Exam 4
Looking at one DNA strand of a restriction site. If the first four bases on one side of the center of a restriction site are 5'-ATGC, what are the next four bases on the same DNA strand (hint: restriction sites have inverted repeats)?
5'-GCAT
The hydrolytic enzymes, used in cloning experiments, which make cuts at distinct sites in DNA are called?
restriction endonucleases
What are the other components of DNA synthesis and how do they participate?
- Single strand binding proteins (SSB's): keep strands apart after they seperate - Helicase enzyme: catalyzes energy dependent strand separation - Topoisomerases enzymes: collectively relax the super coiled DNA that is created by the action of the helicase - Primase enzyme: creates an RNA primer region in lagging strand fragments that provide the starting material to which DNA polymerase III can add segments of DNA - Nucleotide triphosphates (NTP's): provide energy for the action of helicase and topoisomerase - DNA polymerase I: removes the RNA primer nucleotides from the lagging strand segments and replaces them with the appropriate deoxynucleotides. - Ligase: closes the gap between segments of lagging strand DNA.
What are the components of DNA cloning, and what is the role of each of them in the cloning process?
1. A DNA source provides the gene of interest 2. A cloning vector (virus or plasmid) carries a DNA insert. 3. A restriction endonuclease makes specific cuts in DNA 4. A ligase enzyme allows insertion of the DNA segment into the virus or plasmid. 5. A host organism where the plasmid or virus can be grown (amplified).
If the polymerase chain reaction (PCR) is run for seven cycles starting with one DNA strand, the selected DNA segment is amplified how many fold?
128
If a restriction site in a circular plasmid has the sequence GGAATTCC, and the endonucleas cuts between A and A, how many unpaired bases will occur on each strand at the opening that is produced?
2
The condensation of an amino acid with a tRNA to form an amino-acyl-tRNA is driven by the hydrolysis of
2 ATP to 2ADP+2Pi
How many ATP equivalents are used for each addition of one amino acid to the growing peptide chain?
3 ATP equivalents (four phosphoanhydride bonds broken)
During DNA replication the lagging DNA strand is READ starting at its ________ end?
3'
In bacteria a protein with 1200 amino acids might be coded by a sequence of DNA with an open reading frame of _____.
3600
RNA polymerase adds:
5'-NTPs to the 3'-hydroxyl end of the growing RNA strand
How many triplet codons can be produced using four different bases? Which three triplet codons are stop or termination codons and what are their sequences? What is the sequence of the start codon and what amino acid does it code for in prokaryotic organisms?
64 triplet codons can be produced using four different bases. UAA, UAG, and UGA code for stop codons. The start codon sequence is AUG, which codes for methionine in prokaryotic bases.
What is a genomic library and how can such a library be used as the starting point for isolating a gene of interest? What advantage do lambda phages have over plasmids in DNA cloning?
A genomic library is a collection of DNA fragments that make up the full-length genome of an organism. After identifying the chromosome the gene of interest is one, a library could be purchased as a mixed set of host bacteria containing plasmids with inserts that encompassed the whole chromosome. Radioactive probes can be used to hybridize to the gene of interest through small sequences of complementary DNA. The spots on X-ray film darkened by radioactivity correspond to colonies carrying the target gene. Lambda phages can be used for recombinant DNA studies. After infection with the lambda phage, the viral DNA becomes inserted into the host chromosome. It is possible to cut out the middle of lambda phage DNA and insert foreign DNA in its place. The advantage to using bacteriophages is that they can be used to clone large fragments of DNA that will not fit into a productive plasmid. DNA is amplified in the lysogenic pathway. In the lytic phase, many copies of the phage are produced and cell lysis occurs.
What is a point mutation and what is a deletion or substitution mutation? How can deletion or substitution mutations cause a frameshift and what happens when a frameshift occurs?
A point (substitution) mutation occurs when one base is substituted for another in a DNA sequence. A deletion mutation is when one base is deleted from a DNA sequence. A frameshift mutation occurs when a base is inserted or deleted from a sequence and shifts the reading frame. This commonly results in the formation of a shortened protein molecule.
What is a polyribosome and how are these structures related to the simultaneous occurrence of transcription and translation in bacteria?
A polyribosome has multiple ribosomes carrying out protein synthesis on a single mRNA. Transcription and translation can occur simultaneously in bacteria, as they both occur in the cytoplasm.
Which of the following is needed for replication of DNA but NOT for transcription of DNA to RNA?
A primer strand
What does it mean that translation uses a triplet codon of RNA? What does it mean that the genetic code is universal, degenerate, non-overlapping, not punctuated, and comma-less?
A sequence of three bases in mRNA carries the code for one amino acid in the final protein product. The genetic code is universal (same in all organisms), degenerate (several codons specify the same amino acids), non-overlapping (a given stretch of mRNA has only one reading frame and only codes for one protein), not punctuated (read continuously), and comma-less (read continuously).
The signals at the ends of introns in immature mRNA, that code for their removal are:
AG and UU
About what percentage of the human genome codes for proteins or functional RNAs? What is the function of the rest of the human genome?
About 1.5% of the human genome codes for proteins or functional RNAs. The part of the genome that does not code for proteins or functional RNAs may represent remnants of ancient gene duplications or be so-called "selfish DNA" that may represent raw material for the evolution of new genes. Simple sequences of 10-12 base pairs are sometimes repeated millions of times. Long interspersed nuclear elements move by copying themselves and enlarge the genome. They may be remnants of retroviral integrations and some produce active reverse transcriptase. Short interspersed nuclear elements may be implicated in some genetic diseases and cancers. They include Alu elements, which are fragments of a normal gene which can insert in new places in the DNA. Highly expressed genes are present in several hundred copies.
What is the Wobble Hypothesis and how does it explain why some tRNA's can bind to more than one codon? What is special about the third base in some codons?
According to the wobble hypothesis, some aminoacyl-tRNAs recognize more than one codon because pairing of the third base of a codon is less crucial than that of the other two. This explains why some tRNAs can bind to more than one codon; the 5' base on the anticodon triplet, which binds to the 3' base on the mRNA triplet, is not as spatially confined and can exhibit non-standard base pairing.
What are alternate promoters and how and when do they function?
Alternative promoters are sequences located upstream or downstream of a known promoter that are used to direction transcription initiation at a gene. They function when there are changes in the environment such as temperature or nutritional status.
What is the nature of the triple codons that code for amino acids? In this context with respect to the genetic code, what is the meaning of the terms non-overlapping, lacking punctuation and degenerate?
Amino acids are encoded by groups of three bases starting from a fixed point. The genetic code is non-overlapping (codons are separate from each other) and lacking punctuation (no sudden uncoded stops or other directions). The genetic code is degenerate in some cases, meaning some three codons code for the same amino acid.
What is the mechanism of peptidyl transfer and how does the amino nitrogen of the incoming aminoacyl-tRNA attack the peptide unit of the peptidyl-tRNA?
An amino group from the aminoacyl-tRNA in the A site performs a nucleophilic attack on the carbonyl carbon of the aminoacyl-tRNA (peptide) in the P site. A new peptide bond is forged and the link between the peptide in the P site and its tRNA is severed.
With respect to gene expression in prokaryotes, what is an operon? What are operators and what are promoters? How are multiple genes linked in operons? What are the functions of the operator and promoter regions?
An operon is a unit of coordinated genetic expression. Operators and promoters are control sites of an operon. The operator is a segment of DNA that a repressor binds to, while the promoter is the nucleotide sequence that enables a gene to be transcribed. Regulatory genes encode proteins that interact with the operator and promoter sites to stimulate or inhibit transcription. A regulatory gene may or may not be closely linked to the operon where the gene's protein product is working to stimulate or inhibit transcription. Regulatory genes code for repressor proteins. Inducers can displace repressor proteins from the operator site, resulting in an uninhibited operon. Alternatively, a corepressor can bind to the repressor to allow its binding to the operator site.
How do single stranded regions of RNA hybridize to form stem loop structures?
Base pairing occurs between a single-stranded RNA's nucleotides when they are complementary to each other.
How could the acetylation of histone proteins result in the breakdown of histone octamers and lead to increased gene expression?
Coactivators catalyze the addition of acetyl groups to lysine residues in the tails of histone proteins. This decreases the affinity of the histones for DNA, making additional genes accessible for transcription. Additionally, acetylated histones are targets for proteins containing specific binding units called bromodomains. Two classes of large complexes are eventually recruited: chromatic remodeling engines and RNA polymerase II (and its associated factors). These complexes open up sites on chromatin and initiate transcription.
The discovery of restriction enzymes and ligases led directly to which technological advance in molecular biology?
DNA cloning
How can the Solid Phase Synthesis of DNA Oligonucleotides be used to create specific probes and primers?
DNA fragments of a known sequence can be synthesized using the solid-phase phosphite triester method. The first nucleotide is connected to a resin bead for ease of purification. The nucleotide has a free 5' hydroxyl group that reacts with the activated deoxynucleotide compound, deoxyribonucleoside 3'-phosphoramidite. Then, the phosphorus atom is oxidized. Finally, a protecting group is removed from the new 5' hydroxyl group, and the whole cycle can be repeated.
What is a DNA microarray and how can such and array be used to diagnose a disease or measure changes in gene expression?
DNA microarrays consist of an arrayed series of thousands of microscopic spots of DNA oligonucleotides called features. Each of these features contain picomoles of a specific DNA sequence and are known as probes or reporters. These probes can be a short section of a gene or other DNA element that are used to hybridize a cDNA or cRNA sample (called target) under high-stringency conditions. Since an array has so many probes, a microarray experiment can accomplish many genetic tests in parallel.
What is the definition of DNA replication? What is the template, and what is the product? What is the energy source for replication? What is the other product of replication in addition to additional nucleotide unit on the growing DNA chain?
DNA replication is the process by which parental DNA is copied. The template is the strand that is copied, while the product is the new strand synthesized by DNA polymerase. The template can be single or double-stranded DNA. The other product of replication in addition to additional nucleotide units on the growing DNA chain is a phosphate group (PPi).
How do the correct amino acids become attached to tRNA's by aminoacyl-tRNA synthases? What is the energy source for this process? How does the attachment of an amino acid to a tRNA energize the amino acids for the process of forming a protein polymer?
Each amino acid is activated and linked to a specific transfer RNA by an ATP-dependent enzyme called an aminoacyl-tRNA synthetase. This creates an ester linkage between the carboxyl group of an amino acid and the 2' or 3'-hydroxyl group of the adenosine unit of a CCA sequence at the 3' end of the tRNA. ATP is the energy source for this process, as it is coupled to the hydrolysis of ATP to AMP and inorganic pyrophosphate. It takes one ATP going to AMP and two Pi's to charge each amino acid onto its tRNA. It takes two GTP's going to GDP and Pi to drive the elongation phase of protein synthesis. That means it requires the hydrolysis of four phosphoanhydride bonds to forge one peptide bond during protein synthesis.
How can both strands of the DNA serve as templates for RNA synthesis? What does it mean that only selective segments of the DNA are transcribed and how is this accomplished?
Either strand of DNA can serve as a template for RNA synthesis, depending on which genes are being transcribed. Only selective segments of DNA are transcribed; promoters initiate transcription of certain genes.
What added element of regulation is found in human gene expression? What are histones, histone octamers and nucleosomes? How does the present of nucleosomes block gene expression?
Eukaryotic DNA is tightly bound to basic proteins called histones to form chromatin. DNA wraps around an octamer of core histones to form a nucleosome, blocking access to many potential DNA binding sites. The nucleosome consists of a core of eight histone proteins surrounded by DNA. The DNA forms a left handed superhelix as it wraps around the core of histone proteins.
Given that there are only 20 amino acids used for protein synthesis, why are there more than 20 tRNAs? Give that there are 61 codons for amino acids why aren't there 61 tRNAs?
For many of the amino acids there is more than one tRNA. However, there is not a tRNA for all 61 codons; some tRNAs can read up to three triplets. This is particularly true when the 5' base on the anticodon is the nonstandard inosine molecule (wobble).
The protein that moves directionally along a nucleic acid phosphodiester backbone, separating two annealed nucleic acid strands is called?
Helicase
What role does helicase play in strand separation and what is supercoiling of DNA?
Helicase enzyme slides along single stranded DNA, gradually unwinding it. The A1 and B1 domains of the helicase have a cleft between them that closes when ATP is bound, causing domain A1 to slide along the DNA. When ATP is hydrolyzed, the cleft between the two domains opens, pulling the DNA from domain B1 toward domain A1. A consequence of strand separation is that additional strain is introduced into the DNA molecule, causing it to be overwound in surrounding regions. These supercoiled regions of DNA are referred to as topoisomers.
What three components that bond the two strands of DNA together must be overcome for replication to occur?
Hydrogen bonds holding together base pairs, stabilization by aromatic stacking of successive bases, and unwinding of DNA strands.
What is IPTG and how is it used to study lac repression?
IPTG stands for isopropylthiogalactoside and is an artificial analog of 1,6-allolactose. It binds to the lac repressor and causes the repressor protein to dissociate from the operator, which greatly enhances the production of lac mRNA by RNa polymerase. IPTG is not metabolized by E. coli, so its level in the cell can be accurately controlled by researchers.
Which bases pair in DNA and which pair in RNA? How do these bases bond to each other? Why is GC bonding a bit stronger than AT bonding. Why do the base pairs always involve one purine and one pyrimidine?
In DNA, adenine and thymine hydrogen bond (one hydrogen bond between the hydrogen atom of the 6-amino group of adenine and carbonyl oxygen atom of thymine, the other between one of the of the ring nitrogen atoms of adenine and a hydrogen atom attached to a ring nitrogen atom of thymine) while guanine and cytosine hydrogen bond (between a carbonyl oxygen of guanine and an amino group of cytosine, a hydrogen attached to a nitrogen atom on the guanine ring to a nitrogen atom on the cytosine ring, and an amino group of guanine to a carbonyl oxygen of cytosine). In RNA, thymine is replaced with uracil and still hydrogen bonds with adenine. GC bonding is a bit stronger than AT bonding due to aromatic stacking forces (collective affinity of the base pairs) and a greater number of hydrogen bonds. The base pairs always involve one purine and one pyrimidine because if both pairs were purines that would be too far apart, while if they were both pyrimidines they would be too close together.
What is the role of IF 1, IF 2, IF 3, GTP, f-Met-tRNA, 30S ribosome, and the Shine-Dalgarno Sequence on the mRNA in the initiation of translation?
In prokaryotes, a mRNA, fMet-tRNAf, GTP, and 30S ribosomal subunit all come together with the assistance of three initiation factor proteins (IF 1, IF 2, and IF 3) to form a 30S initiation complex that binds specifically to the Shine-Dalgarno sequence near the 5' end of the mRNA. The fMet-tRNA becomes oriented on the first AUG codon and IF 3 diffuses away. Next, the 50S ribosomal subunit is bound, and IF 1 and IF 2 are released. The hydrolysis of GTP to GDP and Pi also occurs in this phase. The complete 70S ribosome is now assembled and elongation can begin. The important of the Shine-Dalgarno sequence is that is results in initiation at the nearest AUG. This insures that protein synthesis will proceed from the correct start codon and in the right reading frame.
How does the site of synthesis and RNA processing differ from prokaryotes to eukaryotes? What do the terms capping, addition of a polyA tail, and splicing mean with respect to RNA processing? What is the site of mRNA synthesis in eukaryotes and what is the site of translation?
In prokaryotes, transcription and translation both take place in the cytoplasm, only one RNA polymerase is involved, and there is little processing of mRNA. In eukaryotes, transcription and processing take place in the nucleus and translation takes place in the cytoplasm. There are three DNA polymerases with divergent functions. RNA polymerase I is located in the nucleoli and transcribes the genes of some ribosomal RNA, RNA polymerase III is located in the nucleoplasm and synthesizes other ribosomal RNA (5S RNA) and tRNA, and RNA polymerase II is located in the nucleoplasm and synthesizes the precursors of mRNA as well as several small RNA molecules, like those responsible for splicing. Eukaryotic RNA is processed by capping at the 5'-end (addition of a 7-methylguanylate residue attached by a triphosphate linkage to the terminal ribose group and often methylation of the last two ribose residues), addition of a poly-A tail (at the 3' end with about 250 adenylate residues), and removal of introns (splicing of exons).
What is 1,6-allolactose and how does its binding to the lac repressor relieve repression?
In the presence of lactose, the binding of 1,6-allolactose (a product of lactose metabolism in E. coli) to the lac repressor induces a conformational change that leads to the dissociation of the repressor protein from the lac operator. RNA polymerase is then free to move through the operator and transcribe the three linked genes of the lac operon.
Where and how are introns removed from eukaryotic RNAs? What is the mechanism of RNA splicing?
Introns are removed from eukaryotic RNA after the poly-A tail and 5' cap are added. The first exon is attacked at its 3' end by the 2'- OH group of a specific adenosine residue (the branch-site adenosine, which forms a lariat intermediate with the 5' ribose of the first intron) within the first intron). The newly formed 3' - OH of the upstream exon then attacks the splice site between the first intron and the downstream (second) exon. The first and second exons are joined, and the first intron containing the lariat structure is released.
What are introns and what are exons? What role does each of these sequences play in normal gene expression in humans? How does the presence of introns facilitate gene shuffling and the creation of new protein activities? How are RNAs containing introns processed in the nucleus prior to export into the cytosol?
Introns are sequences of DNA within the gene that do not code for amino acids in the final protein product. Exons are the coding regions of genes. The presence of introns facilitates gene shuffling and the creation of new protein activities by allowing various domains to be recombined. RNAs containing introns are processed in the nucleus prior to being exported into the cytosol. This is done by splicing at consensus sequences of GU at the 5' end of the intron and AG at the 3' end of the intron. At these sites, the intron is cut out and the ends of the exons are spliced together.
How is eukaryotic mRNA modified at its 3' end?
It is polyadenylated.
What is the chemical nature of N-formyl-methionine? Where is this amino acid derivative found in many prokaryotic proteins and what is the codon for this amino acid? How does this relate to the start site for protein synthesis?
Methionyl-tRNA is formed by a specific methionyl-tRNA synthetase. Then, a transformylase enzyme adds a one carbon formyl group to the alpha-amino group of the methionine residue. The formyl group donor in this reaction is the coenzyme N10-formyl tetrahydrofolate, a derivative of folic acid. In prokaryotic organisms, N-formylmethionyl-tRNA is generally the first amino acid at the N-terminal end of proteins. The codon for this amino acid is AUG. In most prokaryotic genes, an AUG codon downstream from the Shine-Dalgarno sequence is recognized by a special tRNA, tRNAf, carrying N-formylmethionine.
How does transcription differ from replication with respect to initiation, strand separation, and editing?
Only the template strand is transcribed into RNA; the coding strand is not used for this purpose. RNA synthesis can be initiated at specific locations without a primer, unlike DNA synthesis, which requires a primer for initiation. Unlike DNA synthesis, there is no permanent separation of the two DNA strands during transcription; it occurs in open bubbles that hybridize back together. There is no editing or correction of RNA products buy nuclease activity, while there are mechanisms to fix damaged or incorrect DNA.
How is PAGE electrophoresis used to identify DNA restriction fragments?
PAGE electrophoresis (polyacrylamide gel electrophoresis) is a method of separating fragments of DNA that have been cut with restriction endonucleases. The fragments migrate at a rate that is inversely proportional to the log of the number of base pairs (smaller fragments migrate faster than larger fragments).
What is the Polymerase Chain Reaction Method (PCR), and how is it used to amplify small samples of DNA? What is the special ingredient used in PCR that allows for repeated temperature cycling during the amplification of DNA? What are the components of a PCR system?
PCR is a method for amplifying small samples of DNA. The components of a PCR system are the small sample of DNA to be amplified, two oligonucleotide primers (one for each of the two complementary DNA chains being replicated), four deoxynucleotide triphosphates, and Taq polymerase. Taq polymerase is the "special" component of PCR that allows the system to undergo repeated cycles of heating and cooling, as it is a heat stable DNA polymerase isolated from thermophilic bacteria. The protocol for PCR involves 20-30 repeated cycle of heating and cooling. First, a duplex DNA sample is heated to 95 degrees celcius for fifteen seconds to separate the strands. Then, the solution is cooled to 54 degrees celcius and short DNA primers are allowed to hybridize to each end of the gene. Finally, the solution is raised to 72 degrees celcius and Taq DNA polymerase is used to copy each strand. This process is then repeated.
What are phosphodiester bonds, and how do these bonds form the covalent backbone of DNA and RNA?
Phosphodiester bonds link the nucleotides of DNA in a strand together. These form the covalent backbone of DNA and RNA.
In the structure of ATP the inner phosphate group is joined to the 5' carbon of ribose by what type of bond?
Phosphoester
What is a plasmid and how are plasmids used to clone and amplify DNA samples?
Plasmids are small pieces of circular DNA that can replicate independently in a host organism. Plasmids are used to carry and amplify cloned fragments of DNA by inserting foreign DNA into the plasmid vector and allowing host bacteria to be grown for many generations.
What is primer walking and what is shotgun sequencing?
Primer walking is a method of characterizing a long fragment of DNA. First, a DNA fragment with a known base sequence that occurs at one end of a longer region is isolated. That region of the DNA is sequenced. At that point, a new probe can be synthesized that corresponds to the far end of the first DNA sequence. The new probe can then be used to characterize the DNA that is further along in the sequence. The process is repeated until the whole length of the DNA has been sequenced. Shotgun sequencing is another method of sequencing long DNA strands. It is like primer walking but uses random fragments rather than sequential fragments.
What is a "transcription bubble", and what is the role of RNA polymerase in creating this bubble?
Prior to transcription of an area of DNA, the RNA polymerase unwinds about 17 base pairs of template DNA to produce an open :transcription bubble". The DNA is unwound at the rear end of the polymerase as transcription of an area is completed.
What part of the ribosome serves as the site of protein synthesis and what component of the ribosome acts catalytically in protein synthesis? What is the meaning and function of 30S, 50S and 70S ribosome structures?
Prokaryotic organisms have a 70S ribosome, while eukaryotes have a 80S ribosome. In 70S ribosomes, a smaller 30S subunit consists of a 16S component and about 21 different proteins. The 50S subunit consists of one 23S rRNA and one 5S rRNA, and at least 31 different proteins. The large and small ribosomal subunits can exist independently and are brought together on the messenger RNA when protein synthesis occurs. The interaction of 16S rRNA and mRNA helps the 30S subunit to recognize the starting end of mRNA. The 50S subunit joins with the 30S subunit, mRNA, and charged tRNA to form the 70S initiation complex. The site of peptide bond synthesis lies within a central cavity in the 50S ribosome. The 23S rRNA is a ribozyme and is responsible for catalyzing peptide bond formation between successive amino acids.
What are the multiple functions of the RNA polymerase enzyme complex in transcription?
RNA polymerase searches DNA for initiation sites (promoters), unwinds a short stretch of double-helical DNA to produce a single-stranded template, reads the template, selecting correct ribonucleoside triphosphates and synthesizing phosphodiester bonds, detects termination signals that specify where a transcript ends, and interacts with activator and repressor proteins that modulate the rate of transcription initiation over a wide range.
How are the components necessary for RNA synthesis similar to those required for DNA replication? How do transcription requirements differ from those needed for replication?
RNA synthesis is similar to DNA replication in that the direction of synthesis is 5' to 3', the mechanism of elongation is similar, and the synthesis is driven forward by the hydrolysis of pyrophosphate. However, RNA polymerase does not require a primer and lacks the nuclease capability mused by DNA polymerase to excise mismatched nucleotides.
What feature is found in DNA synthesis but NOT in RNA synthesis?
Repair mechanisms which remove incorrect bases
What are Restriction Fragment Length Polymorphisms and how are such fragments used to identify genetic mutations? Does the mutation of interest have to cause the change in fragment length directly, or can it be a change that is linked to the mutation but not involved in the actual mutation?
Restriction Fragment Length Polymorphisms are mutations within restriction sites that change the sizes of restriction fragments and thus the positions of bands in Southern-blot analyses (polymorphism = existence of genetic diversity in a population). The mutation may cause disease or be closely linked to one that does.
The process of cutting DNA strands at specific sites is accomplished by?
Restriction endonucleases
What are restriction endonucleases and what do they do to DNA?
Restriction endonucleases are hydrolytic enzymes that make cuts in distinct sites of DNA.
What are sticky ends and blunt ends in restriction sites? How do sticky ends lend themselves to cloning and what has to be done to make blunt ends usable for cloning experiments?
Restriction enzymes can leave sticky ends, which can hybridize with each other, or cut evenly, leaving a blunt cut. Sticky ends in cloning allow the ends of DNA to recognize each other by base pairing. They then stick together and can be joined through the use of a ligase enzyme. DNA lacking cohesive ends can be given cohesive ends by the addition of chemically synthesized linkers using T4 ligase. The linkers are attached to the ends of the DNA fragments then cleaved with a restriction endonuclease.
What are restriction sites? In what sense are restriction sites palindromes and in what sense are they not palindromes? What does it mean that the sequences of the two DNA chains within a restriction site are inverted, identical copies of each other?
Restriction sites are the positions where the restriction enzymes are cut. They contain a two-fold axis of symmetry; they are inverted, identical copies of one another. Restriction sites are sometimes called palindromes because they are inverted identical copies of one another. However, the palindromes are on two different chains. For example, the palindrome GGAAGG would have GGA on one chains and AGG on the other. EcoR1 Restriction Endonuclease: GAATTC CTTAAG
What enzyme can be used to make cDNA? What is cDNA and how was it used in the cloning of the insulin gene?
Reverse transcriptase can be used to make cDNA. cDNA, or double-stranded copy DNA, is the DNA complementary to a given RNA. It is possible to use cDNA in an E.coli expression system to produce mRNA, and then to translate that mRNA into the corresponding protein. The cDNA is cloned into a plasmid that contains a highly active bacterial promoter upstream from the inserted cDNA. The plasmid is transformed into E. coli. and the bacteria are grown and plated to give hundreds of single colonies which synthesize the desired protein. These can be identified by Western blotting. This method was used to clone the human insulin gene.
Two genes are said to be highly homologous if they
Share significant similarity in their sequence
What is the name of the sequence on the mRNA, which is upstream from the initiation codon and which helps to establish the correct reading frame?
Shine Delgarno
What type of genetic mutation causes sickle cell anemia? What dramatic change occurs in Red Blood Cells in sickle cell anemia?
Sickle cell anemia is caused by a substitution mutation. This results in an amino acid change at position 6 in the beta chain of hemoglobin (glutamate to valine) that changes the solubility of hemoglobin. The hemoglobin forms long rod-like structures that distort the shape of the cell. Ultimately, these distorted cells burst and the hemoglobin is degraded, causing anemia (the reduced capacity to transport oxygen in the blood).
Using the _____ method a specific DNA fragment of interest can be detected with a radioactive DNA probe.
Southern Blot
What is Southern Blotting and how is it used to identify specific fragments of DNA? What is a DNA probe? What is Northern Blotting and how does it differ from Southern Blotting? What is Western Blotting?
Southern blotting is a technique that uses a DNA probe, which is DNA that contains a sequence of interest that specifically hybridizes to a DNA sample containing a complementary sequence. In Southern Blotting, the DNA sample is cleaved with a restriction endonuclease and undergoes electrophoresis. The separated DNA fragments are transferred to a sheet of nitrocellulose paper, where is is hybridized to the radioactive DNA probe. The sample is then washed and placed in contact with X-ray film. The DNA sample band that is hybridized to the radioactive probe can be identified by its reaction with th X-ray film. In Northern Blotting, RNA is analyzed rather than DNA. Western blotting uses specific antibodies to identify proteins of interest. E. coli is infected with the plasmid carrying the gene of interest. Individual bacterial colonies are grown up on an agar plate. The bacterial colonies are then lysed to expose the protein, and the protein spots are transferred to a nitrocellulose filter. A radiolabeled antibody, specific for the protein of interest, is added. The filter is placed on X-ray film, and the dark spots on the film are used to identify the colonies containing the protein.
During the elongation of the growing peptide how does elongation factor TU deliver the aminoacyl-tRNA to the A site?
TU delivers the amino acid to the A site in a GTP-dependent reaction.
In what direction are the template strands of DNA read, and in what direction are the new DNA strands synthesized?
Template strands are read in the 3' to 5' direction and new strands are synthesized in the 5' to 3' direction.
What is the meaning of "melting" of hybridized strands of DNA? What factors determine the melting temperature?
The "melting" point of hybridized strands of DNA is the point at which half of the double-stranded DNA is changed to single-stranded DNA. Factors the determine the melting temperature are the GC content of the nucleic acid sample, the sodium ion concentration (sodium shields the negative charges of the sugar-phosphate backbone from interacting with each other, increasing stability), and the DNA hybrid length.
What are the 3' and 5' ends of DNA and RNA?
The 3' end of DNA and RNA is the end of the strand that contains the 3' hydroxyl group of the terminal deoxyribose. The 5' end is the end with the phosphate residue of the first nucleotide.
What is the Catabolite Activator Protein (CAP) and how does that protein function in the regulation of the lac operon? Why is the expression of the lac operon low when glucose is present in high levels? How is cAMP involved in the activation of the lac operon?
The CAP activates transcription of the lac operon by directly contacting RNA polymerase. When the level of cyclic AMP (a hunger signal) rises in the cell, cAMP stimulates the transcription of many catabolic operons by binding to the CAP. Cultures of E. coli growing in the presence of glucose do not synthesize beta-galactosidase, even when lactose is present in the growth media. The synthesis of beta-galactosidase only occurs after glucose has been depleted. Thus, the presence of glucose in the growth media suppressed the expression of the lac operon. When glucose is high in the growth medium, it enters the cell and inhibits the synthesis of cAMP, reducing the level of the cAMP-CAP complex that is needed for efficient binding of RNA polymerase to the lac operon.
In what direction is the DNA template read for transcription and in what direction is the RNA synthesized? In what way are the basic chemical mechanisms used for replication and transcription the same?
The DNA template is read for transcription in the 3' to 5' direction. RNA is synthesized in the 5' to 3' direction. The basic chemical mechanisms used for replication and transcription are the same; the 3' hydroxyl group of the terminal ribose attacks the alpha phosphate residue of the incoming nucleotide.
What is the nature and function of the E. coli lac operon? What genes are present in the lac operon and how are those genes linked to each other and to the upstream control region? What is the location of the lac repressor gene?
The E. coli lac operon consists of a series of clustered genes that are under coordinate regulation by a closely linked upstream control region. It consists of three genes that code for three enzymes: beta-galactosidase, galactoside permease, and thiogalactoside transacetylase. Upstream from these genes are an operator and a promoter region. Interactions between these control sites and regulatory proteins determine the degree of Lac gene expression. Closely linked to the operon is a regulatory gene with its own promoter. This regulatory (repressor) gene produces a protein that inhibits Lac gene expression.
What is the structure of the RNA polymerase and what special role does the sigma subunit play in the initiation of transcription?
The RNA polymerase complex consists of five subunits. Four of these subunits are always part of the complex (a2bb'), which the sigma subunit is a transient component of the complex. The sigma unit's function is to recognize promoter sites. It decreases RNA polymerase's affinity for general regions of DNA by a factor of 10^4.
What is Shine-Dalgarno sequence, and what is its role in the initiation of protein synthesis? At what codon is protein synthesis generally initiated?
The Shine-Dalgarno sequence is a DNA sequence upstream from the first AUG codon that binds to a complementary region on the 16S component of the 30S ribosome. The first AUG codon downstream from the Shine-Dalgarno sequence is recognized by tRNA carrying N-formylmethionine.
How does the Shine-Dalgarno sequence function in the initiation of protein synthesis? Where is the Shine-Dalgarno sequence located with respect to the AUG codon where translation begins? How does this sequence participate in the assembly of the ribosome complex?
The Shine-Dalgarno sequence, which is purine rich and located upstream from the first AUG codon, functions in the initiation of protein synthesis by binding to a complementary region on the 16S component of the 30S ribosome. The first AUG codon downstream from the Shine-Dalgarno sequence is recognized by a tRNA carrying N-formylmethionine.
What is the role of the anticodon on a tRNA and how does this anticodon interact with the codons on the messenger RNA?
The anticodon on a tRNA is the template-recognition site. It hybridizes to the complementary triplet codon on the messenger RNA.
What is the apparent chemical paradox in the synthesis of the lagging DNA strand and how is that problem solved? How does the DNA polymerase III function in lagging strand synthesis, and what components are involved repeatedly for lagging strand synthesis but not for leading strand synthesis? What are Okazaki fragments?
The apparent chemical paradox in the synthesis of the lagging DNA strand is that the new flow of the synthesis is in the 3' to 5' direction, but the DNA polymerase reaction only runs in the 5' to 3' direction. This problem is solved by replicating the lagging strand in short pieces called Okazaki fragments in the 5' to 3' direction, then linking them together with ligase later. DNA polymerase III is a dimer with two equivalent regions of catalytic activity that is responsible for replicating both strands of DNA. However, in lagging strand synthesis, one dimeric section travels down short sections which have looped around so they are also read 3' to 5'. The discontinuous replication of lagging strand DNA is primed by short stretches of RNA, which are synthesized by a primase enzyme. The primer RNA is later removed by DNA polymerase I, which also fills in the gap with the appropriate deoxynucleotides.
How does the cAMP-CAP complex function in stimulating the expression of the lac operon? How does that complex influence the binding of RNA polymerase?
The binding of the cAMP-CAP complex to a specific site in the promoter region of an inducible catabolic operon enhances the binding of RNA polymerase and the initiation of transcription.
What is the central dogma of molecular biology? What does it mean that DNA serves as a template for DNA and RNA synthesis, but not as a precursor or substrate for DNA and RNA synthesis? Similarly, what does it mean that RNA serves as a template for protein synthesis, but not as a precursor or substrate for protein synthesis?
The central dogma of molecular biology concerns the flow of information from DNA, the hereditary storage of genetic information, to RNA, and then from RNA to protein. The arrows in the scheme do not signify chemical conversion; they represent the information transfer.
With respect to DNA replication what does the central dogma of molecular biology tell us?
The central dogma tells us that in replication, the DNA is not converted into new DNA; rather, the parental DNA strands serve as templates for the synthesis of new DNA.
What is the direction of reading the template strand of mRNA and how is this different than the reading of DNA in replication and transcription?
The direction of reading the template strand of mRNA is 5' to 3', in contrast to replication and transcription in which DNA and RNA polymerases read in the 3' to 5' direction.
During the elongation process how does elongation factor G help move the growing peptidyl-tRNA from the A site to the P site and also help move the deacylated tRNA to the E site?
The elongation factor G catalyzes the translocation of the peptidyl-tRNA from the A site into the P site and the deacylated tRNA into the E site through a GTP-dependent reaction.
Which codon is generally the first one read, and for which amino acid does it code at the beginning of a prokaryotic protein? For which amino acid does this triplet code within the internal sequence of a protein?
The first codon to be read is typically AUG, which codes for methionine.
What are five basic requirements for the synthesis of new DNA?
The five basic requirements for the synthesis of new DNA are a DNA template that is copied and directs the synthesis of the complementary DNA sequence, a primer that is the initial sequence of DNA that provides a 3' hydroxyl that attacks the incoming nucleotide, four deoxynucleotide triphosphates that provide the nucleoside monophosphate unit that is added to the growing chain of DNA, a DNA polymerase enzyme that catalyzes the process of DNA elongation, and metal ions (like magnesium) and several other protein factors.
How does growth on lactose stimulate the expression of the lac operon? What is the mechanism by which the presence of lactose reduces the repression of the lac operon?
The growth of E. coli on lactose results in a dramatic increase of production of the three genes that are encoded in the lactose operon; otherwise, they are barely detectable. In the presence of lactose, the binding of 1,6-allolactose (a product of lactose metabolism in E. coli) to the lac repressor induces a conformational change that leads to the dissociation of the repressor protein from the lac operator. RNA polymerase is then free to move through the operator and transcribe the three linked genes of the lac operon.
The critical ingredient in PCR which allows for multiple rounds of DNA synthesis is?
The heat resistant DNA polymerase
What is role of an origin of replication in initiating DNA synthesis, and how do these elements generally function in initiating DNA synthesis in bacteria vs humans?
The initiation of DNA replication begins at a special sequence called the origin of replication. Prokaryotic chromosomes have a single origin of replication, while eukaryotes have multiple. Replication proceeds bidirectionally from the origin, which yields two sites where DNA is synthesized called replication forks.
What is the lac repressor protein and how does it function?
The lac repressor protein can exist as a dimer of 37-kd subunits. Two dimers often come together to form a tetramer. In the absence of lactose, the repressor binds very tightly and rapidly to the operator. It inserts an alpha helix into the major groove of operator DNA, preventing transcription.
What is the leading strand of DNA and what is the lagging strand? What is the net flow of leading strand synthesis and what is the net flow of lagging strand synthesis? Given that the chemistry of DNA strand synthesis is always 5' to 3', how can the net flow of lagging strand synthesis be 3' to 5'?
The leading strand is the strand of DNA that is synthesized in a continuous fashion, whereas the lagging strand is synthesized in fragments in a discontinuous fashion. The net flow of the leading strand is 5' to 3' while the new flow of the lagging strand is 3' to 5'. However, synthesis on the lagging strand actually occurs with small segments of DNA in the 5' to 3' direction which are then linked together.
The critical ingredient in the Sanger method for DNA sequencing which results in the termination of DNA synthesis at various points is _____?
The one dideoxy-NTP
What is the function of an origin of replication in a plasmid? What functions do antibiotic resistant genes serve in a plasmid? How can a reporter gene be used to verify that a plasmid is present in a bacteria?
The origin of replication in a plasmid allows it to replicate autonomously. Antibiotic resistant genes in plasmids serve as a way to select bacteria that contain the genes of interest, as the organisms that do not have the plasmid will die when grown in a media containing the bacteria. To verify that there is indeed a DNA insert in a plasmid, it is common to use a plasmid containing a reporter gene, which has a disrupted product when the plasmid contains the insert (example: lacZ gene no longer codes for beta-galactosidase when foreign DNA is inserted into the middle of it).
What is the process of DNA replication? What is semiconservative replication? Which DNA strands serve as templates for replication?
The process of DNA replication is the copying of a parental copy of DNA to produce two copies of progeny DNA. In semiconservative replication, in the first generation of DNA synthesis each daughter molecule of DNA contains one parental strand and one newly synthesized strand. In each round of DNA replication, each parent strand synthesizes a complementary daughter strand. In DNA replication, a parent strand serves as a template strand for replication.
During DNA synthesis in what direction is the template strand read, and in what direction is the product DNA synthesized? How does this directionality relate to the overall mechanism of DNA synthesis? What hydroxyl group on the 3' end of the growing DNA strand attacks the incoming deoxynucleotide? What is the leaving group from this reaction, and what happens to that leaving group? How does this energize DNA formation?
The synthesis of DNA proceeds from the 5' to 3' end, though it is read in the 3' to 5' direction. The 3' hydroxyl group of the terminal deoxyribose attacks the inmost (alpha) phosphate residue of the incoming deoxynucleotide triphosphate. The leaving group of this reaction is a pyrophosphate group, which is hydrolyzed by phosphatase enzyme to release energy. This energizes DNA formation by allowing a phosphodiester bond that attaches the remaining phosphate to the growing chain to form.
How do the DNA template strand and coding strand compare to the RNA product? What does it mean the template strand and the RNA product are antiparallel? Which DNA strand has essentially the same sequence as the RNA product (except for T vs U)?
The template strand and the RNA product are antiparallel, so they run in opposite directions. The coding strand of DNA has essentially the same sequence as the RNA product. The template strand has the complementary sequence.
In what direction is the template strand copied and in what direction is the product RNA synthesized?
The template strand is synthesized in the 5' to 3' direction and read by RNA polymerase in the 3' to 5' direction.
How does the central dogma apply to transcription?
The template strand of DNA provides the information for messenger RNA synthesis. The messenger RNA and various transfer RNA molecules act as intermediaries between DNA and protein. While the flow of information from DNA to RNA involves the same "language" (a sequence of bases encodes information in both type of nucleic acid), the flow of information from mRNA to protein involves a translation from one language to another (a sequence of three base units in RNA is converted to a sequence of single amino acids in the protein product).
What is the mechanism of termination of transcription and what role does the RNA product play in this process?
The termination of transcription in prokaryotic organisms occurs when the RNA product forms a stable hairpin structure followed by a sequence of uridine residues. As some sites, transcription is terminated in an alternate way by the action of a rho protein.
What three types of RNA are involved in translation? What is the role of each of these three RNA's in protein synthesis? What is the size range of each of these RNA types? How does ribosomal RNA function along with various proteins to provide both the site of protein synthesis and the catalytic activity that accomplishes protein synthesis? Is protein synthesis catalyzed by ribosomal RNA or by a ribosomal protein?
The three kinds of RNA involved in translation are mRNA, which serves as the template for protein synthesis; tRNA, which carriers amino acids in an activated form to the ribosome for peptide-bond formation; and rRNA, which constitutes the major component of ribosomes. The average length of an mRNA molecule is about 1.2 kilobases, while the average length of tRNA and molecules is 25 kd. rRNA molecules contain about 3.7 kb (23S), 1.7 kb (16S), or 0.12 kb (5S). rRNA associates with a set of proteins to form ribosomes, which move along an mRNA molecule and catalyze protein assembly. Ribosomes are also responsible for binding tRNA and other necessary accessory molecules.
What are the three stop codons and how does a stop codon lead to the termination of peptide synthesis?
The three stop codons are UAG, UAA, and UGA. Releasing or termination proteins recognize these codons. The termination factors hydrolyze the bond between the protein and the tRNA. After hydrolysis severs the link to the tRNA, the protein diffuses away and the ribosomal complex dissociates from the mRNA.
What three types of RNA function in translation and what is the role of each of these RNA types?
The three types of RNA that function in translation are messenger RNA, which are the template for protein synthesis, transfer RNA, which carries amino acids in an activated form to the ribosome for peptide-bond formation, and ribosomal RNA, which constitutes the major component of ribosomes and play a catalytic and structural role in protein synthesis.
What is the traditional Sanger dideoxy sequencing method? How does this method use an in vitro DNA replication system to create DNA fragments that end at specific nucleotides? What are the components of this system and what "special" component allows the system to be used for DNA sequencing?
The traditional Sanger dideoxy sequencing method uses a template DNA strand, a primer molecule (complementary to the template DNA, with a 3' hydroxyl to be added on to), 4 deoxynucleotide triphosphates (radioactively labeled), 1 dideoxynucleotide triphosphate, DNA polymerase, Mg2+, and several other protein factors to sequence DNA. Four reaction mixtures are made, each with a different dideoxy-nucleotide triphosphate. The appropriate amount of a dideoxynucleotide triphosphate (the "special" component that allows for DNA sequencing) in the reaction mixture causes DNA elongation to be terminated at positions where that particular nucleotide occurs.
What are the features of double stranded DNA that must be overcome in order to accomplish semiconservative DNA replication? What are the unique challenges that must be overcome to separate base pairs, to break aromatic stacking forces and to unwind the two DNA strands from each other?
The two parental strands of DNA must be completely unwound and separated in the process of replication; all hydrogen bonds between the parental strands must be broken. The DNA helix is stabilized by the energy of aromatic stacking between successive bases in the helix, so this type of stabilization but be broken.
How can four bases be used to generate 64 triplet codons? Which three of the codons are stop/termination codons?
There are 64 possible codons that can be generated with four different bases combined in any order to form triplets (4^3). Three of these code for stop codons: UAA, UGA, and UAG.
What topoisomerases are involved in strand separation, and what is their role and mechanism?
Topoisomerases function to first increase and then to eventually relieve the strain of supercoiled DNA. Topoisomerase II (DNA gyrase) catalyzes an ATP-driven formation of negative supercoils, making the DNA more susceptible to unwinding by helicase. Topoisomerase I cleaves one DNA strand of supercoiled DNA, rotates in a controlled fashion around the other strand, and then religates the cleaved strand. This results in the partial or complete relaxation of supercoiled DNA.
What is a consensus sequence and what role do such sequences play in the initiation of transcription? What type of promoter sequences are found upstream from the start site of transcription in the DNA?
Transcription is initiated on the DNA template starting downstream from promoter sites that contain consensus sequences. Consensus sequences are similar to each other in base composition and sequence, but an individual promoter sequence may vary from the consensus sequence by one or two bases. Prokaryotes have a TTGACA sequence -35 bases upstream and the Pribnow box -10 bases upstream from the transcription start site. Eukaryotes have a CAAT box -75 bases upstream and a TATA box -25 bases upstream from the transcription start site.
What is a 5' triphosphate tag and how is it created?
Transcription is initiated without a primer; the usual first residue is a 5'-GTP or ATP. The first residue transcribed retains a 5' triphosphate tag.
What type of sequence information in the mRNA product leads to the termination of transcription?
Transcription is terminated by various signals. In E. coli, a stable hairpin followed by a uracil sequence leads to dissociate of the RNA from the template DNA.
What is the process of DNA transcription? Which DNA strands serve as templates for transcription? Which strand of DNA is copied during transcription? What does it mean that the coding strand is parallel to the product RNA and has a similar sequence? What does it mean the template strand is antiparallel to the template strand?
Transcription is the process of copying DNA sequence information into RNA sequence information. The coding (or sense) strand of DNA has the same directionality and sequences as the product RNA with U instead of T residues. In contrast, the template (or antisense) strand serves as a template for transcription. It is complementary and antiparallel to the product RNA.
How and where is transcription initiated? What is a promoter region of DNA and how does this region interact with the RNA polymerase complex? What is a consensus sequence? How do variations from the standard consensus sequence influence the efficiency of the promoter?
Transcription is usually initiated at promoter regions (initiation sites) on the DNA template. These promoter regions interact with the RNA polymerase complex and are recognized by the sigma subunit. The holoenzyme (all five subunits) slides along the DNA unit it encounters a promoter region. A consensus sequence is a sequence of DNA having similar structure and function in different organisms. Variations from the standard consensus sequence can increase or decrease the rate at which its gene is transcribed (sequences with less variation from the consensus sequence are typically more efficient promoters).
In addition to mRNA what other types of RNA are produced by transcription?
Transfer RNA, which brings amino acids to ribosomes during translation; ribosomal RNA, which along with ribosomal proteins makes up the ribosomes; and small nuclear RNA, which form complexes with proteins that are using in RNA processing in eukaryotes.
How do tRNA's serve as a link between the mRNA message and the protein product?
Transfer RNAs function as carriers of amino acids; they also contain anticodon regions that recognize distinct codons in the mRNA.
What are the characteristic features of translation? How many and what types of nucleic acid and protein factors are involved in translation?
Translation is mediated by the interplay of more than a hundred macromolecules, including mRNA, rRNAs, tRNAs, aminoacyl-tRNA synthetases, and protein factors. It takes place on ribosomes and the translation of mRNA occurs in the 5' to 3' direction. The proteins are synthesized in the amino-to-carboxyl direction. The start signal on prokaryotic mRNA is AUG or GUG preceded by a purine-rich sequence that can base-pair with 16S rRNA.
What is translation? How does translation fit into the general pattern of the central dogma? Why is the term translation appropriate for this process?
Translation is the mechanism in which the nucleotide sequence information in mRNA is used to produce the amino acid sequence of a protein. In the central dogma, the mRNA carries nucleotide sequence information that can be translated into a sequence of amino acids in the final protein product.
What are the requirements for RNA polymerase and how do these requirements differ from those for DNA polymerase?
Unlike DNA polymerase, DNA polymerase does not require a primer. It requires a DNA template (which can be double-stranded or single-stranded), all four ribonucleoside triphosphates, a variety of ancillary proteins, and a divalent metal ion like magnesium.
When a DNA probe is generated based on a protein sequence, why would the probe most likely be heterogeneous?
When a DNA probe is generated based on a protein sequence, the probe most likely would be heterogeneous because of the degeneracy of the genetic code.
What functions do the A, P and E sites of the 50S ribosome play in protein synthesis? To which site does the peptidyl-tRNA bind? To which site does the incoming aminoacyl-tRNA bind?
With a tRNA attached to the growing peptide chain in the P site, an aminoacyl-tRNA binds to the A site. A peptide bond is formed when the amino group of the aminoacyl-tRNA attacks the ester carbonyl group of the peptidyl-tRNA in the P site. Upon peptide-bond formation, the tRNAs and mRNA must be translocated for the next cycle to being. The deacylated tRNA moves to the E site and subsequently leaves the ribosome. Then, the peptidyl-tRNA moves from the A site into the P site. The codons of the mRNA form base pairs with the anticodons of the aminoacyl-tRNAs.
What is X-gal and how does it help facilitate the assay of beta-galactosidase?
X-gal is an artificial substrate for beta-galactosidase that is convenient to assay because its cleavage produces a colored product.
In addition to hydrogen bonding, the stability of the double helix between complementary DNA chains is promoted by?
aromatic stacking
Which deoxy-nucleotides are required for replication? What enzyme complex is involved? What is a primer, and how are primers involved in DNA synthesis?
dATP, dGTP, dTTP, and dCTP are required for replication. The enzyme complex involved is DNA polymerase. A primer is a short single strand of RNA or DNA that serves as a starting point for DNA synthesis. A primer is needed to replicate single-stranded DNA; the template DNA must be bound to a primer strand that has a free 3'-hydroxyl group.
What is a ddT-BigDye Terminator, and how do such fluorescent nucleotide derivatives allow for the sequencing of all four nucleotides in one lane of a gel system?
ddT-BigDye terminator consists of dideoxythymidine triphosphate with a dye covalently attached on the side of the pyrimidine ring that is not involved in hydrogen bonding. This compound can pair with an "A" in the template DNA chains and cause chain termination of the new DNA strand. The use of fluorescent nucleotide derivatives allows for the sequencing of all four nucleotides in one lane of a gel system and allows for the sequencing of longer segments of DNA. Each of the derivative fluoresces has a different wavelength so that each one has a distinctive color by which it can be recognized.
In eukaryotes, a mature mRNA that is ready for export to the cytosol contains ____.
exons, but normally no introns
The inhibition of lac operon expression is accomplished by the binding of a _____ protein:
lac repressor
During protein synthesis in Eukaryotic organisms the first amino added to the amino terminal end of a protein is usually?
methionine
All of the following terms are normally associated with recombinant DNA cloning except
proteases
Which type of RNA catalyzes the synthesis of proteins?
rRNA
If a known mRNA can be isolated, it is possible to synthesize the complementary DNA sequence using the enzyme?
reverse transcriptase
What is the general size range of tRNAs. What is the anticodon and what is its function. To what end of the tRNA do amino acids become attached and what is the linkage? What sort of unusual bases are found in tRNAs?
tRNAs consist of about 70-80 nucleotides. An anticodon is a region on transfer RNA that recognizes distinct codons in the mRNA. At the top of the tRNA structure is a step that is made up of both ends of the tRNA. The 3' hydroxyl group on this stem ends in the sequence CCA, and serves as the site of amino acid attachment (ester linkage). tRNA contains uridine rings and pseudouridine, a derivative with the same basic ring system as uridine but a C-C bond between the base and ribose instead of an N-glycosidic bond.