MMBIO 240 Exam 1 Review

energy coupling in biological processes

(1) Transfer of energy from catabolism to anabolism, or transfer of energy from exergonic process to endergonic process. (2) Free energy (from ATP hydrolysis) is coupled or functionally linked to the energy needs of another chemical reaction.

Which of the following DNA molecules (only one strand is shown) will most likely denature at the lowest temperature?

(C's and A's)

ion exchange chromatography

(Charge, DNA is negative) A chromatography process that separates ions and polar molecules based on their affinity to the ion exchanger. It works on almost any kind of charged molecule—including large proteins, small nucleotides, and amino acids. The ion exchanger could be beads with negative (anion) or positive (cation) charge.

peptide bond

(holds amino acids) A peptide bond is a chemical bond formed between two molecules when the carboxyl group of one molecule reacts with the amino group of the other molecule, releasing a molecule of water (H2O). This is a dehydration synthesis reaction (also known as a condensation reaction), and usually occurs between amino acids

chromatography

(separates by dif. speeds)A set of laboratory techniques for the separation of mixtures. The mixture is dissolved in a fluid called the mobile phase, which carries it through a structure holding another material called the stationary phase. The various constituents of the mixture travel at different speeds, causing them to separate. The separation is based on differential partitioning between the mobile and stationary phases

SDS-PAGE

(separation by mass) (Polyacrylamide gel electrophoresis (PAGE), describes a technique widely used in biochemistry, forensics, genetics, molecular biology and biotechnology to separate biological macromolecules, usually proteins or nucleic acids, according to their electrophoretic mobility. Mobility is a function of the length, conformation and charge of the molecule. As with all forms of gel electrophoresis, molecules may be run in their native state, preserving the molecules' higher-order structure, or a chemical denaturant may be added to remove this structure and turn the molecule into an unstructured linear chain whose mobility depends only on its length and mass-to-charge ratio. For nucleic acids, urea is the most commonly used denaturant. For proteins, sodium dodecyl sulfate (SDS) is an anionic detergent applied to protein samples to linearize proteins and to impart a negative charge to linearized proteins. This procedure is called SDS-PAGE. In most proteins, the binding of SDS to the polypeptide chain imparts an even distribution of charge per unit mass, thereby resulting in a fractionation by approximate size during electrophoresis. The sample to analyze is optionally mixed with a chemical denaturant if so desired, usually SDS for proteins or urea for nucleic acids. SDS is an anionic detergent that denatures secondary and non-disulfide-linked tertiary structures, and additionally applies a negative charge to each protein in proportion to its mass. Urea breaks the hydrogen bonds between the base pairs of the nucleic acid, causing the constituent strands to separate. Heating the samples to at least 60 °C further promotes denaturation.

electrophoresis

(size). A process which enables the sorting of molecules based on size. Using an electric field, molecules (such as DNA) can be made to move through a gel made of agar or polyacrylamide. The electric field consists of a negative charge at one end which pushes the molecules through the gel, and a positive charge at the other end that pulls the molecules through the gel. The molecules being sorted are dispensed into a well in the gel material. The gel is placed in an electrophoresis chamber, which is then connected to a power source. When the electric current is applied, the larger molecules move more slowly through the gel while the smaller molecules move faster. The different sized molecules form distinct bands on the gel

Which of the following is the most likely overall charge of the amine group on alanine at pH 7?

1

After SDS-PAGE, you estimate that molecular weight of a protein is 22 kD. The average molecular weight of an amino acid is 110. An accurate estimate (is this an oxymoronic phrase?) of the number of peptide bonds in this protein is:

199

You perform an assay on the activity of the JAK tyrosine kinase using a 40 microliter (μl) aliquot of a lysate prepared from cells labeled for six minutes with radioactive cysteine. The total volume of the lysate is 5 milliliters (ml). In the aliquot, you measure 47 micromoles (μmoles) of ADP created in 2 minutes. In the lysate, you measure the amount of all proteins and determine the presence of 13 micrograms (μg) of protein in 75 microliters of lysate. Which of the following is the best calculation of the specific activity of the kinase in the 5 ml lysate? Your answer has the units of μmole/min/μg protein.

3.4

lac repressor protein

A DNA-binding protein which inhibits the expression of genes coding for proteins involved in the metabolism of lactose in bacteria. The lac repressor interacts/binds with DNA at a helixturn-helix motif in the protein

Southern Blotting

A Southern blot is a method used in molecular biology for detection of a specific DNA sequence in DNA samples. Southern blotting combines transfer of electrophoresis-separated DNA fragments to a filter membrane and subsequent fragment detection by probe hybridization. The method is named after its inventor, the British biologist Edwin Southern. Other blotting methods (i.e., western blot, northern blot, eastern blot, southwestern blot) that employ similar principles, but using RNA or protein, have later been named in reference to Edwin Southern's name. Hybridization of the probe to a specific DNA fragment on the filter membrane indicates that this fragment contains DNA sequence that is complementary to the probe. The transfer step of the DNA from the electrophoresis gel to a membrane permits easy binding of the labeled hybridization probe to the size-fractionated DNA. It also allows for the fixation of the target-probe hybrids, required for analysis by autoradiography or other detection methods. Southern blots performed with restriction enzyme-digested genomic DNA may be used to determine the number of sequences (e.g., gene copies) in a genome. A probe that hybridizes only to a single DNA segment that has not been cut by the restriction enzyme will produce a single band on a Southern blot, whereas multiple bands will likely be observed when the probe hybridizes to several highly similar sequences (e.g., those that may be the result of sequence duplication). Modification of the hybridization conditions (for example, increasing the hybridization temperature or decreasing salt concentration) may be used to increase specificity and decrease hybridization of the probe to sequences that are less than 100% similar.

molecular or gel exclusion (size) chromatography

A chromatographic method in which molecules in solution are separated by their size, and in some cases molecular weight

deoxyribonuclease (Dnase)

A deoxyribonuclease (DNase, for short) is any enzyme that catalyzes the hydrolytic cleavage of phosphodiester linkages in the DNA backbone, thus degrading DNA. Some DNases cut, or "cleave", only residues at the ends of DNA molecules (exodeoxyribonucleases, a type of exonuclease). Others cleave anywhere along the chain (endodeoxyribonucleases, a subset of endonucleases). Some are fairly indiscriminate about the DNA sequence at which they cut, while others, including restriction enzymes, are very sequence-specific. Some cleave only double-stranded DNA; others are specific for single-stranded molecules; and still others are active toward bot

lysate

A fluid containing the contents of lysed cells.

agarose gel electrophoresis

A method of gel electrophoresis used in biochemistry, molecular biology, genetics, and clinical chemistry to separate a mixed population of macromolecules such as DNA or proteins in a matrix of agarose.

affinity chromatography

A method of separating biochemical mixtures based on a highly specific interaction such as that between antigen and antibody, enzyme and substrate, or receptor and ligand. The stationary phase is typically a gel matrix. Usually the starting point is an undefined heterogeneous group of molecules in solution, such as a cell lysate, growth medium or blood serum. The molecule of interest will have a well known and defined property, and can be exploited during the affinity purification process. The process itself can be thought of as an entrapment, with the target molecule becoming trapped on a solid or stationary phase or medium. The other molecules in the mobile phase will not become trapped as they do not possess this property. The stationary phase can then be removed from the mixture, washed and the target molecule released from the entrapment in a process known as elution. Possibly the most common use of affinity chromatography is for the purification of recombinant proteins.

affinity chromatography

A method of separating biochemical mixtures based on a highly specific interaction such as that between antigen and antibody, enzyme and substrate, or receptor and ligand

nucleoside

A nucleoside consists simply of a nucleobase (also termed a nitrogenous base) and a 5-carbon sugar (either ribose or deoxyribose), whereas a nucleotide is composed of a nucleobase, a five-carbon sugar, and one or more phosphate groups. In a nucleoside, the base is bound to either ribose or deoxyribose via a beta-glycosidic linkage. Examples of nucleosides include cytidine, uridine, adenosine, guanosine, thymidine and inosine.

nucleotide

A nucleotide is made of a nucleobase (also termed a nitrogenous base), a five-carbon sugar (either ribose or 2-deoxyribose, depending on if it is RNA or DNA), and one or, depending on the definition, more than one phosphate groups. Authoritative chemistry sources such as the ACS Style Guide and IUPAC Gold Book clearly state that the term nucleotide refers only to a molecule containing one phosphate. However, common usage in molecular biology often extends this definition to include molecules with two or three phosphate groups.Thus, the term "nucleotide" generally refers to a nucleoside monophosphate, but a nucleoside diphosphate or nucleoside triphosphate could be considered a nucleotide as well

plasmid

A plasmid is a small DNA molecule within a cell that is physically separated from a chromosomal DNA and can replicate independently. They are most commonly found in bacteria as smaller circular, double-stranded DNA molecules.

On average, which restriction enzyme (a.k.a., restriction nuclease) would cut a random DNA sequence into a greater number of fragments?

A restriction nuclease that recognizes a four-nucleotide pair sequence

cloning vector

A small piece of DNA, taken from a virus, a plasmid, or the cell of a higher organism, that can be stably maintained in an organism, and into which a foreign DNA fragment can be inserted for cloning purposes.

coiled-coil motif

A structural motif in proteins in which 2-7[1] alpha-helices are coiled together like the strands of a rope (dimers and trimers are the most common types). Many coiled coil-type proteins are involved in important biological functions such as the regulation of gene expression, e.g. transcription factors

palindrome

A word or an expression that is spelled the same backward and forward, used for restriction enzymes in cutting DNA

In PCR of the ND4 gene, the annealing temperature is 50 C. If the annealing temperature was set to 72 C, what is the most likely result?

Amplification will be inefficient.

ATP binding site

An ATP binding site is a protein micro-environment where ATP is captured and hydrolyzed to ADP, releasing energy that is utilized by the protein to "do work" by changing the protein shape and/or making the enzyme catalytically active.

Northern blotting

An adaptation of the Southern blot procedure used to detect specific sequences of RNA by hybridization with complementary DNA or RNA probes

phosphatase

An enzyme that catalyzes the hydrolysis of organic phoshates

kinase

An enzyme that catalyzes the transfer of the gamma phosphate group from ATP to a specified molecule.

Band

As used in molecular biology, a band is the detected molecules. If separating by size, then a band is the detected molecules of a distinct size. If separating by charge, then a band is molecules of a certain charge. In SDS-PAGE of proteins from a cell, many bands are since there are many proteins of different sizes. A band in SDS-PAGE contains thousands of proteins of the same size. In DNA gel electrophoresis, a band contains thousands of DNA molecules of the same size

Colony blot hybridization

Colony blot hybridization is applied to DNA or RNA released from blotted microbial colonies. The microbial colonies are transferred (blotted) to a membrane. The cells are lysed in place to release the nucleic acids. The RNA or DNA (after denaturation) is fixed to the filter and hybridized with a labelled probe. Blocking reagent may be added prior to the probe to prevent unspecific binding. Excess probe is washed away and the membrane is visualized by UV or autoradiography. Colony blot hybridization can be used for screening clones or bacterial isolates.

DNA ligase

DNA ligase is a specific type of enzyme, a ligase, that facilitates the joining of DNA strands together by catalyzing the formation of a phosphodiester bond. DNA ligase is used in both DNA repair and DNA replication. In addition, DNA ligase has extensive use in molecular biology laboratories for recombinant DNA experiments. Purified DNA ligase is used in gene cloning to join DNA molecules together to form recombinant DNA.

A protein has glutamine residues at various locations in the primary structure. The C-terminal amino acid in the protein is a glutamine. You calculate the ratio of C-terminal radioactivity (radioactivity due to the C-terminal glutamine incorporated into the polypeptides) to the total radioactivity of the entire polypeptides (total radioactivity due to each of the five possible glutamine locations) over time, starting with an experiment with two minutes of growth in the presence of radioactive glutamine and ending with a separate experiment with 80 minutes of growth in the presence of radioactive glutamine. With increasing time of exposure of the cells to the radioactive glutamine, the ratio of C-terminal radioactivity to total radioactivity in the isolated proteins should:

Decrease to a final value of 0.20 (1/5 of radioactivity is due to the C-terminal glutamine residues)

D vs L amino acids

Every amino acid (except glycine) can occur in two isomeric forms, because of the possibility of forming two different enantiomers (stereoisomers) around the central carbon atom. By convention, these are called L- and D- forms, analogous to left-handed and right-handed configurations. Only L-amino acids are manufactured in cells and incorporated into proteins. Some D-amino acids are found in the cell walls of bacteria, but not in bacterial proteins.

Combined DNA Index System (CODIS)

FBI's index for criminals

Which of the following oligonucleotides would best be used as one of the primers in a PCR reaction with a template that contains the sequence, ATGCCTAGGTC? [note: most primers are 15-22 nucleotides in length to ensure unique and specific binding. In this case, shorter sequences are used to make the problem less tedious. If you can figure out what to do for a 5 nucleotide primer, you can figure out what to do for an 18 nucleotide primer.] AGTCC

GACCT or GGCAT

gel filtration or size exclusion chromatography

Gel filtration chromatography is a separation based on size. It is also called molecular exclusion or gel permeation chromatography. In gel filtration chromatography, the stationary phase consists of porous beads with a well-defined range of pore sizes

motif

In a chain-like biological molecule, such as a protein or nucleic acid, a structural motif is a supersecondary structure, which also appears in a variety of other molecules. Motifs do not allow us to predict the biological functions: they are found in proteins and enzymes with dissimilar functions. Motifs are between domains and secondary structure in complexity

Agarose gel electrophoresis of DNA is similar to SDS-PAGE of proteins because

In both cases you can plot of molecular weight versus distance to create a standard curve.

A water-soluble globular protein is most likely to have the highest proportion of which of the following amino acid residues buried in the interior of the protein three-dimensional structure?

Isoleucine

homogenate

Mixture of all of the components of the cell, but no intact cell

chaperonin

Newly made proteins usually must fold from a linear chain of amino acids into a three-dimensional form. Chaperonins belong to a large class of molecules that assist protein folding, called molecular chaperones. The potential energy to assist in protein folding is supplied by structural changes imposed by adenosine triphosphate (ATP) binding and/or hydrolysis

denaturation

Nucleic acid denaturation occurs when hydrogen bonding between nucleotides is disrupted, and results in the separation of previously annealed strands. For example, denaturation of DNA due to high temperatures results in the disruption of Watson and Crick base pairs and the separation of the double stranded helix into two single strands. Nucleic acid strands are capable of re-annealling when "normal" conditions are restored, but if restoration occurs too quickly, the nucleic acid strands may re-anneal imperfectly resulting in the improper pairing of bases.

Primary structure

Peptide bonds link amino acids into polypeptide chains. Amino acid side chains confer the unique chemical properties of polypeptides

polymerase chain reaction (PCR)

Polymerase chain reaction (PCR) is a technique used in molecular biology to amplify a single copy or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence

denatured

Proteins or nucleic acids that have lost their quaternary structure, tertiary structure and secondary structure which is present in their native state. In vitro, molecules can be denatured by application of some external stress or compound such as a strong acid or base, a concentrated inorganic salt, an organic solvent (e.g., alcohol or chloroform), radiation or heat. If proteins in a living cell are denatured, this results in disruption of cell activity and possibly cell death. Protein denaturation is also a consequence of cell death. Denatured proteins can exhibit a wide range of characteristics, from conformational change and loss of solubility to aggregation due to the exposure of hydrophobic groups.

The radioactive labeling of the protein in the glutamine labeling experiment is best identified as an example of

Pulse/ chase (labeling)

pyrophosphate

Pyrophosphate is a phosphorus oxyanion. The group is also called diphosphate or dipolyphosphate, although this should not be confused with phosphates. The diphosphate anion P2O74- is abbreviated PPi and is formed by the hydrolysis of ATP into AMP in cells. For example, when a nucleotide is incorporated into a growing DNA or RNA strand by a polymerase, pyrophosphate (PPi) is released.

renaturation

Renaturation in molecular biology refers to the reconstruction of a protein or nucleic acid (such as DNA) to their original 3D form especially after denaturation. This process is therefore the inverse of denaturation. Reconstructing the denatured nucleic acid or protein into its original form is done by the process of renaturation. For instance, a heat-denatured DNA can revert to its original form by cooling slowly the two strands which, by chance, finding complementary regions and reform into a double-stranded helix. A denatured protein may be restored following denaturation although it is not as likey to occur as can be done with denatured nucleic acids. One way through which a denatured protein is restored to its original form is by removing the SDS and denaturing agents following denaturation

reverse transcriptase PCR (RT-PCR)

Reverse transcription polymerase chain reaction (RT-PCR), a variant of polymerase chain reaction (PCR), is a technique commonly used in molecular biology to detect RNA expression. RT-PCR is often confused with real-time polymerase chain reaction (qPCR) by students and scientists alike, but they are separate and distinct techniques. While RT-PCR is used to qualitatively detect gene expression through creation of complementary DNA (cDNA) transcripts from RNA, qPCR is used to quantitatively measure the amplification of DNA using fluorescent dyes. qPCR is also referred to as quantitative PCR, quantitative real-time PCR, and real-time quantitative PCR. Although RT-PCR and the traditional PCR both produce multiple copies of particular DNA isolates through amplification, the applications of the two techniques are fundamentally different. Traditional PCR is used to exponentially amplify target DNA sequences. RT-PCR is used to clone expressed genes by reverse transcribing the RNA of interest into its DNA complement through the use of reverse transcriptase. Subsequently, the newly synthesized cDNA is amplified using traditional PCR.

hairpin structure

Stem-loop intramolecular base pairing is a pattern that can occur in single-stranded DNA or, more commonly, in RNA. The structure is also known as a hairpin or hairpin loop. It occurs when two regions of the same strand, usually complementary in nucleotide sequence when read in opposite directions, base-pair to form a double helix that ends in an unpaired loop. The resulting structure is a key building block of many RNA secondary structures.

Which of the following is NOT a normal component of a ribonucleic acid?

Sulfur

The term "DNA cloning" could refer to a number of contextually correct meanings. Which one of the following is a correct definition of "DNA cloning"?

The act of making many identical copies of a DNA molecule

specific activity (enzyme)

The activity of an enzyme per milligram of total protein (expressed in ìmol/time/mg of all protein). Specific activity gives a measurement of enzyme purity in the mixture. It is the moles of product formed by an enzyme in a given amount of time (milliseconds, seconds, minutes) under given conditions per milligram of total protei

a-helix

The alpha helix (a-helix) is a common motif in the secondary structure of proteins and is a righthand-coiled or spiral conformation (helix) in which every backbone N-H group donates a hydrogen bond to the backbone C=O group of the amino acid located three or four residues earlier along the protein sequence

annealing

The creation of a region of double-stranded nucleic acid when single-stranded DNA or RNA molecules basepair with DNA or RNA molecules via hydrogen bonds. The term is often used to describe the binding of a DNA probe, or the binding of a primer to a DNA strand during polymerase chain reaction.

Tertiary Structure

The final three dimensional shape of a single polypeptide protein

green fluorescent protein (GFP)

The green fluorescent protein (GFP) is a protein composed of 238 amino acid residues (26.9 kDa) that exhibits bright green fluorescence when exposed to light in the blue to ultraviolet range. In cell and molecular biology, the GFP gene is frequently used as a reporter of expression. It has been used in modified forms to make biosensors, and many animals have been created that express GFP, which demonstrates a proof of concept that a gene can be expressed throughout a given organism, in selected organs, or in cells of interest. GFP can be introduced into animals or other species through transgenic techniques, and maintained in their genome and that of their offspring. To date, GFP has been expressed in many species, including bacteria, yeasts, fungi, fish and mammals, including in human cells.

Trypsin is a protease that breaks peptide bonds. Trypsin interacts with the substrate at the active site. In the active site, binding to the substrate is stabilized by hydrophobic interactions between the side chains of the amino acids in the active site and the substrate. Trypsin is a monomer. Trypsin is available in high quantity in the pancreas, a glandular organ in the digestive system and endocrine system of vertebrates. Trypsin aids in digestion. You assay the action of another molecule, the tumor associated trypsin inhibitor (TATI), on trypsin isolated from the pancreas of H. sapiens. The elevated presence of this inhibitor in humans is associated with some types of ovarian and kidney cancers. TATI acts as a competitive inhibitor. Which one of following statements about TATI is most probably true?

The inhibitor increases Km but has no effect on Vmax.

origin of replication

The origin of replication (also called the replication origin) is a particular sequence in a genome at which replication is initiated. This can either involve the replication of DNA in living organisms such as prokaryotes and eukaryotes, or that of DNA or RNA in viruses, such as double-stranded RNA viruses.

A LHON mutation in another NADH dehydrogenase subunit gene causes the loss of a Mae III restriction enzyme site. Compared to normal DNA digested with Mae III, the DNA from a patient with LHON digested with Mae III would be expected to:

Travel a shorter distance during gel electrophoresis

coomasie blue protein stain

a family of dues commonly used to stain proteins in SDS-PAGE gels. The gels are soaked in dye, and excess stain is then eluted with a solvent "destaining,"

ligand

a molecule that binds to another (usually larger) molecule

substrate

a molecule upon which an enzyme acts

coenzyme

a nonprotein that is necessary for an enzyme to function

amide buffer solution

a system capable of resisting changes in pH, consisting of a conj. acid-base pair in which the ratio of proton acceptor to proton donor is near unity

phosphorylation

addition of a phosphate group, it is carried out through the action of enzymes known as phosphotransferases or kinases

competitive inhibitor

an enzyme inhibitor that binds to the active site of the enzyme which prevents the binding of the substrate it "competes," with the substrate

Hershey Chase

bacteriophage, with protein coat and DNA code

pulse-chase

cells are exposed to a labeled compound. The labeled compound is incorporated into the molecule or pathway being studied.

in situ

in its original place

In which of the following purification methods are proteins separated on the basis of their charge?

ion exchange chromatography

methylation

methylation is a form of alkylation with a methyl group, rather than a larger carbon chain replacing a hydrogen atom

If you performed an SDS-PAGE using an aliquot of a cell lysate, you would separate the proteins in the lysate on the basis of their

molecular weight

Protein folding

non-covalent bonds are the major force determining 2nd, 3rd and 4th protein structures

You separate the macromolecule (shown above in this block of questions) by size from other macromolecules using agarose gel electrophoresis and transfer macromolecules from the gel to a membrane. Using a radioactive single strand DNA probe, you detect the macromolecule shown above in this block of questions. This experimental protocol is best described as

northern blotting

Consider the macromolecule shown above in this block of questions. The 5' end of this macromolecule is found ___________.

on the right end as drawn.

Which of the following types of bonds or interactions are LEAST likely to be involved in stabilizing the three-dimensional folding of most proteins?

phosphodiester bonds

Secondary structures

protein secondary structures include a-helices, B-sheets, loops and turns. Secondary structures fold into motifs and domains

allosteric protiens

proteins wiht mult. ligand-binding sites such that a ligand binding at one site affets a ligand binding at another site

Protein structure

structure studies include the use of x-ray crystallography and NMR

hydrolysis

the chemical breakdown of a compound due to reaction with water

Quaternary structure

the final 3-D shape of a protein with multiple polypeptides (subunits)

Protein analysis and purification

the physical and chemical properties of polypeptides are used to purify proteins and to gain understanding of protein structure

gylosylation

the reaction is which a carbohydrate (glycosyl donor) is attached to a hydroxyl or other functional group of another molecule

dephosphorylation

the removal of a phosphate group from an organic compound by hydrolysis

dimer

two monomers joined by bonds that can be either strong or weak, covalent or intermolecular. The term homodimer is used when the two molecules are identical and heterodimer when they are not

in vitro

within a glass, observable within a test tube

in vivo

within the living body