is able to stimulate the initiation of transcription at the core promoter. Transcription factors accomplish this feat through the action of intermediaries known as co-activators. Co-activators are large complexes that consist of numerous subunits. Co-activators can be broadly divided into two functional groups: (1) those that interact with components of the basal transcription machinery (the general transcription factors and RNA polymerase II) and (2) those that act on chromatin, converting it from a state that is relatively inaccessible to the transcription machinery to a state that is much more transcription "friendly." Sometimes these various types of co-activators work together in an orderly manner to activate the transcription of particular genes in response to specific intracellular signals. Given the large number of transcription factors encoded in the genome, and the limited diversity of co-activators, each co-activator complex operates in conjunction with a wide variety of different transcription factors. The co-activator CBP, for example, participates in the activities of hundreds of different transcription factors.

Contrast the events of nucleotide excision repair and base excision repair

11. Nucleotide excision repair (NER) operates by a cut-and-patch mechanism that removes a variety of bulky lesions, including pyrimidine dimers and nucleotides to which various chemical groups have become attached. Two distinct NER pathways can be distinguished: A. A transcription-coupled pathway in which the template strands of genes that are being actively transcribed are preferentially repaired. Repair of a template strand is thought to occur as the DNA is being transcribed, and the presence of the lesion may be signaled by a stalled RNA polymerase. This preferential repair pathway ensures that those genes of greatest importance to the cell, which are the genes the cell is actively transcribing, receive the highest priority on the "repair list." B. A slower, less efficient global genomic pathway that corrects DNA strands in the remainder of the genome. Although recognition of the lesion is probably accomplished by different proteins in the two NER pathways, the steps that occur during repair of the lesion are thought to be very similar. One of the key components of the NER repair machinery is TFIIH, a huge protein that also participates in the initiation of transcription. The discovery of the involvement of TFIIH established a crucial link between transcription and DNA repair, two processes that were previously assumed to be independent of one another. Included among the various subunits of TFIIH are two subunits (XPB and XPD) that possess helicase activity; these enzymes separate the two strands of the duplex in preparation for removal of the lesion. The damaged strand is then cut on both sides of the lesion by a pair of endonucleases, and the segment of DNA between the incisions is released. Once excised, the gap is filled by a DNA polymerase and the strand is sealed by DNA ligase.

Describe the cascade of events responsible for the sudden changes in gene expression in a bacterial cell following the addition of lactose. How does this compare with the events that occur in response to the addition of tryptophan?

A bacterial cell lives in direct contact with its environment, which may change dramatically in chemical composition or temperature from one moment to the next. At certain times, a particular food source may be present, while at other times that compound is absent. The lac operon is a cluster of genes that regulates production of the enzymes needed to degrade lactose in bacterial cells. The lac operon is an example of an inducible operon, in which the presence of a key metabolic substance (in this case, lactose) induces transcription of the operon, allowing synthesis of the proteins encoded by the structural genes. The lac operon contains three tandem structural genes: the z gene, which encodes beta-galactosidase; the y gene, which encodes galactoside permease, a protein that promotes entry of lactose into the cell; and the a gene, which encodes thiogalactoside transacetylase, an enzyme whose physiologic role is unclear. If lactose is present in the medium, the disaccharide enters the cell via limiting amounts of galactoside permease where it binds to the lac repressor, changing the conformation of the repressor and making it unable to attach to the DNA of the operator. This frees RNA polymerase to transcribe the operon followed by translation of the three encoded proteins. Thus, in an inducible operon the repressor protein binds to the DNA only in the absence of lactose, which functions as the inducer. As the concentration of lactose in the medium decreases, the disaccharide dissociates from its binding site on the repressor molecule, which allows the repressor to again bind to the operator and repress transcription. In a repressible operon, such as the tryptophan (or trp) operon, the repressor is unable to bind to the operator DNA by itself (the reverse of what happens with the lac repressor). Instead, the repressor is active as a DNA-binding protein only when complexed with a specific factor, such as tryptophan, which functions as a corepressor. In the absence of tryptophan, the conformation of the repressor does not allow binding to the operator sequence, thus permitting RNA polymerase to bind to the promoter and transcribe the structural genes of the trp operon, leading to production of the enzymes that synthesize tryptophan; in the lac operon, if lactose is absent, the lac operon is inactive, again the reverse of what happens in the trp operon. Once tryptophan becomes available, the enzymes of the tryptophan biosynthetic pathway are no longer required. Under these conditions, the increased concentration of tryptophan leads to the formation of the tryptophan-repressor complex, which binds to the operator and blocks transcription. Lactose is a disaccharide composed of glucose and galactose whose oxidation can provide the cell with metabolic intermediates and energy. The first step in the catabolism (i.e., degradation) of lactose is the hydrolysis of the bond that joins the two sugars (a beta-galactoside linkage), a reaction catalyzed by the enzyme beta-galactosidase. When bacterial cells are growing under minimal conditions, the cells have no need for beta-galactosidase. Under minimal conditions, an average cell contains fewer than five copies of beta-galactosidase and a single copy of the corresponding mRNA. Within a few minutes after adding lactose to the culture medium, cells contain approximately 1000 times the number of beta-galactosidase molecules. The presence of lactose has induced the synthesis of this enzyme. Tryptophan is an amino acid required for protein synthesis. In humans, tryptophan is an essential amino acid; it must come from the diet. In contrast, bacterial cells can synthesize tryptophan in a series of reactions requiring the activity of multiple enzymes. Cells growing in the absence of tryptophan activate the genes encoding these enzymes. If, however, tryptophan should become available in the medium, the cells no longer have to synthesize this amino acid, and, within a few minutes, production of the enzymes needed to synthesize tryptophan is repressed. In bacteria, the genes that encode the enzymes needed to synthesize tryptophan are clustered together on the chromosome in a functional complex called an operon. A typical bacterial operon consists of structural genes, a promoter region, an operator region, and a regulatory gene. Structural genesencode the enzymes themselves.The promoteris the site where the RNA polymerase binds to the DNA prior to beginning transcription. The operatortypically resides adjacent to or overlaps with the promoter and serves as the binding site for a protein, usually a repressor. The repressor is an example of a gene regulatory protein—a protein that recognizes a specific sequence of base pairs within the DNA and binds to that sequence with high affinity. The regulatory geneencodes the repressor protein. The key to operon expression lies in the sequence of the operator and the presence or absence of a repressor. When the repressor binds the operator, it prevents RNA polymerase from initiating transcription. The capability of the repressor to bind the operator and inhibit transcription depends on the conformation of the repressor, which is regulated allosterically by a key compound in the metabolic pathway, such as lactose or tryptophan. It is the concentration of this key metabolic substance that determines whether the operon is active or inactive at any given time.

Distinguish between the effects of a base substitution in the DNA on a non-overlapping and an overlapping code.

A conclusion as to whether the genetic code was overlapping or non-overlapping could be inferred from studies of mutant proteins, such as the mutant hemoglobin responsible for sickle cell anemia. In sickle cell anemia, as in most other cases that were studied, the mutant protein was found to contain a single amino acid substitution. If the code is overlapping, a change in one of the base pairs in the DNA would be expected to affect three consecutive codons and, therefore, three consecutive amino acids in the corresponding polypeptide. If, however, the code is non-overlapping and each nucleotide is part of only one codon, then only one amino acid replacement would be expected. These and other data indicated that the code is non-overlapping.

What is the consequence of having the DNA of the lagging-strand template looped back on itself as in Figure 13.13a?

A polymerase III molecule moves from one site on the lagging-strand template to another site that is closer to the replication fork by "hitching a ride" with the DNA polymerase that is moving in that direction along the leading-strand template. Thus, even though the two polymerases are moving in opposite directions with respect to the linear axis of the DNA molecule, they are, in fact, part of a single protein complex. The two tethered polymerases can replicate both strands by looping the DNA of the lagging-strand template back on itself, causing this template to have the same orientation as the leading-strand template. Both polymerases then can move together as part of a single replicative complex without violating the "5' → 3' rule" for synthesis of a DNA strand. Once the polymerase assembling the lagging strand reaches the 5' end of the Okazaki fragment synthesized during the previous round, the lagging-strand template is released and the polymerase begins work at the 3' end of the next RNA primer toward the fork. This model is often referred to as the "trombone model" because the looping DNA repeatedly grows and collapses during the replication of the lagging strand, reminiscent of the movement of the brass "loop" of a trombone as it is played.

What is the difference between an HDAC and an HAT?

Acetyl groups are added to specific lysine residues on the core histones by a family of enzymes called histone acetyltransferases (HATs). In the late 1990s, it was discovered that a number of coactivators possessed HAT activity. If the HAT activity of these coactivators was eliminated by mutation, so too was their ability to stimulate transcription. The discovery that coactivators contain HAT activity provided a crucial link between histone acetylation, chromatin structure, and gene activation. Once bound to the DNA, the activator recruits a coactivator to a region of the chromatin that is targeted for transcription. Once positioned at the target region, the coactivator acetylates the core histones of nearby nucleosomes, which exposes a binding site for a chromatin remodeling complex. The combined actions of these various complexes increase the accessibility of the promoter to the components of the transcription machinery, which assembles at the site where transcription will be initiated. The state of acetylation of chromatin is a dynamic property; just as there are enzymes (HATs) to add acetyl groups, there are also enzymes to remove them. Removal of acetyl groups is accomplished by histone deacetylases (HDACs). Whereas HATs are associated with transcriptional activation, HDACs are associated with transcriptional repression. HDACs are present as subunits of larger complexes described as corepressors. Corepressors, as mentioned above, are similar to coactivators, except that they are recruited to specific genetic loci by transcriptional factors (repressors) that cause the targeted gene to be silenced rather than

Explain the principle of separation when using affinity columns.

Affinity chromatography takes advantage of the unique structural properties of the desired protein; this contrasts with anion exchange and gel filtration, which used the protein's bulk properties to effect purification or fractionation. The desired protein can be specifically withdrawn from solution, while all others remain behind in solution. To do so the proteins interact with specific substances: enzymes interact with substrates, receptors with ligands, antibodies with antigens, etc. If one covalently attaches such interacting molecules to the column's inert, immobilized material (matrix) and passes the protein mixture through column, the protein that binds the column-linked molecule is retained. After the contaminants have passed through the column and eluted out the bottom, the protein can then be displaced from matrix binding sites by changing ionic composition and/or pH of column solvent. One can achieve near-total purification of the desired protein in a single step, unlike the other procedures that separate on the basis of size or charge. In recent years, affinity chromatography has been widely used to purify proteins that have been genetically modified to include a specific protein "tag" on its N- or C-terminus. Common tags include, glutathione-S-transferase (GST) tags or a His tag (composed of a string of 6 x histidine). His-tagged proteins are purified using a resin onto which nickel ions have been immobilized, while GST-tagged proteins are purified based on their affinity for glutathione-immobilized resin.

How does the number of RNA polymerases distinguish prokaryotes and eukaryotes? What is the relationship between a pre-RNA and a mature RNA?

Bacteria, such as E. coli, contain a single type of RNA polymerase composed of five subunits 5 that are tightly associated to form a core enzyme. On the other hand, it was discovered in 1969 that eukaryotic cells have three distinct transcribing enzymes in their cell nuclei. Each of these enzymes is responsible for synthesizing a different group of RNAs. While no prokaryote has been found with multiple RNA polymerases, the simplest eukaryotes (yeast) have the same three nuclear types that are present in mammalian cells. This difference in number of RNA polymerases is another sharp distinction between prokaryotic and eukaryotic cells. All three major types of eukaryotic RNAs - mRNAs, rRNAs, and tRNAs - are derived from precursor RNA molecules that are considerably longer than the final RNA product. The initial precursor RNA is equivalent in length to the full length of the DNA transcribed and is called the primary transcript, or pre-RNA. The corresponding segment of DNA from which a primary transcript is transcribed is called a transcription unit. Primary transcripts do not exist within the cell as naked RNA but become associated with proteins even as they are synthesized. Primary transcripts typically have a fleeting existence, being processed into smaller, functional RNAs by a series of "cut-and-paste" reactions. RNA processing requires a variety of small RNAs (90 to 300 nucleotides long) and their associated proteins.

How does the effect of a nonsense mutation differ from that of a frameshift mutation? Why?

Because the three termination codons can be readily formed by single base changes from many other codons, one might expect mutations to arise that produce stop codons within the coding sequence of a gene. Mutations of this type, termed nonsense mutations, have been studied for decades and are responsible for roughly 30 percent of inherited disorders in humans. Premature termination codons, as they are also called, are also commonly introduced into mRNAs during splicing. Nonsense mutations result in proteins whose synthesis is terminated before it should be and thus the proteins are shorter than normal and they possess reduced or no functionality. The proteins often retain more function if the mutation is closer to the C-terminus of the protein. The particular sequence of codons in the mRNA that is utilized by a ribosome (i.e., the reading frame) is fixed at the time the ribosome attaches to the initiation codon at the beginning of translation. Some of the most destructive mutations are ones in which a single base pair is either added to or deleted from the DNA. Consider the effect of an addition of one or two nucleotides or a deletion of one or two nucleotides in a given sequence. The ribosome moves along the mRNA in the incorrect reading frame from the point of mutation through the remainder of the coding sequence. Mutations of this type are called frameshift mutations. Frameshift mutations lead to the assembly of an entirely abnormal sequence of amino acids from the point of the mutation.

What general strategy can be used to successfully purify a protein from a sample by FPLC?

Before one can obtain information about a protein's fine structure or function, it must be isolated in a relatively pure state. Because most cells contain thousands of different proteins, the purification of a single species can be a challenging mission, particularly if the protein is present in the cell at low concentration. Purification of a protein is generally accomplished by the stepwise removal of contaminants. Two proteins may be very similar in one property, like overall charge, but very different in others, like molecular size or shape. Thus, complete purification of a given protein usually requires the use of successive techniques that take advantage of different properties of the proteins being separated. Start with methods that work best with high protein concentrations, then move to more sensitive ones. Purification is measured as an increase in specific activity (the ratio of the amount of that protein to the total amount of the protein present in the sample). This is called an assay. Catalytic activity may be used as an assay to monitor purification, if the protein is an enzyme. Alternatively, assays can be based on immunologic properties, electrophoretic properties, electron microscopic, or other criteria. Measurements of total protein in a sample can be made using various properties (dye binding, Bradford assay, etc.). Chromatography is a term for a variety of techniques in which a mixture of dissolved components is fractionated as it moves through some type of a porous matrix. In solvent based chromatographic techniques, components in a mixture can become associated with 1 of 2 alternative phases: A mobile phase, consisting of a moving solvent and a matrix through which the solvent is moving (often packed into a column). The proteins to be fractionated are dissolved in a solvent and then passed through the column. The materials that make up the immobile phase contain sites to which the proteins in solution can bind or pores it can either pass through or is excluded from. As individual protein molecules interact with the materials of the matrix, their progress through the column is retarded. Thus, the greater the affinity of a particular protein for the matrix material, the slower is its passage through column. Because different proteins in the mixture have different affinities for the matrix, they are retarded to different degrees and are thus separated from each other. As the solvent passes through the column and drips out the bottom, it is collected as fractions in a series of tubes. Those proteins in the mixture with the least affinity for the column appear in the first fractions to emerge from the column. Those proteins with more affinity for the matrix emerge later or may bind more permanently to the column.

Why are tRNAs referred to as adaptor molecules? What aspects of their structure do tRNAs have in common?

Decoding the information in an mRNA is accomplished by transfer RNAs, which act as adaptors. On one hand, each tRNA is linked to a specific amino acid (as an aa-tRNA), while on the other hand, that same tRNA is able to recognize a particular codon in the mRNA. The interaction between successive codons in the mRNA and specific aa-tRNAs leads to the synthesis of a polypeptide with an ordered sequence of amino acids. A number of distinct similarities present in all the different tRNAs became evident. All tRNAs were roughly the same length (between 73 and 93 nucleotides) and all had a significant percentage of unusual bases that were found to result from enzymatic modifications of one of the four standard bases after it had been incorporated into the RNA chain, that is, post transcriptionally. In addition, all tRNAs had sequences of nucleotides in one part of the molecule that were complementary to sequences located in other parts of the molecule. Because of these complementary sequences, the various tRNAs become folded in a similar way to form a structure that can be drawn in two dimensions as a cloverleaf. The unusual bases, which are concentrated in the loops, disrupt hydrogen bond formation in these regions and serve as potential recognition sites for various proteins. All mature tRNAs have the triplet sequence CCA at their 3' end. Transfer RNAs fold into a unique and defined tertiary structure. X-ray diffraction analysis has shown that tRNAs are constructed of two double helices arranged in the shape of an L. Those bases that are found at comparable sites in all tRNA molecules are particularly important in generating the L-shaped tertiary structure. The common shapes of tRNAs reflect the fact that all of them take part in a similar series of reactions during protein synthesis. However, each tRNA has unique features that distinguish it from other tRNAs. It is these unique features that make it possible for an amino acid to become attached enzymatically to the appropriate (cognate) tRNA. Transfer RNAs translate a sequence of mRNA codons into a sequence of amino acid residues. The part of the tRNA that participates in the complementary interaction of the tRNA with the codon of the mRNA is a stretch of three sequential nucleotides, called the anticodon, which is located in the middle loop of the tRNA molecule. This loop is invariably composed of seven nucleotides, the middle three of which constitute the anticodon. The anticodon is located at one end of the L-shaped tRNA molecule opposite from the end at which the amino acid is attached.

How to clone a plasmid?

For example, DNA molecules from 2 different sources are treated with a restriction enzyme that makes staggered cuts in the DNA duplex. Staggered cuts leave short, single-stranded tails that act as "sticky ends" that can bind to a complementary single-stranded tail on another DNA molecule to restore a double-stranded molecule. Often one DNA fragment is a bacterial plasmid, a small, circular, double-stranded DNA molecule that is separate from the main bacterial chromosome. The other DNA fragment comes from human cells after treatment with the same restriction enzyme used to open the plasmid. When the human DNA fragments and plasmid are incubated together along with DNA ligase, the 2 types of DNAs are H bonded to one another by sticky ends and then ligated to form circular DNA recombinants. The first recombinant DNAs were made by the basic method above (1972) - Paul Berg, Herbert Boyer, Annie Chang, & Stanley Cohen (Stanford & U. of Ca., SF,); the birth of modern genetic engineering. Once this is done, you have produced a large number of different recombinant molecules, each of which has a bacterial plasmid and a piece of human DNA incorporated into a circular DNA structure. If you want to isolate a single human gene from the human genome like the one that encodes insulin, you have to be able to separate this specific fragment from all of the others. This is done by a process called DNA cloning. The foreign DNA to be cloned is inserted into the plasmid to form a recombinant DNA, and the bacteria are transformed and grown in culture. The plasmids used for DNA cloning are modified versions of those found in bacterial cells; like the natural counterparts from which they are derived these plasmids contain: (1) an origin of replication and (2) one or more genes that make the recipient cell resistant to one or more antibiotics; antibiotic resistance allows researchers to select for those cells that contain the recombinant plasmid. Recombinant DNAs with different foreign DNAs are added to bacterial culture where they can be taken up by bacterial transformation as demonstrated by Avery, MacLeod, & McCarty. This phenomenon forms the basis for cloning plasmids in bacterial cells. Once taken up, the plasmid replicates autonomously within the recipient and is passed on to the progeny during cell division. Those bacteria containing a recombinant plasmid can be selected from the others by growing the cells in the presence of the antibiotic whose resistance is conferred by one or more genes on the plasmid. This procedure eliminates bacteria that have not taken up plasmids.

Explain the principle of separation when using gel-filtration columns.

Gel filtration chromatography - separates proteins (or nucleic acids) primarily on the basis of their effective size (hydrodynamic radius). Like ion-exchange chromatography, the separation material consists of tiny beads that are packed into a column through which the protein solution slowly passes. The materials used in gel filtration are composed of cross-linked polysaccharides (dextran or agarose) of different porosity, which allow the proteins to diffuse in and out of the beads differentially. For example: the protein being purified (Mw = 125 kD molecular mass) is mixed with contaminating proteins (Mw = 250 and 75 kD) of similar shape, one much larger and one much smaller. One could pass the mixture through a column with beads that allow entry of globular proteins of <200 kD into their interiors (e.g. Sephadex G-150). As it passes through the column bed, a 250 kD protein cannot enter the beads and stays dissolved in the moving solvent phase. As a result, the 250 kD protein elutes as soon as the preexisting solvent in column (the bed volume) has dripped out. In contrast, the other 2 proteins can diffuse into the interstices within the beads and are retarded in their passage through the column. As more and more solvent moves through column, the proteins move down its length and out the bottom, but they do so at different rates. Among those proteins that enter the beads, smaller species are retarded to a greater extent than larger ones. Thus, the 125 kD protein is eluted in a purified state, while the 75 kD protein still remains in the column.

Explain the principle of separation when using ion-exchange columns

Ion-exchange chromatography - it is unlikely that many proteins in a partially purified preparation have the same overall charge, because they are large, polyvalent electrolytes; their ionic charge is a basis of purification. The overall charge of a protein is the sum of all the individual charges of its component amino acids. Because the charge of each amino acid depends on the pH of the medium, the charge of each protein also depends on the medium's pH. As the pH is lowered, negatively charged groups become neutralized and positively charged groups become more numerous; the opposite occurs as the pH is increased. For each protein, there is a pH (the isoelectric point) at which negative and positive charges are equal (the protein's charge is neutral - cf. isoelectric point). Most proteins have an isoelectric point below 7. Ionic charge is used as a basis for purification in a variety of techniques, including ion-exchange chromatography. Ion-exchange chromatography depends on the ionic bonding of proteins to an inert matrix material, like cellulose, containing covalently linked charged groups. Two of the most commonly employed ion-exchange resins are: (a) diethylaminoethyl (DEAE)-cellulose (positively charged); binds negatively charged molecules; it is an anion exchanger and (b) Carboxymethyl (CM)-cellulose (negatively charged); binds positively charged molecules; a cation exchanger.

How do the two exonuclease activities of DNA polymerase I differ from one another? What are their respective roles in replication?

It was subsequently shown that all of the bacterial DNA polymerases possess exonuclease activity. Exonucleases can be divided into 5'→ 3' and 3' → 5' exonucleases, depending on the direction in which the strand is degraded. DNA polymerase I has both 3' → 5' and 5' → 3' exonuclease activities, in addition to its polymerizing activity (Figure 13.16). These three activities are found in different domains of the single polypeptide. Thus, remarkably, DNA polymerase I is three different enzymes in one. The two exonuclease activities have entirely different roles in replication. Most nucleases are specific for either DNA or RNA, but the 5' → 3' exonuclease of DNA polymerase I can degrade either type of nucleic acid. Initiation of Okazaki fragments by the primase leaves a stretch of RNA at the 5' end of each fragment, which is removed by the 5' → 3' exonuclease activity of DNA polymerase I. As the enzyme removes ribonucleotides of the primer, its polymerase activity simultaneously fills the resulting gap with deoxyribonucleotides. The last deoxyribonucleotide incorporated is subsequently joined covalently to the 5' end of the previously synthesized DNA fragment by DNA ligase. On occasion, the polymerase incorporates an incorrect nucleotide, resulting in a mismatched base pair, that is, a base pair other than A-T or G-C. It is estimated that an incorrect pairing of this sort occurs once for every 105-106 nucleotides incorporated, a frequency that is 103-104 times greater than the spontaneous mutation rate of approximately 10-9. The second of the two exonuclease activities of DNA polymerase I, the 3' → 5' activity, is part of the reason that the spontaneous mutation rate is so low. When an incorrect nucleotide is incorporated by DNA polymerase I, the enzyme stalls and the end of the newly synthesized strand has an increased tendency to separate from the template and form a single-stranded 3' terminus. When this occurs, the frayed end of the newly synthesized strand is directed into the 3'→5' exonuclease site, which removes the mismatched nucleotide. This job of "proofreading" is one of the most remarkable of all enzymatic activities and illustrates the sophistication to which biological molecular machinery has evolved. The 3' → 5' exonuclease activity removes approximately 99 out of every 100 mismatched bases, raising the fidelity to about 10-7-10-8. In addition, bacteria possess a mechanism called mismatch repair that operates after replication and corrects nearly all of the mismatches that escape the proofreading step. Together these processes reduce the overall observed error rate to about 10-9. Thus, the fidelity of DNA replication can be traced to three distinct activities: (1) accurate selection of nucleotides, (2) immediate proofreading, and (3) post-replicative mismatch repair.

Describe the different levels at which gene expression is regulated to allow a -globin gene with the following structure to direct the formation of a protein that accounts for over 95 percent of the protein of the cell. exon 1—intron—exon 2—intron—exon 3

Of all the hundreds of different types of cells in the human body, only those in the lineage leading to red blood cells produce the protein hemoglobin. Moreover, hemoglobin accounts for more than 95 percent of a red blood cell's protein, yet the genes that encode the two hemoglobin polypeptides represent less than one-millionth of the developing cell's total DNA. Not only does the cell have to find this genetic needle in the chromosomal haystack, it has to regulate its expression to such a high degree that production of these few polypeptides becomes the dominant synthetic activity of the cell. Because the chain of events leading to the synthesis of a particular protein includes a number of discrete steps, there are several levels at which control might be exercised. There are four distinct levels of the regulation of gene expression in eukaryotic cells at four distinct levels: 1. Transcriptional control mechanisms determine whether a particular gene can be transcribed and, if so, how often. 2. Processing control mechanisms determine the path by which the primary mRNA transcript (pre-mRNA) is processed into a messenger RNA that can be translated into a polypeptide. 3. Translational control mechanisms determine whether a particular mRNA is actually translated and, if so, how often and for how long a period. 4. Posttranslational control mechanisms regulate the activity and stability of proteins.

What is the major difference between bacteria and eukaryotes that allows a eukaryotic cell to replicate its DNA in a reasonable amount of time?

One remarkable feature of bacterial replication is its rate. The replication of an entire bacterial chromosome in approximately 40 minutes at 37°C requires that each replication fork move about 1000 nucleotides per second, which is equivalent to the length of an entire Okazaki fragment. Thus, the entire process of Okazaki fragment synthesis, including formation of an RNA primer, DNA elongation and simultaneous proofreading by the DNA polymerase, excision of the RNA, its replacement with DNA, and strand ligation, occurs within a few seconds. Although it takes E. coli approximately 40 minutes to replicate its DNA, a new round of replication can begin before the previous round has been completed. Consequently, when these bacteria are growing at their maximal rate, they double their numbers in about 20 minutes. Replication in E. coli begins at only one site along the single, circular chromosome. Cells of higher organisms may have a thousand times as much DNA as this bacterium, yet their polymerases incorporate nucleotides into DNA at much slower rates. To accommodate these differences, eukaryotic cells replicate their genome in small portions, termed replicons. Each replicon has its own origin from which replication forks precede outward in both directions. In a human cell, replication begins at about 10,000 to 100,000 different replication origins. The existence of replicons was first demonstrated in autoradiography experiments in which single DNA molecules were shown to be replicated simultaneously at several sites along their length.

What are two different ways that co-activators can influence gene expression?

One-way co-activators can influence gene expression is to interact with the core transcriptional machinery to stimulate its activity. An example of a co-activator that works by this mechanism is the complex known as "mediator", which works directly on polymerase II. The other way that co-activators can influence gene expression is to modify chromatin to make it easier to transcribe the gene in question. Co-activators that work by this second mechanism include histone acetyltransferases and chromatin remodeling complexes.

Explain the how enzymatic amplification of DNA by PCR works and provide a simple protocol for a PCR experiment.

Polymerase Chain Reaction (PCR) was developed by Kary Mullis (Cetus Corporation, 1983) and is now widely used to amplify specific DNA regions without the need for bacterial cells. There are many different PCR protocols that have been used for a multitude of different applications in which anywhere from one to a large population of related DNAs can be amplified. PCR amplification is readily adapted to RNA templates by first converting them to complementary DNAs (cDNA) using reverse transcriptase (c.f. 'violation of central dogma'). The basic procedure and simplest protocol employs a heat-stable DNA polymerase (Taq polymerase), originally isolated from Thermus aquaticus, a bacterium that lives in hot springs at temperatures around 70-85°C, close to the boiling point of water. Mix a DNA sample with all 4 deoxyribonucleotides (dNTPs) and an aliquot of Taq polymerase. Also add a large excess of 2 short synthetic DNA fragments (oligonucleotides = 'primers') that are complementary to DNA sequences on both strands and flang your gene of interest (GOI). These short oligonucleotides serve as primers to which nucleotides are added during the following replication steps. Then heat mixture to ~95°C, hot enough to melt (denature) DNA in the mix and separate DNA molecules into their 2 component strands. The mix is then cooled to ~60°C, which allows the primers to hybridize (bind) to the target DNA strands. The temperature is then raised to ~72°C, which allows the thermophilic polymerase to add complementary nucleotides to the 3' end of the primers. As the polymerase extends the primers, it selectively copies the target DNA and forms new complementary DNA strands. The temperature is then raised again to ~95°C, causing newly formed and original strands to separate from each other. The sample is then cooled to allow the synthetic primers in the mixture to bind once again to the target DNA, which is now present at twice the original amount. This cycle is repeated over and over again, doubling the amount of the specific DNA region flanked by the bound primers with each cycle. This generates billions of copies of this one specific DNA region from minute amounts in just a few hours using a thermal cycler. The thermal cycler automatically changes the temperature of the reaction mixture, allowing each step in the cycle to take place.

What is a riboswitch?

Proteins such as the lac and trp repressors are not the only gene regulatory molecules that are influenced by interaction with small metabolites. A number of bacterial mRNAs have been identified that can bind a small metabolite, such as glucosamine or adenine, with remarkable specificity. The metabolite binds to a highly structured 5' noncoding region of the mRNA. Once bound to the metabolite, these mRNAs, or riboswitches, as they are called, undergo a change in their folded conformation that allows them to alter the expression of a gene involved in production of that metabolite. Thus, riboswitches act by means of a feedback mechanism similar to the alternative RNA structures that regulate attenuation in the trp operon. Most riboswitches suppress gene expression by blocking either termination of transcription or initiation of translation. Like the repressors that function in conjunction with operons, riboswitches allow cells to adjust their level of gene expression in response to changes in the available levels of certain metabolites. Given that they act without the participation of protein cofactors, riboswitches are likely another legacy from an ancestral RNA world.

What is the role of pyrophosphate hydrolysis?

RNA polymerase catalyzes the reaction in which ribonucleoside triphosphate substrates (NTPs) are cleaved into nucleoside monophosphates as they are polymerized into a covalent chain. Reactions leading to the synthesis of nucleic acids (and proteins) are inherently different from those of intermediary metabolism. Whereas some of the reactions leading to the formation of small molecules, such as amino acids, may be close enough to equilibrium that a considerable reverse reaction can be measured, those reactions leading to the synthesis of nucleic acids and proteins must occur under conditions in which there is virtually no reverse reaction. This condition is met during transcription with the aid of a second reaction catalyzed by a different enzyme, a pyrophosphatase. In this case, the pyrophosphate (PPi) produced in the first reaction is hydrolyzed to inorganic phosphate (Pi). The hydrolysis of pyrophosphate releases a large amount of free energy and makes the nucleotide incorporation reaction essentially irreversible.

Describe the events that occur at an origin of replication during the initiation of replication in yeast cells. What is meant by replication being bidirectional?

Replication in E. coli begins at only one site along the single, circular chromosome. Cells of higher organisms may have a thousand times as much DNA as a bacterium, yet their polymerases incorporate nucleotides into DNA at much slower rates. To accommodate these differences, eukaryotic cells replicate their genome in small portions, termed replicons. Each replicon has its own origin from which replication forks precede outward in both directions. In a human cell, replication begins at about 10,000 to 100,000 different replication origins. The existence of replicons was first demonstrated in autoradiography experiments in which single DNA molecules were shown to be replicated simultaneously at several sites along their length. The mechanism by which replication is initiated in eukaryotes has been a focus of research over the past decade. The greatest progress in this area has been made with budding yeast because the origins of replication can be removed from the yeast chromosome and inserted into bacterial DNA molecules, conferring on them the ability to replicate either within a yeast cell or in cellular extracts containing the required eukaryotic replication proteins. Because these sequences promote replication of the DNA in which they are contained, they are referred to as autonomous replicating sequences (ARSs). Those ARSs that have been isolated and analyzed share several distinct elements. The core element of an ARS consists of a conserved sequence of 11 base pairs, which functions as a specific binding site for an essential multi-protein complex called the origin recognition complex (ORC). If the ARS is mutated so that it is unable to bind the ORC, initiation of replication cannot occur. Once replication is initiated in bacteria and in eukaryotes at replicon origins, it proceeds outward from the origin in both directions, that is, bidirectionally. The sites where the pair of replicated segments come together and join the non-replicated DNA are termed replication forks. Each replication fork corresponds to a site where (1) the parental double helix is undergoing strand separation, and (2) nucleotides are being incorporated into the newly synthesized complementary strands. The two replication forks move in opposite directions. This is what was meant by bidirectional replication.

What is the major class of enzymes whose discovery and use has made the formation of recombinant DNA molecules possible?

Restriction enzymes were discovered in the 1970s in bacteria; they are nucleases that recognize short nucleotide sequences in duplex DNA and cleave the DNA backbone at specific sites on both duplex strands. They were called this because they function in bacteria to destroy viral DNAs that might enter the cell, thereby restricting the potential growth of the viruses. The bacterium protects its own DNA from nucleolytic attack by methylating the bases at susceptible sites, a chemical modification that blocks the action of the enzyme. Enzymes from several hundred different prokaryotic organisms have been isolated; together, they recognize >100 different nucleotide sequences. Most restriction enzymes recognize restriction sites that are 4 - 6 nucleotides long and make cuts at specific sites on both duplex strands; these sites are characterized by a particular type of internal symmetry. The DNA segment has twofold rotational symmetry, because it can be rotated 180° without a change in base sequence. If one reads the sequence in the same direction (3' to 5' or 5' to 3') on either strand, the base order observed is the same; a sequence with this type of symmetry is called a palindrome. When for example EcoR1 attacks the palindrome, it breaks each strand at the same site in the sequence. Some restriction enzymes cleave bonds directly opposite one another on the 2 strands producing blunt ends; others, like EcoR1, make staggered cuts. The discovery and purification of restriction enzymes have been invaluable in the advances made by molecular biologists in recent years. Because a particular sequence of 4 to 6 nucleotides occurs quite frequently simply by chance, any type of DNA is susceptible to fragmentation by these enzymes. The use of restriction enzymes allows the DNA of the human genome, or that of any other organism, to be dissected into a precisely defined set of specific fragments. Once the DNA from a particular individual is digested with a restriction enzyme, the fragments generated can be fractionated on the basis of length by gel electrophoresis. Different enzymes cleave the same DNA prep into different sets of fragments, and the sites within the genome that are cleaved by various enzymes can be identified and ordered into a restriction map.

Why do you use SDS?

SDS-PAGE is usually done in the presence of a negatively charged detergent, sodium dodecyl sulfate (SDS), which binds in large numbers to all types of protein molecules. SDS molecules denature the proteins and causes them to lose their activity. The electrostatic repulsion between SDS molecules unfolds all of the proteins into a similar rod-like shape, eliminating differences in shape as a factor in separation. The number of protein-bound SDS molecules is roughly proportional to the protein's molecular mass (~1.4 g SDS/g protein). Thus, all proteins have an equivalent charge density and are driven through the gel with the same force, regardless of size. However, because the polyacrylamide is highly cross-linked, larger proteins are held up to a greater degree than are smaller proteins. Thus, proteins are separated by SDS-PAGE on the basis of a single property; their molecular mass. SDS-PAGE can also be used to determine the molecular mass of the various proteins in addition to separating the proteins in the mixture. This is done by comparing the positions of the protein bands on the gel to those produced by proteins of known size.

What are the different roles of a single ubiquitin versus multiple ubiquitins when attached to a target protein?

Single ubiquitins typically act as signals to control the sorting and trafficking of proteins. In contrast, when multiple ubiquitins are added to a protein, this acts as a trigger to begin degradation of the protein by targeting it to the proteasome.

Distinguish between a synonymous and a nonsynonymous base change.

Spontaneous mutations causing single base changes in a gene often will not produce a change in the amino acid sequence of the corresponding protein. A change in nucleotide sequence that does not affect amino acid sequence is said to be synonymous, whereas a change that causes an amino acid substitution is said to be non-synonymous. Synonymous changes are much less likely to change an organism's phenotype than are non-synonymous changes. Consequently, non-synonymous changes are much more likely to be selected for or against by natural selection. Now that the genomes of related organisms, such as those of chimpanzees and humans, have been sequenced, we can look directly at the sequences of homologous genes and see how many changes are synonymous or non-synonymous. Genes possessing an excess of non-synonymous substitutions in their coding regions are likely to have been influenced by natural selection, while those possessing an excess of synonymous substitutions in their coding regions would be less likely to have been influenced by natural selection.

Why do the DNA molecules depicted in Figure 13.7a fail to stimulate the polymerization of nucleotides by DNA polymerase I? What are the properties of a DNA molecule that allow it to serve as a template for nucleotide incorporation by DNA polymerase I?

The DNA molecules depicted in Figure 13.7a fail to stimulate the polymerization of nucleotides by DNA polymerase I because they lack a primer. The enzyme cannot initiate the formation of a DNA strand. Rather, it can only add nucleotides to the 3' hydroxyl terminus of an existing strand. Studies of the enzymes responsible for replicating DNA (DNA polymerases) soon indicated that all of these polymerases, both prokaryotic and eukaryotic, have the same two basic requirements: a template DNA strand to copy and a primer strand to which nucleotides can be added. The enzyme cannot initiate the formation of a DNA strand. Rather, it can only add nucleotides to the 3' hydroxyl terminus of an existing strand. The strand that provides the necessary 3' OH terminus is called a primer.

What is the nature of the reaction in which nucleotides are incorporated into a growing RNA strand?

The formation of an elongation complex is followed by release of the sigma factor. RNA polymerase moves along the template DNA strand toward its 5' end (i.e., in a 3'—> 5' direction). As the polymerase progresses, the DNA is temporarily unwound, and the polymerase assembles a complementary strand of RNA that grows from its 5' terminus in a 3' direction. Once the polymerase has moved past a particular stretch of DNA, the DNA double helix re-forms. Consequently, the RNA chain does not remain associated with its template as a DNA-RNA hybrid (except for about nine nucleotides just behind the site where the polymerase is operating). Even though polymerases are relatively powerful motors, these enzymes do not necessarily move in a steady, continuous fashion but may pause at certain locations along the template or even backtrack before resuming their forward progress. A number of elongation factors have been identified that enhance the enzyme's ability to traverse these various roadblocks. In some cases, as occurs following the rare incorporation of an incorrect nucleotide, a stalled polymerase must backtrack and then digest away the 3' end of the newly synthesized transcript and resynthesize the missing portion before continuing its movement. The ability of RNA polymerase to recognize and remove a misincorporated nucleotide is referred to as "proofreading." This corrective function is carried out by the same active site within the enzyme that is responsible for nucleotide incorporation.

How is it that alternative splicing can effectively increase the number of genes in the genome?

The genes of complex plants and animals contain numerous introns and exons, and the introns must be precisely removed to allow transport through the nuclear pore. However, the pattern of intron removal can be subject to dramatic regulation, allowing for multiple protein products from the same gene. The particular splicing pathway that is followed may depend on the particular stage of development, or the particular cell type or tissue being considered. In the simplest case, a specific exon can either be retained or spliced out of the transcript. An example of this type of alternative splicing occurs during synthesis of fibronectin, a protein found in both blood plasma and the extracellular matrix. Fibronectin produced by fibroblasts and retained in the matrix is encoded by an mRNA that contains two extra exons compared to the version of the protein produced by liver cells and secreted into the blood. The extra peptides are encoded by portions of the pre-mRNA that are retained during processing in the fibroblast but are removed during processing in the liver cell. Thus, by using different combinations of exons in different cells or tissues, multiple proteins can be produced from one gene.

What is the role of a promoter in gene expression? Where are the promoters for bacterial polymerases located?

The site on the DNA to which an RNA polymerase molecule binds prior to initiating transcription is called the promoter. Cellular RNA polymerases are not capable of recognizing promoters on their own but require the help of additional proteins called transcription factors. In addition to providing a binding site for the polymerase, the promoter contains the information that determines which of the two DNA strands is transcribed and the site at which transcription begins. Bacterial promoters are located in the region of a DNA strand just preceding the initiation site of RNA synthesis. The nucleotide at which transcription is initiated is denoted as +1 and the preceding nucleotide as -1. Those portions of the DNA preceding the initiation site (toward the 3′ end of the template) are said to be upstream from that site. Those portions of the DNA succeeding it (toward the 5′ end of the template) are said to be downstream from that site. Analysis of the DNA sequences just upstream from a large number of bacterial genes reveals that two short stretches of DNA are similar from one gene to another. One of these stretches is centered at approximately 35 bases upstream from the initiation site and typically occurs as the sequence TTGACA. This TTGACA sequence (known as the -35 element) is called a consensus sequence, which indicates that it is the most common version of a conserved sequence, but that some variation occurs from one gene to another. The second conserved sequence is found approximately 10 bases upstream from the initiation site and occurs at the consensus sequence TATAAT. This site in the promoters responsible for identifying the precise nucleotide at which transcription begins.

Explain the principle of the yeast two-hybrid system.

The yeast two-hybrid system technique is most widely used to search for protein-protein interactions and was invented in 1989 by Stanley Fields and Okkyu Song. The technique depends on the expression of a reporter gene, like beta-galactosidase (lacZ), whose activity is readily monitored by a test that detects a color change when the enzyme is present in a yeast cell population. The lacZ gene expression in this system is activated by a particular protein, a TF, that contains 2 domains, a DNA-binding domain and an activation domain. The DNA-binding domain mediates binding to the promoter and the activation domain mediates interaction with other proteins involved in the activation of gene expression. Both domains must be present for transcription to occur. To employ the technique, 2 different types of recombinant DNA molecules are prepared: (1) One has a DNA segment encoding the TF's DNA-binding domain linked to a DNA segment encoding the "bait" protein X (the protein already characterized and for which potential binding partners are sought) and (2) the other DNA has a DNA segment encoding the TF's activation domain linked to DNA encoding an unknown protein Y and the assumption is that Y is a protein capable of binding the "bait" protein. Such DNAs (or cDNAs) are prepared from mRNAs by reverse transcriptase and if both recombinant DNAs are expressed in a yeast cell this lead to the production of hybrid proteins. If produced in the cell alone, neither the X- nor Y-containing hybrid proteins activate lacZ transcription. However, if both of these particular recombinant DNAs are introduced into the same yeast cell, the X and Y proteins can interact with one another to reconstitute a functional TF that transcribes the lacZ gene. The transcription of lacZ that results can be detected by cell's ability to make beta-galactosidase. This technique allows researchers to "fish" for proteins encoded by unknown genes that are capable of interacting with the "bait" protein.

Why are there no heavy bands in the top three centrifuge tubes of Figure 13.3a?

Those three centrifuge tubes show the results expected after the first, second, and third generations if semiconservative replication was occurring. At the beginning of the first replication, all of the DNA would have been found in the heavy bands. After the first replication had occurred, each duplex should consist of one heavy DNA strand and one light DNA strand in the case of semiconservative replication. There should be no fully heavy DNA after the first, second or third replications. Once they were gone, they could not be reconstituted

What are some of the structural properties that tend to be found in several groups of transcription factors?

Transcriptional control is orchestrated by a large number of proteins, called transcription factors. These proteins can be divided into two functional classes: general transcription factors that bind at core promoter sites in association with RNA polymerase, and sequence-specific transcription factors that bind to various regulatory sites of particular genes. This latter group of transcription factors can act either as transcriptional activators that stimulate transcription of the adjacent gene or as transcriptional repressors that inhibit transcription. Transcriptional activators, have the job of binding to specific regulatory sites within the DNA and initiating the recruitment of a large number of protein complexes that bring about the actual transcription of the gene itself. Single genes are usually controlled by many different DNA regulatory sites that bind a combination of different transcription factors. Conversely, a single transcription factor may bind to numerous sites around the genome, thereby controlling the expression of a host of different genes. Each type of cell has a characteristic pattern of gene transcription, which is determined by the particular complement of transcription factors contained in that cell. The control of gene transcription is complex, regulated by the presence of multiple binding sites for transcription factors and the binding affinity for these transcription factors to each sequence. Transcription factors have preferred, high-affinity binding sites. A region upstream of a given gene might contain one or more preferred high-affinity sites or might contain sites with a slightly different sequence that bind with lower affinity. The binding of multiple transcription factors is usually required to activate transcription. In a sense, the regulatory region of a gene can be thought of as a type of integration center for that gene's expression. Cells exposed to different stimuli respond by synthesizing different transcription factors, which bind to different sites in the DNA. The extent to which a given gene is transcribed depends on the particular combination of transcription factors bound to upstream regulatory elements. Given that roughly 5 to 10 percent of genes encode transcription factors, it is apparent that a virtually unlimited number of possible combinations of interactions among these proteins are possible. When this is coupled to the fact that the binding sites for these factors vary from gene to gene, the combination of the presence or absence of a given factor and variable binding affinity for each factor allows for precise variation in gene expression patterns between cells of different type, different tissue, different stage of development, and different physiologic state. The three-dimensional structure of numerous DNA-protein complexes has been determined by X-ray crystallography and NMR spectroscopy, providing a basic portrait of the way that these two macromolecules interact with one another. Like most proteins, transcription factors contain different domains that mediate different aspects of the protein's function. Transcription factors typically contain at least two domains: a DNA-binding domain that binds to a specific sequence of base pairs in the DNA, and an activation domain that regulates transcription by interacting with other proteins. In addition, many transcription factors contain a surface that promotes the binding of the protein with another protein of identical or similar structure to form a dimer. The formation of dimers has proved to be a common feature of many different types of transcription factors and plays an important role in regulating gene expression.

How does the distinct biochemistry of reverse transcriptase make viruses like HIV more vulnerable to nucleotide analogs?

Unlike DNA polymerase, which is highly selective for the correct nucleotide, reverse transcriptase is far less selective and can easily incorporate the wrong nucleotide during formation of the DNA copy. This not only means that HIV and other RNA based viruses have a high mutation rate, it also means that they can incorporate nucleotide analogs that don't normally occur in cells, such as azidothymidine.

Describe the role of the DNA helicase, the SSBs, the beta-clamp, the DNA gyrase, and the DNA ligase during replication in bacteria.

Unwinding the duplex and separating the strands require the aid of two types of proteins that bind to the DNA, a helicase (or DNA unwinding enzyme) and single-stranded DNA-binding (SSB) proteins. DNA helicases unwind a DNA duplex in a reaction that uses energy released by ATP hydrolysis to move along one of the DNA strands, breaking the hydrogen bonds that hold the two strands together and exposing the single-stranded DNA templates. E. coli has at least 12 different helicases for use in various aspects of DNA (and RNA) metabolism. One of these helicases, the product of the dnaB gene, serves as the major unwinding machine during replication. The DnaB helicase consists of six subunits arranged to form a ring-shaped protein that encircles a single DNA strand. Initiation of replication begins in E. coli when multiple copies of the DnaA protein bind to the origin of replication (oriC) and separate (melt) the DNA strands at that site. The DnaB helicase is then loaded onto the single-stranded DNA of the lagging strand of oriC, with the help of the protein DnaC. The DnaB helicase then travels in a 5' → 3' direction along the lagging-strand template, unwinding the helix as it proceeds. DNA polymerase III, the enzyme that synthesizes DNA strands during replication in E. coli, is part of a large "replication machine" called the DNA polymerase III holoenzyme. One of the non-catalytic components of the holoenzyme, called the -clamp, keeps the polymerase associated with the DNA template. DNA polymerases (like RNA polymerases) possess two somewhat contrasting properties: (1) they must remain associated with the template over long stretches if they are to synthesize a continuous complementary strand, and (2) they must be attached loosely enough to the template to move from one nucleotide to the next. These contrasting properties are provided by the doughnut-shaped -clamp that encircles the DNA and slides along it. As long as it is attached to a - "sliding clamp," a DNA polymerase can move from one nucleotide to the next without diffusing away from the template. The polymerase on the leading-strand template remains tethered to a single -clamp during replication. In contrast, when the polymerase on the lagging-strand template completes the synthesis of an Okazaki fragment, it disengages from the -clamp and is cycled to a new -clamp that has been assembled at an RNA primer-DNA template junction located closer to the replication fork. Cells contain enzymes, called topoisomerases, that can change the state of supercoiling in a DNA molecule. One enzyme of this type, called DNA gyrase, a type II topoisomerase, relieves the mechanical strain that builds up during replication in E. coli. DNA gyrase molecules travel along the DNA ahead of the replication fork, removing positive supercoils. DNA gyrase accomplishes this feat by cleaving both strands of the DNA duplex, passing a segment of DNA through the double-stranded break to the other side, and then sealing the cuts, a process that is driven by the energy released during ATP hydrolysis. Eukaryotic cells possess similar enzymes that carry out this required function. The enzyme that joins the Okazaki fragments into a continuous strand is called DNA ligase.

What is a polyribosome? How does its formation differ in prokaryotes and eukaryotes?

When a messenger RNA in the process of being translated is examined in the electron microscope, a number of ribosomes are invariably seen to be attached along the length of the mRNA thread. This complex of ribosomes and mRNA is called a polyribosome, or polysome. Each of the ribosomes initially assembles from its subunits at the initiation codon and then moves from that point toward the 3' end of the mRNA until it reaches a termination codon. As each ribosome moves away from the initiation codon, another ribosome attaches to the mRNA and begins its translation activity. The rate at which translation initiation occurs varies with the mRNA being studied; some mRNAs have a much greater density of associated ribosomes than others. The simultaneous translation of the same mRNA by numerous ribosomes greatly increases the rate of protein synthesis within the cell. Recent studies utilizing cryoelectron tomography have suggested that the three-dimensional arrangement and orientation of ribosomes within a "free" (i.e., non-membrane bound) polysome are quite highly ordered. The ribosomes that comprise such a polysome are densely packed and have adopted a "double-row" array. Moreover, each of the individual ribosomes is oriented so that its nascent polypeptide (red or green filaments) is situated at its outer surface facing the cytosol. It is suggested that this orientation maximizes the distance between nascent chains, thereby minimizing the likelihood that the nascent chains will interact with one another and, possibly, aggregate. It is presumed that these polysomes had been engaged in the synthesis of membrane and/or organelle proteins at the time the cell was fixed. The ribosomes within each polysome appear to be organized at the surface of the ER membrane into a circular loop or spiral. Unlike eukaryotic cells, in which transcription occurs in the nucleus and translation occurs in the cytoplasm with many intervening steps, the corresponding activities in bacterial cells are tightly coupled. Thus, protein synthesis in bacterial cells begins on mRNA templates well before the mRNA has been completely synthesized. The synthesis of an mRNA proceeds in the same direction as the movement of ribosomes translating that message, that is, from the 5' to 3' end. Consequently, as soon as an RNA molecule has begun to be produced, the 5' end is available for attachment to ribosomes. Thus, transcription and translation occur in different compartments in eukaryotic cells (transcription in the nucleus and translation in the cytoplasm). However, in prokaryotes, transcription and translation can occur at the same time on the same strand of mRNA, since these two processes occur in the same compartment in prokaryotes.

During translation elongation, it can be said that an aminoacyl-tRNA enters the A site, a peptidyl-tRNA enters the P site, and a deacylated tRNA enters the E site. Explain how each of these events occurs.

With the charged initiator tRNA in place within the P site, the ribosome is available for entry of the second aminoacyl-tRNA into the vacant A site, which is the first step in elongation. Before the second aminoacyl-tRNA can effectively bind to the exposed mRNA codon in the A site, it must combine with a protein elongation factor bound to GTP. This particular elongation factor is called EF-Tu (or Tu) in bacteria and eIF1A in eukaryotes. EF-Tu is required to deliver aminoacyl-tRNAs to the A site of the ribosome. Although any aminoacyl-tRNA-Tu-GTP complex can enter the site, only one whose anticodon is complementary to the mRNA codon situated in the A site will trigger the necessary conformational changes within the ribosome that causes the tRNA to remain bound to the mRNA in the decoding center. Once the proper aminoacyl-tRNA-Tu-GTP is bound to the mRNA codon, the GTP is hydrolyzed and the Tu-GDP complex is released, leaving the newly arrived aa-tRNA situated in the ribosome's A site. Regeneration of Tu-GTP from the released Tu-GDP requires another elongation factor, EF-Ts. At the end of the first step, the two amino acids, attached to their separate tRNAs, are juxtaposed to one another and precisely aligned to chemically interact. The second step in the elongation cycle is the formation of a peptide bond between these two amino acids. Peptide bond formation is accomplished as the amine nitrogen of the aa-tRNA in the A site carries out a nucleophilic attack on the carbonyl carbon of the amino acid bound to the tRNA of the P site, displacing the P-site tRNA. As a result of this reaction, the tRNA bound to the second codon in the A site has an attached dipeptide, whereas the tRNA in the P site is deacylated. Peptide bond formation occurs spontaneously without the input of external energy. The reaction is catalyzed by peptidyl transferase, a component of the large subunit of the ribosome. The formation of the first peptide bond leaves one end of the tRNA molecule of the A site still attached to its complementary codon on the mRNA and the other end of the molecule attached to a dipeptide. The tRNA of the P site is now devoid of any linked amino acid. The following step, called translocation, is characterized by a small (6°) ratchet-like motion of the small subunit relative to the large subunit. As a result of this ratcheting motion, the ribosome moves three nucleotides (one codon) along the mRNA in the 5'—> 3' direction. Translocation is accompanied by the movement of (1) the dipeptidyl-tRNA from the A site to the P site of the ribosome, and (2) the deacylated tRNA from the P site to the E site. As these movements occur, both of these tRNAs remain hydrogen-bonded to their codons in the mRNA. An intermediate stage in the translocation process has been visualized by cryoelectron microscopy, which shows the tRNAs occupying partially translocated "hybrid states." In these hybrid states, the anticodon ends of the tRNAs still reside in the A and P sites of the small subunit, while the acceptor ends of the tRNAs have moved into the P and E sites of the large subunit. The tRNAs are said to occupy A/P and P/E hybrid sites, respectively. The shift from the "classic," non-ratcheted state to the hybrid, ratcheted state occurs spontaneously, that is, without the involvement of other factors. It appears that the ribosome can spontaneously oscillate back and forth between these two states. Once it has shifted to the hybrid state, a GTP-bound elongation factor (EF-G in bacteria and eEF2 in eukaryotes) binds to the ribosome, stabilizing the ribosome in the ratcheted state and thereby preventing the movement of the tRNAs back to the classic A/A and P/P conformation. Then, hydrolysis of the bound GTP generates a conformational change that moves the mRNA and associated anticodon loops of the tRNAs relative to the small ribosomal subunit, which places the bound tRNAs in the E/E and P/P states, leaving the A site empty. At the same time, the ribosome is reset to the non-ratcheted state. Following this reaction, EF-G-GDP dissociates from the ribosome.