Cell Bio (BIOS367) Exam 1

high speed

80,000 X 1 hour - microsomes - small vesicles

One cell can be labelled with multiple fluorescent markers, allowing visualization of

multiple structures

As a signal sequence emerges from the ribosome and binds to the SRP, a conformational change in the SRP exposes a ____ for the SRP receptor

binding site

Transgenic plant - "golden rice"

"Golden rice" has been engineered to contain beta-carotene, as an approach address vitamin A deficiency in the developing world DNA technology allows the production of rice grains with high levels of b-carotene. To help reduce vitamin A deficiency in the developing world, a strain of rice, called "golden rice," was developed in which the edible part of the grain (called the endosperm) contains large amounts of β-carotene, which is converted in the human gut to vitamin A. (A) Rice plants, like most other plants, can synthesize β-carotene in their leaves from an abundant precursor (geranylgeranyl pyrophosphate) found in all plant tissues. However, the genes that code for two of the enzymes that act early in this biosynthetic pathway are turned off in the endosperm, preventing the production of β-carotene in rice grains. To produce golden rice, the genes for these two enzymes were obtained from organisms that produce large amounts of β-carotene: one from maize and the other from a bacterium. Using DNA technology, these genes were connected to a promoter that drives gene expression in rice endosperm. This engineered DNA was then used to generate a transgenic rice plant that expresses these enzymes in endosperm, resulting in rice grains that contain high levels of β-carotene. Compared to the milled grains of wild-type rice, the grains of the transgenic rice are a deep yellow/orange due to the presence of β-carotene

Membrane-bending Proteins Deform Bilayers (3 main ways)

(A) Bilayer without protein bound (B) A hydrophobic region of the protein can insert as a wedge into one monolayer to pry lipid head groups apart. Such regions can either be amphiphilic helices as shown or hydrophobic hairpins (C) The curved surface of the protein can bind to lipid head groups and deform the membrane or stabilize its curvature. (D) A protein can bind to and cluster lipids that have large head groups and thereby bend the membrane

Four ways of restricting the lateral mobility of specific plasma membrane proteins

(A) The proteins can self-assemble into large aggregates (B) Proteins tethered to extracellular molecules (C) Proteins tethered to intracellular molecules (D) Proteins tethered to proteins of another cell

Proteins Can Move Between Compartments in Different Ways

*Protein translocation:* direct transport of a protein across a membrane from the cytosol to a topologically distinct space *Gated transport:* protein and RNA move between the cytosol and the nucleus through nuclear pore complexes in the nuclear envelope *Vesicular transport:* membrane enclosed vesicles transport proteins from one topologically equivalent space to another

The Conventional Light Microscope Can Resolve Details ___ Apart

0.2 μm

resolution formula

0.61 x wavelength/numerical aperture θ = half the angular width of the cone of rays collected (max width is 180 degrees, so max value is 1) n = refractive index of the medium separating the specimen from the objective and condenser lenses

low speed

1000X gravity 10 min - whole cells - nuclei - cytoskeletons

very high speed

150,000 X gravity 3 hours - ribosomes - viruses - large macromolecules

medium speed

20,000 X gravity 20 min - mitochondria - lysosomes - peroxisomes

Proteins Can Be Separated by SDS Polyacrylamide-Gel Electrophoresis

A heterogeneous mix of proteins are treated with SDS (a detergent) and mercaptoethanol. This results in unfolded proteins that are coated with negatively charged SDS. The bigger the protein, the more SDS is bound, and the greater the negative charge The prepared samples are applied to a gel for electrophoresis. As they migrate through the gel, they are separated by size

The signal-recognition particle (SRP)

A mammalian SRP is a rodlike ribonucleoprotein complex containing six protein subunits (brown) and one RNA molecule (blue). The SRP RNA forms a backbone that links the protein domain containing the signal sequence-binding pocket to the domain responsible for slowing translation.

Fluorescence recovery after photobleaching (FRAP)

A strong focused pulse of laser light will extinguish, or bleach, fluorescent proteins. By selectively photobleaching a set of fluorescently tagged protein molecules within a defined region of a cell, the microscopist can monitor recovery over time, as the remaining fluorescent molecules move into the bleached regio

___ Are Found on the Surface of All Eukaryotic Plasma Membranes

Glycolipids Glycolipids are similar to phospholipids, but have carbohydrate rather than phosphate at the hydrophilic end

Mitochondrial Protein Import Is Powered by

ATP Hydrolysis (Mitochondrial hsp70), a Membrane Potential (TIM23 complex), and Redox Potential (Mia 40 - reduction and reoxidation - once a disulfide bond is made, the folding protein can no longer slide back into the cytosol)

affinity chromatography

Affinity chromatography uses an insoluble matrix that is covalently linked to a specific ligand, such as an antibody molecule or an enzyme substrate, that will bind a specific protein. Enzyme molecules that bind to immobilized substrates on such columns can be eluted with a concentrated solution of the free form of the substrate molecule, while molecules that bind to immobilized antibodies can be eluted by dissociating the antibody-antigen complex with concentrated salt solutions or solutions of high or low pH. High degrees of purification can be achieved in a single pass through an affinity column.

Most Proteins Synthesized in the Rough ER Are Glycosylated by the Addition of a Common N-Linked* Oligosaccharide *asparagine

Almost as soon as a polypeptide chain enters the ER lumen, it is glycosylated on target asparagine amino acids. The precursor oligosaccharide is attached only to asparagine side chains in the sequences Asn-X-Ser and Asn-X-Thr (where X is any amino acid except proline). These sequences occur much less frequently in glycoproteins than in nonglycosylated cytosolic proteins. Evidently there has been selective pressure against these sequences during protein evolution, presumably because glycosylation at inappropriate sites would interfere with protein folding. The five sugars in the gray box form the core region of this oligosaccharide. For many glycoproteins, only the core sugars survive the extensive oligosaccharide trimming that takes place in the Golgi apparatus. (B) The precursor oligosaccharide is transferred from a dolichol lipid anchor to the asparagine as an intact unit in a reaction catalyzed by a transmembrane oligosaccharyl transferase enzyme complex. One copy of this enzyme is associated with each protein translocator in the ER membrane. Oligosaccharyl transferase contains 13 transmembrane a helices and a large ER lumenal domain that contains binding sites for the nascent protein and dolichol-oligosaccharide. The asparagine binds a tunnel that penetrates the enzyme interior. There, the amino group of the asparagine is twisted out of the plane that stabilizes the otherwise poorly reactive amide bond, activating it for reaction with the dolichol-oligosaccharide.

Single-Molecule Localization Microscopy (SMLM)

Also Delivers Superresolution (resolution beyond the 200 nm diffraction limit) Single fluorescent molecules can be located with great accuracy Sparse subsets of fluorescent molecules are individually switched on briefly and then bleached. The exact positions of all these well-spaced molecules can be gradually added together and built up into an image at superresolution

Polyacrylamide Gel Electrophoresis (SDS-PAGE)

An electrophoresis apparatus, in which a polyacrylamide gel is sandwiched between two glass plates, with each end of the gel immersed in a buffer connected to an electrode. Individual polypeptide chains form a complex with negatively charged molecules of sodium dodecyl sulfate (SDS) and therefore migrate as a negatively charged SDS-protein complex through a porous gel of polyacrylamide. Because smaller polypeptides move more quickly through the gel, this technique can be used to determine the approximate mass of a polypeptide chain as well as the subunit composition of a protein complex.

Fluorescence and the fluorescence microscope

An orbital electron of a fluorochrome molecule can be raised to an excited state after the absorption of a photon. Fluorescence occurs when the electron returns to its ground state and emits a photon of light at a longer wavelength Too much exposure to light or too bright a light can destroy the fluorochrome molecule in a process called photobleaching. In the fluorescence microscope, a filter set consists of two barrier filters (1 and 3) and a dichroic (beam-splitting) mirror High-numerical-aperture objective lenses are especially important in this type of microscopy because, for a given magnification, the brightness of the fluorescent image is proportional to the fourth power of the numerical aperture

Improperly Folded Proteins Are Exported from the ER and Degraded in the Cytosol

As the defective proteins are translocated out of the ER, they are tagged with *ubiquitin*, which marks them for degradation by a multi-protein complex called the *proteasome*

The Lipid Bilayer Is a Two-dimensional Fluid

Because the lipids are not covalently bound to one another, they are free to move independently

Specific Proteins Can Be Detected by

Blotting with Antibodies (Western Blotting)

The presence of the protein of interest can be detected by following its

biochemical activity (ex: via chromatography)

how does mass spectrometry work to figure out an AA sequence?

By identifying the amino acid composition of many random fragments of a protein, tandem mass spectrometry can deduce the amino acid sequence.

Fluorescent Biosensors Can Monitor

Cell Signaling ex: Visualizing intracellular Ca2+ concentrations by using a fluorescent indicator: neuron treated with a marker that fluoresces differently depending on the local concentration of calcium (red for high Ca+ conc in dendrites)

____ bind to the unfolded mitochondrial proteins while they are in the cytosol to keep them unfolded

Chaperone proteins (hsp70)

Translocation Across the ER Membrane Does Not Always Require Ongoing Polypeptide Chain Elongation

Co-translational and post-translational protein translocation: Ribosomes bind to the ER membrane during co-translational translocation. By contrast, cytosolic ribosomes complete the synthesis of a protein and release it prior to post-translational translocation. The released protein is kept unfolded in the cytosol by chaperones that dissociate before the protein is translocated across the membrane. In both cases, the protein is directed to the ER by an ER signal sequence

____ Reveal Whether Two Mutations Are in the Same Gene or Different Genes

Complementation Tests ex: two albino parents produced normally colored offspring, indicating the parents have mutations in two different genes This restoration of the wild-type plumage indicates that the two white breeds lack color because of recessive mutations in different genes

plasma membrane: lipid bilayer and mosaic of lipids and proteins

Hydrophobic ("water fearing") nonpolar "tails" form the center (interior) of the membrane. This interior makes the plasma membrane relatively impermeable to most water-soluble molecules. No covalent bonds between the phospholipids!

An Entire Genome Can Be Represented in a

DNA Library Human genomic libraries containing DNA fragments that represent the whole human genome can be constructed using restriction nucleases and DNA ligase. Such a genomic library consists of a set of bacteria, each carrying a different fragment of human DNA. For simplicity, only the colored DNA fragments are shown in the library; in reality, all of the different gray fragments will also be represented.

PCR can use ____ as the initial template. Very little template is needed!

DNA or RNA PCR can be used to obtain either genomic or cDNA clones. (A) To use PCR to clone a segment of chromosomal DNA, total genomic DNA is first purified from cells. PCR primers that flank the stretch of DNA to be cloned are added, and many cycles of PCR are completed. Because only the DNA between (and including) the primers is amplified, PCR provides a way to obtain selectively any short stretch of chromosomal DNA in an effectively pure form. (B) To use PCR to obtain a cDNA clone of a gene, total mRNA is first purified from cells. The first primer is added to the population of mRNAs, and reverse transcriptase is used to make a DNA strand complementary to the specific RNA sequence of interest. The second primer is then added, and the DNA molecule is amplified through many cycles of PCR.

PCR Is Also Used for _____ Applications

Diagnostic and Forensic ex: PCR can be used to detect the presence of a viral genome in a nasal sample. Because of its ability to amplify enormously the signal from a single molecule of nucleic acid, PCR is an extraordinarily sensitive method for detecting trace amounts of virus in a sample of saliva, blood, or other tissue, without the need to purify the virus. For the coronavirus SARS-CoV-2, the virus that causes COVID-19, the genome is a single-strand molecule of RNA, as illustrated here. Typically, only a short segment of the viral genome (100-200 nucleotides) is amplified. Although it is possible to visualize the amplified DNA by gel electrophoresis as shown here, it is usually detected by rapid optical methods described later in the chapter (see Figure 8-64). Many other viruses that infect humans—such as HIV—are detected using this strategy

PCR

Each cycle of PCR includes three steps: (1) The double-stranded DNA is heated briefly to separate the two strands (2) The DNA is exposed to a large excess of a pair of specific primers—designed to bracket the region of DNA to be amplified—and the sample is cooled to allow the primers to hybridize to complementary sequences in the two DNA strands. (3) This mixture is incubated with DNA polymerase and the four deoxyribonucleoside triphosphates so that DNA can be synthesized, starting from the two primers. To amplify the DNA, the cycle is repeated many times by reheating the sample to separate the newly synthesized DNA strands The technique depends on the use of a special DNA polymerase isolated from a thermophilic bacterium; this polymerase is stable at much higher temperatures than eukaryotic DNA polymerases, so it is not denatured by the heat treatment shown in step 1. The enzyme therefore does not have to be added again after each cycle.

Gene Products Can Be Ordered in Pathways by ____

Epistasis Analysis Using genetics to determine the order of function of genes: ex: In normal cells, secretory proteins are loaded into vesicles, which fuse with the plasma membrane to secrete their contents into the extracellular medium. Two mutants, A and B, fail to secrete proteins. In mutant A, secretory proteins accumulate in the ER. In mutant B, secretory proteins accumulate in the Golgi. In the double mutant AB, proteins accumulate in the ER; this indicates that the gene defective in mutant A acts before the gene defective in mutant B in the secretory pathway.

____ Lines Are a Widely Used Source of Homogeneous Cells

Eukaryotic Cell

____ in the proteins lining the channel weakly stick to one another, forming a mesh that prevents random proteins from diffusing in or out.

FG repeats

The ER Assembles Most Lipid Bilayers

Fatty acids are attached to glycerol 3-phosphate to form a phospholipid on the *cytosolic* side of the ER membrane

Intact Tissues Are Usually____ Before Microscopy

Fixed and cut into thin Sections Use a microtome Very rapidly frozen samples can also be sectioned, and these better preserve the structure of cells in their native state.

Specific Molecules Can Be Located in Cells by

Fluorescence Microscopy

Protein Dynamics Can Be Followed in Living Cells via

Fluorescence recovery after photobleaching (FRAP)

Protein interactions Can Be Followed in Living Cells via

Fluorescence resonance energy transfer (FRET)

Nuclear Export Works Like Nuclear Import, but in Reverse

For proteins being exported out of the nucleus, binding of Ran-GTP is required to load the cargo onto a nuclear export receptor

Protein Sequence and Structure Provide Clues About Protein

Function

______ determines whether the protein is folded properly or not: if the protein is still incompletely folded, the enzyme transfers a new glucose from _____ to the N-linked oligosaccharide, renewing the protein's affinity for calnexin and retaining it in the ER

Glucosyl transferase UDP-glucose

_____ Provides a Powerful but Simple Way to Detect Specific Nucleotide Sequences

Hybridization (denaturation and renaturation)

_____ Cell Lines Are Factories That Produce Monoclonal Antibodies

Hybridoma ex: An antibody producing B-lymphocyte can be fused with an immortalized B-lymphocyte to form an immortal cell line that produces a specific antibody

Membrane spanning regions can be predicted from the amino acid sequence of a protein by calculating hydrophobicity

Hydrophobic regions in dark green Hydrophilic regions in light green Using hydropathy plots to localize potential a-helical membrane-spanning segments in a polypeptide chain: A positive value indicates that free energy is required for transfer to water (that is, the segment is hydrophobic) Peaks in the hydropathy index appear at the positions of hydrophobic segments in the amino acid sequence. Glycophorin (A) has a single membrane-spanning α helix and one corresponding peak in the hydropathy plot. Bacteriorhodopsin (B) has seven membrane-spanning α helices and seven corresponding peaks in the hydropathy plot.

what can affect an SDS PAGE?

If the protein contains a large amount of carbohydrate, however, it will move anomalously on the gel, and its apparent mass estimated by SDS-PAGE will be misleading. Other modifications, such as phosphorylation, can also cause small changes in a protein's migration in the gel.

Some Membrane Proteins Acquire a Covalently Attached Glycosylphosphatidylinositol (GPI) Anchor

If this protein winds up at the cell surface by vesicular transport, the entire protein is exposed, and can be released by cleavage of the anchor. GPI-anchored proteins are targeted to the ER membrane by an N-terminal signal sequence (not shown), integrated into the membrane, and processed by signal peptidase similarly to a single-pass transmembrane protein (see Figure 12-27). Immediately after the completion of protein synthesis, the precursor protein remains anchored in the ER membrane by a hydrophobic C-terminal sequence of 15-20 amino acids; the rest of the protein is in the ER lumen. Within less than a minute, a transamidase enzyme in the ER cleaves the protein from its membrane-bound C-terminus and simultaneously attaches the new C-terminus to an amino group on a preassembled GPI intermediate. The sugar chain contains an inositol attached to the lipid from which the GPI anchor derives its name. It is followed by a glucosamine and three mannoses. The terminal mannose links to a phosphoethanolamine that provides the amino group to attach the protein through an amide bond. The signal that specifies this modification is contained within the hydrophobic C-terminal sequence and a few amino acids adjacent to it on the lumenal side of the ER membrane; if this signal is added to other proteins, they too become modified in this way. Because of the covalently linked lipid anchor, the protein remains membrane-bound, with all of its amino acids exposed initially on the lumenal side of the ER and eventually on the exterior of the plasma membrane.

Is a Rapid Affinity Purification Method

Immunoprecipitation

equilibrium sedimentation

In equilibrium sedimentation, subcellular components move up or down when centrifuged in a gradient until they reach a position where their density matches that of their surroundings. Although a sucrose gradient is shown here, denser gradients, which are especially useful for protein and nucleic acid separation, can be formed from cesium chloride. The final bands, at equilibrium, can be collected

gel filtration chromatography

In gel-filtration chromatography, the small beads that form the matrix are inert but porous. Molecules that are small enough to penetrate into the matrix beads are thereby delayed and travel more slowly through the column than do larger molecules that cannot penetrate. Beads of cross-linked polysaccharide (dextran, agarose, or acrylamide) are available commercially in a wide range of pore sizes, making them suitable for the fractionation of molecules of various masses, from less than 500 daltons to more than 5 × 106 daltons

ion-exhange chromatography

In ion-exchange chromatography, the insoluble matrix carries ionic charges that retard the movement of molecules of opposite charge. Matrices used for separating proteins include diethylaminoethylcellulose (DEAE-cellulose), which is positively charged, and carboxymethylcellulose (CM-cellulose) and phosphocellulose, which are negatively charged. Analogous matrices based on agarose or other polymers are also frequently used. The strength of the association between the dissolved molecules and the ion-exchange matrix depends on both the ionic strength and the pH of the solution that is passing down the column, which may therefore be varied systematically to achieve an effective separation.

Macromolecules Can Be Segregated Without a Surrounding Membrane, Forming "Condensates"

In other words, membrane bound compartments are not the only way to organize cellular components scaffold proteins and RNA can recruit "clients" ex: nucleolus for txn and ribosome assembly

_____ can be used to locate genes on isolated chromosomes

In situ hybridization

two-dimensional gel electrophoresis (isoelectric focusing)

In the first step, proteins are separated according to their intrinsic charge by migration through a pH gradient. Proteins will be separated by their isolelectic point, the pH at which they have no net charge In the second step, the separated proteins are subject to SDS gel electrophoresis in a direction that is at a right angle to the first step, separating proteins by size Separation of protein molecules by isoelectric focusing. At low pH (high H+ concentration), the carboxylic acid groups of proteins tend to be uncharged (-COOH) and their nitrogen-containing basic groups fully charged (for example, -NH3+), giving most proteins a net positive charge. At high pH, the carboxylic acid groups are negatively charged (-COO-) and the basic groups tend to be uncharged (for example, -NH2), giving most proteins a net negative charge. At its isoelectric point, a protein has no net charge because the positive and negative charges balance. Thus, when a tube containing a fixed pH gradient is subjected to a strong electric field in the appropriate direction, each protein species migrates until it forms a sharp band at its isoelectric point, as shown.

Cells Can Confine Proteins and Lipids to Specific Domains Within a Membrane

In this drawing of an epithelial cell, protein A (in the apical domain of the plasma membrane) and protein B (in the basal and lateral domains) can diffuse laterally in their own domains but are prevented from entering the other domain, at least partly by the specialized cell-cell junction called a tight junction. Lipid molecules in the outer (extracellular) monolayer of the plasma membrane are likewise unable to diffuse between the two domains; lipids in the inner (cytosolic) monolayer, however, are able to do so (not shown). The basal lamina is a thin mat of extracellular matrix that separates epithelial sheets from other tissues

Transmembrane α Helices Often Interact with One Another

In this example, each helix has a hydrophilic side. Together, they assemble to form a hydrophilic pore that goes through the membrane Polar and charged amino acids contained in transmembrane helices are energetically disfavored in the hydrophobic environment of the lipid bilayer. They become buried in the interface between spatially adjacent helices in folded membrane proteins. In membrane protein complexes, these contacts can occur between helices from different protein subunits. In this way, multipass membrane proteins can provide a hydrophilic path across the hydrophobic barrier of the bilayer.

velocity sedimentation

In velocity sedimentation, subcellular components sediment at different speeds according to their size and shape when layered over a solution containing sucrose. To stabilize the sedimenting bands against convective mixing caused by small differences in temperature or solute concentration, the tube contains a continuous shallow gradient of sucrose, which increases in concentration toward the bottom of the tube (typically from 5 to 20% sucrose). After centrifugation, the different components can be collected individually, most simply by puncturing the plastic centrifuge tube with a needle and collecting drops from the bottom, as illustrated here

Antibodies Can Be Used to Detect

Specific Proteins

DNA Cloning Allows Any Protein to Be Produced in

Large Amounts

Membrane Proteins Can Be Solubilized and Purified in Detergents

Like phospholipids and glycolipids, the detergents shown here have a hydrophilic region and a hydrophobic region Three commonly used detergents are sodium dodecyl sulfate (SDS), an anionic detergent, and Triton X-100 and β-octylglucoside, two nonionic detergents

Transmembrane Proteins Contain Hydrophobic Segments That Are Recognized Like Signal Sequences

Many single-pass membrane proteins use their transmembrane segment to direct insertion into the ER membrane. The transmembrane segment is recognized by SRP (not shown) and delivered via the SRP receptor (not shown) to the Sec61 translocator at the ER membrane. The transmembrane segment then inserts into the lateral gate of the Sec61 translocator in one of two orientations. (A) Some transmembrane segments insert into the lateral gate such that the N-terminal domain is retained on the cytosolic side of Sec61. This orientation is favored for proteins whose N-terminal domains are very long or folded, and for transmembrane segments whose flanking amino acids have a net positive charge on the N-terminal side. (B) Some transmembrane segments insert into the lateral gate such that the C-terminal flanking region is retained on the cytosolic side of Sec61. In this case, the N-terminal flanking region is thought to translocate across the membrane through the Sec61 channel. This orientation is favored for transmembrane segments whose flanking amino acids have a net positive charge on the C-terminal side.

_____ Provides a Highly Sensitive Method for Identifying Proteins, based on the mass to charge ration of their fragments

Mass Spectrometry

Fluorescence resonance energy transfer (FRET)

One fluorescent protein can excite another, if they are close enough To determine whether (and when) two proteins interact inside a cell, the proteins are first produced as fusion proteins attached to different color variants of green fluorescent protein (GFP). The fluorochromes must be quite close together—within about 1-5 nm of one another—for FRET to occur

Biological Specimens Require Special Preparation for Electron Microscopy

Steps include fixation, sectioning, staining. Examination of living specimens is not practical.

standard mass spectrometry (MS)

Mass spectrometers used in biology contain an ion source that generates gaseous peptides or other molecules under conditions that render most molecules positively charged. The two major types of ion source are MALDI and electrospray, as described in the text. Ions are accelerated into a mass analyzer, which separates the ions on the basis of their mass and charge by one of three major methods: (1) Time-of-flight (TOF) analyzers determine the mass-to-charge ratio of each ion in the mixture from the rate at which it travels from the ion source to the detector. (2) Quadrupole mass filters contain a long chamber lined by four electrodes that produce oscillating electric fields that govern the trajectory of ions; by varying the properties of the electric field over a wide range, a spectrum of ions with specific mass-to-charge ratios is allowed to pass through the chamber to the detector, while other ions are discarded. (3) Ion traps contain doughnut-shaped electrodes producing a three-dimensional electric field that traps all ions in a circular chamber; the properties of the electric field can be varied over a wide range to eject a spectrum of specific ions to a detector.

Membrane Proteins Can Be Associated with the Lipid Bilayer in Various Ways

Most membrane proteins are thought to extend across the bilayer as (1) a single α helix, (2) as multiple α helices, or (5) as a rolled-up β sheet (a β barrel Some of these single-pass and multipass proteins have a covalently attached fatty acid chain inserted in the cytosolic lipid monolayer Other membrane proteins are exposed at only one side of the membrane (3, 4). These classes include glycosyltransferases that carry out glycosylation reactions in the Golgi apparatus (3) and SNARE proteins that catalyze membrane fusion (6) Some of these are anchored to the cytosolic surface by an amphiphilic α helix that partitions into the cytosolic monolayer of the lipid bilayer through the hydrophobic face of the helix (7) Others are attached to the bilayer solely by a covalently bound lipid chain—either a fatty acid chain or a prenyl group in the cytosolic monolayer or, (8) via an oligosaccharide linker, to phosphatidylinositol in the noncytosolic monolayer—called a GPI anchor (9, 10) Finally, membrane-associated proteins are attached to the membrane only by noncovalent interactions with other membrane proteins.

___ Can Be Used to Determine Protein Structure in Solution

NMR *based off hydrogens

Lipid Droplets Are Surrounded by a Phospholipid Monolayer

Neutral lipids are deposited between the two monolayers of the endoplasmic reticulum (ER) membrane forming a lenslike structure between the two monolayers. The lens forms as triacylglycerides and cholesterol esters are made and accumulate in the ER membrane and self-aggregate *Multiple copies of the transmembrane protein seipin assemble into a ring together with a lipid droplet assembly factor*. In the presence of triacylglycerides, seipin dissociates from the assembly factor, which migrates to the cytosolic monolayer of the ER membrane where it facilitates a process in which the droplet buds, fills up with nonpolar lipids, and pinches off as a unique organelle that is surrounded by a single monolayer of phospholipids and associated proteins In some cells, such as adipocytes, droplets fuse and can reach a gigantic size

Confocal Microscope

Optical Sections by Excluding Out-of-Focus Light By scanning the beam of light across the specimen, a very sharp two-dimensional image of the exact plane of focus is built up that is not significantly degraded by light from other regions of the specimen Several optical sections can then be assembled into a 3D reconstruction

Genes Can Be Cloned in Vitro Using

PCR (Polymerase Chain Reaction)

peroxisomes

Peroxisomes are membrane bound organelles that use Molecular Oxygen and Hydrogen Peroxide to Perform Oxidation Reactions

Translocated Polypeptide Chains Fold and Assemble in the Lumen of the Rough ER. This includes formation of disulfide bonds formed between cysteine residues.

Proteins that contain free sulfhydryl (SH) groups on cysteines are oxidized during protein folding to incorporate disulfide (S—S) bonds. *Protein disulfide isomerase (PDI)* contains an intramolecular disulfide bond that accepts electrons from a free sulfhydryl group in the substrate protein to be oxidized. This leads to the formation of an intermolecular mixed disulfide bond between PDI and its substrate. A second free sulfhydryl group in the substrate then donates its electrons to the mixed disulfide bond, resulting in an oxidized substrate and reduced PDI. Reoxidation of PDI is carried out by other ER enzymes (not shown).

Sorting Signals and Sorting Receptors Direct

Proteins to the Correct Cell Address

Genetically Engineered Tags Provide an Easy Way to

Purify Proteins

Purified DNA Molecules Can Be Specifically Labeled with

Radioisotopes or Chemical Markers (enzymes or fluorescent molecules) in Vitro ex: A purified DNA polymerase enzyme can incorporate radiolabeled nucleotides as it synthesizes new DNA molecules. In this way, radiolabeled versions of any DNA sequence can be prepared in the laboratory.

The cycling between the GDP and GTP bound forms of the Ran protein is controlled by a cytosolic protein and a nuclear localized protein. The nuclear protein ____ converts it to the GTP bound form, which can dislodge the cargo protein (previous slide). The cytosolic protein ____ converts it to the GDP bound form, which cannot dislodge the cargo protein. In this way, the cargo is only dislodged in the nucleus

Ran GEF Ran GAP

Once in the nucleus, the cargo/receptor complex is disrupted when ___ binds to the receptor, dislodging the cargo

Ran-GTP

_____ make it possible to move experimentally from gene to protein and from protein to gene.

Recombinant DNA techniques

how can resolution be increased, other than oil immersion?

Resolution can also be increased by using shorter wavelength light. It's possible to use UV light, but special lenses are needed, and the light is not visible to the human eye.

_____ Cut Large DNA Molecules into Specific Fragments

Restriction Nucleases restriction enzymes often work as dimers, and the DNA sequence that each restriction enzyme recognizes and cleaves is often symmetrical around a central point. Here, both strands of the DNA double helix are cut at specific points within the target sequence (orange). Some enzymes, such as HaeIII, cut straight across the double helix and leave two blunt-ended DNA molecules; with others, such as EcoRI and HindIII, the cuts on each strand are staggered. These staggered cuts generate "sticky ends"—short, single-strand overhangs that help the cut DNA molecules join back together through complementary base-pairing. This rejoining of DNA molecules becomes important for DNA cloning, as we discuss later. Restriction nucleases are usually obtained from bacteria, and their names reflect their origins; for example, the enzyme EcoRI comes from Escherichia coli. Hundreds of different restriction enzymes are commercially available.

Signal Sequences Were First Discovered in Proteins Imported Into the

Rough ER In a test tube, cytosol is mixed with mRNA that codes for a secreted protein. Two versions of this reaction are performed: one lacking and the other containing rough microsomes derived from the ER. In both reactions, ribosomes in the cytosol translate the mRNA to produce a protein. The protein produced in the reaction lacking microsomes was observed to be slightly larger than the protein produced in the reaction containing microsomes. This difference in size was shown to be due to a small segment of protein at the N-terminus that was selectively removed only in the reaction containing microsomes. Additional analysis showed that the protein produced in the presence of microsomes was located in the microsome lumen. This collection of results was used to formulate the signal hypothesis. This sequence at the N-terminus was postulated to represent a signal sequence. When the ER signal sequence emerges from the ribosome, it directs the ribosome to a translocator on the ER membrane that forms a pore in the membrane through which the polypeptide is translocated. A signal peptidase is closely associated with the translocator and clips off the signal sequence during translation, and the mature protein is released into the lumen of the ER immediately after its synthesis is completed. The translocator is closed until the ribosome has bound, so that the permeability barrier of the ER membrane is maintained at all times.

Images of Surfaces Can Be Obtained by

Scanning Electron Microscopy

_____ is a transmembrane phospholipid translocator that is not specific for particular phospholipid head groups and therefore equilibrates the different phospholipids between the two monolayers. *They do not require ATP*

Scramblase

BLAST search

Sequence databases can be searched to find similar amino acid or nucleic acid sequences. the higher the alignment score, the better the match. The significance of the alignment is reflected in the Expectation (E) value, which specifies how often a match this good would be expected to occur by chance. The lower the E value, the more significant the match; the extremely low value here (e-111) indicates certain significance. E values much higher than 0.1 are unlikely to reflect true relatedness. For example, an E value of 0.1 means there is a 1 in 10 likelihood that such a match would arise solely by chance.

____ Direct the Import of Proteins Into Peroxisomes

Short Signal Sequences

Translocation Into Mitochondria Depends on

Signal Sequences and Protein Translocators

Linked Blocks of Polymorphisms Have Been Passed Down from Our Ancestors

Single-nucleotide polymorphisms (SNPs) are sites in the genome where two or more alternative choices of a nucleotide are common in the population. Most such variations in the human genome occur at locations where they do not significantly affect a gene's function.

Transport Into the Inner Mitochondrial Membrane Occurs Via Several Routes

TIM23 translator complex --> TOM complex *transmembrane results* multipass inner membrane proteins --> TIM22 complex *multipass results* OXA complex (after TIM23 and TOM) *inner membrane results*

Two Signal Sequences Direct Proteins to the Thylakoid Membrane in Chloroplasts

TOC & TIC complexes

tandem mass spectrometry (MS/MS)

Tandem mass spectrometry typically involves two mass analyzers separated by a collision chamber containing an inert, high-energy gas. The electric field of the first mass analyzer is adjusted to select a specific peptide ion, called a precursor ion, which is then directed to the collision chamber. Collision of the peptide with gas molecules causes random peptide fragmentation, primarily at peptide bonds, resulting in a highly complex mixture of fragments containing one or more amino acids from throughout the original peptide. The second mass analyzer is then used to measure the masses of the fragments (called product or daughter ions). With computer assistance, the pattern of fragments can be used to deduce the amino acid sequence of the original peptide.

PCR is used in forensic science to distinguish one individual from another.

The DNA sequences analyzed are short tandem repeats (STRs) composed of sequences such as CACACA... or GTGTGT.... STRs are found in various positions (loci) in the human genome. The number of repeats in each STR locus is highly variable in the population, ranging from 4 to 40 in different individuals. Because of the variability in these sequences, an individual will usually inherit a different number of repeats at each STR locus from his mother and from his father; two unrelated individuals, therefore, rarely contain the same pair of sequences at a given STR locus. (A) PCR using primers that recognize unique sequences on either side of one particular STR locus produces a pair of bands of amplified DNA from each individual, one band representing the maternal STR variant and the other representing the paternal STR variant. The length of the amplified DNA, and thus its position after gel electrophoresis, will depend on the exact number of repeats at the locus. (B) In the schematic example shown here, the same three STR loci are analyzed in samples from three suspects (individuals A, B, and C), producing six bands for each individual. Although different people can have several bands in common, the overall pattern is quite distinctive for each person. The band pattern can therefore serve as a DNA fingerprint to identify an individual nearly uniquely. The fourth lane (F) contains the products of the same PCR amplifications carried out on a hypothetical forensic DNA sample, which could have been obtained from a single hair or a tiny spot of blood left at a crime scene. The more loci that are examined, the more confident one can be about the results. When examining the variability at 5-10 different STR loci, the odds that two random individuals would share the same fingerprint by chance are approximately 1 in 10 billion. In the case shown here, individuals A and C can be eliminated from inquiries, while B is a clear suspect. A similar approach is used routinely in paternity testing.

Endoplasmic Reticulum (ER)

The ER extends as a network of tubules and sheets throughout the entire cytosol, so that all regions of the cytosol are close to some portion of the ER membrane The ER Is Structurally and Functionally Diverse

How ER signal sequences and SRP direct ribosomes to the ER membrane

The SRP and its receptor act in concert. The SRP binds to both the exposed ER signal sequence and the ribosome, thereby causing translation to slow. The SRP receptor in the ER membrane, which in animal cells is composed of two different polypeptide chains, binds the SRP-ribosome complex and directs it to the translocator. The SRP (in complex with SRP receptor) then moves away from its binding site on the ribosome, which is then occupied by the translocator in the ER membrane. SRP then releases the signal sequence, which inserts into the translocator to initiate polypeptide chain transfer across the lipid bilayer. The SRP and SRP receptor dissociate from each other and are recycled for the next round of protein targeting. Although not shown in the figure, one of the SRP proteins and both chains of the SRP receptor contain GTP-binding domains. Conformational changes that occur during cycles of GTP binding and hydrolysis (discussed in Chapter 15) ensure that SRP preferentially binds a signal sequence in the cytosol and releases it only after SRP successfully engages SRP receptor at the ER membrane. The energy of GTP hydrolysis is therefore used to impart directionality to the cycle of SRP-mediated protein targeting.

Many Membrane Proteins Are Glycosylated

The carbohydrate modifications face the outside of the cell Note that the polypeptide chain traverses the lipid bilayer as a right-handed α helix and that the oligosaccharide chains and disulfide bonds are all on the noncytosolic surface of the membrane. As shown, sulfhydryl groups within a transmembrane protein can form either intrachain disulfide bonds with each other or interchain disulfide bonds with sulfhydryl groups in other proteins. The sulfhydryl groups in the cytosolic domain of the protein do not normally form disulfide bonds because the reducing environment in the cytosol maintains these groups in their reduced (-SH) form

Solubilizing a membrane protein with a mild nonionic detergent

The detergent disrupts the lipid bilayer and brings the protein into solution as protein-lipid-detergent complexes. The phospholipids in the membrane are also solubilized by the detergent, as lipid-detergent micelles

chemicals used to solubilize proteins for SDS Polyacrylamide-Gel Electrophoresis

The detergent sodium dodecyl sulfate (SDS) and the reducing agent b-mercaptoethanol.

The insertion of a multipass transmembrane protein into the ER membrane

The events up to the insertion of the first transmembrane segment follow the steps for single-pass membrane proteins. The orientation of this first transmembrane segment depends on the characteristics of the transmembrane segment and flanking regions just as for single-pass membrane proteins. When the next transmembrane segment emerges from the ribosome, it inserts into the lateral gate of Sec61 in an orientation opposite to that of the first transmembrane segment, then moves into the lipid bilayer. Each successive transmembrane segment is similarly inserted into the membrane via the lateral gate in an orientation opposite to that of the transmembrane segment that immediately preceded it. This proceeds until all transmembrane segments have been inserted into the membrane.

The Cortical Cytoskeleton Gives Membranes Mechanical Strength and Restricts Membrane Protein Diffusion

The filaments are thought to provide diffusion barriers that divide the membrane into small domains, or corrals. Spectrin heterodimers (enlarged in the drawing on the right) are linked together into a netlike meshwork by junctional complexes (enlarged in the drawing on the left). Each spectrin heterodimer consists of two antiparallel, loosely intertwined, flexible polypeptide chains called α and β. The two spectrin chains are attached noncovalently to each other at multiple points, including at both ends. Both the α and β chains are composed largely of repeating domains. Two spectrin heterodimers join end-to-end to form tetramers. The junctional complexes are composed of short actin filaments (containing 13 actin monomers) and the proteins band 4.1 and adducin, as well as a tropomyosin molecule that probably determines the length of the actin filaments. The cytoskeleton is linked to the membrane through two transmembrane proteins: a multipass protein called band 3 and a single-pass protein called glycophorin. The spectrin tetramers bind to some band 3 proteins via ankyrin molecules, and to glycophorin and band 3 (not shown) via band 4.1 proteins.

Mitochondrial Proteins Are Imported Post-translationally (not co-translationally) as

Unfolded Polypeptide Chains

The Fluidity of a Lipid Bilayer Depends on Its Composition

Unsaturated fatty acid tails are bent, making it more difficult to pack them together, which makes the membrane more fluid, and thinner

Lipid Anchors Control the Membrane Localization of Some Signaling Proteins.

The lipid anchor can be added and removed so that membrane association is transient, and in response to a signal

The Asymmetry of the Lipid Bilayer Is Functionally Important

The lipid composition of the two monolayers is different

Genes Can Be Cloned Using Bacteria

The plasmid is cut open with a restriction nuclease (in this case, one that produces staggered ends) and is mixed with the DNA fragment to be cloned (which has been prepared with the same restriction nuclease). DNA ligase and ATP are added. The staggered ends base-pair, and DNA ligase seals the nicks in the DNA backbone, producing a complete recombinant DNA molecule The resulting recombinant plasmid DNA is then introduced into a bacterium, where it is replicated many millions of times as the bacterium multiplies

The Polypeptide Chain Passes Through a Signal Sequence-gated Aqueous Channel in the Translocator

The protein can exit the channel into the lumen of the ER, or exit sideways into the ER membrane

Oligosaccharides Are Used as Tags to Mark the State of Protein Folding

The role of *N-linked glycosylation in ER protein folding*: The ER membrane-bound *chaperone protein calnexin* binds to incompletely folded proteins containing one terminal glucose on N-linked oligosaccharides, trapping the protein in the ER. Removal of the terminal glucose by a glucosidase releases the protein from calnexin. *A glucosyl transferase* is the crucial enzyme that determines whether the protein is folded properly or not: if the protein is still incompletely folded, the enzyme transfers a new glucose from UDP-glucose to the N-linked oligosaccharide, renewing the protein's affinity for calnexin and retaining it in the ER. The cycle repeats until the protein has folded completely. Calreticulin functions similarly, except that it is a soluble ER resident protein. Another ER chaperone, ERp57 (not shown), collaborates with calnexin and calreticulin in retaining an incompletely folded protein in the ER. ERp57 recognizes free sulfhydryl groups, which are a sign of incomplete disulfide bond formation. The longer a protein spends in this cycle without folding correctly, the more likely it is that ER-resident mannosidase enzymes (not shown) remove the terminal mannoses from the N-linked oligosaccharide. The trimmed oligosaccharide with reduced mannoses is recognized by other ER lectins that route the polypeptide for degradation. Thus, only proteins that fold promptly and exit the ER avoid trimming by mannosidases and escape degradation.

column chromatography

The sample, a solution containing a mixture of different molecules, is applied to the top of a cylindrical glass or plastic column filled with a permeable gel matrix, such as cellulose. A large amount of solvent is then passed slowly through the column and collected in separate tubes as it emerges from the bottom. Because various components of the sample travel at different rates through the column, they are fractionated into different tubes.

Sequential use of a cleaved ER signal sequence and transmembrane segment during membrane protein insertion

The signal sequence allows the initiation of translocation, while the transmembrane segment anchors the protein in the membrane

GFP (green fluorescent protein)

The structure of GFP highlights the eleven β strands that form the staves of a barrel, buried within which is the active chromophore (dark green) The chromophore is formed post-translationally from the protruding side chains of two amino acid residues in a series of autocatalytic steps has tyrosine and serine

Indirect immunocytochemistry

This detection method is very sensitive because many molecules of the secondary antibody recognize each primary antibody. The secondary antibody is covalently coupled to a marker molecule that makes it readily detectable. Commonly used marker molecules include fluorescent dyes (for fluorescence microscopy) and colloidal gold spheres (for electron microscopy).

Some Proteins Are Integrated Into the ER Membrane by a Post-translational Mechanism

This is useful for proteins anchored in the membrane by their C terminus ("tail") In this post-translational pathway for the insertion of tail-anchored membrane proteins into the ER, a soluble pre-targeting complex captures the hydrophobic C-terminal transmembrane segment (red) after it emerges from the ribosomal exit tunnel and loads it onto the Get3 targeting factor. The resulting complex is targeted to the ER membrane by interaction with the Get1-Get2 receptor complex, which functions as a membrane protein insertion machine. After the tail-anchored protein is released from Get3 and inserted into the ER membrane, Get3 is recycled back to the cytosol. This targeting cycle is conceptually similar to protein targeting by SRP. Although not shown in the figures, both Get3 and SRP bind and hydrolyze nucleoside triphosphates to provide directionality to the targeting cycle. ATP is used by Get3, and GTP is used by SRP.

The Bacterial CRISPR System Has Been Adapted to Edit Genomes in a Wide Variety of Species

This system allows a specific mutation to be introduced to the genome Use of CRISPR to study gene function in a wide variety of species: The Cas9 protein (artificially expressed in the species of interest) binds to a guide RNA, designed by the experimenter and also expressed. The portion of RNA in light blue is needed for associations with Cas9; that in dark blue is specified by the experimenter to match a position on the genome. The only other requirement is that the adjacent genome sequence includes a short PAM (protospacer adjacent motif, not shown) that is needed for Cas9 to cleave. This sequence allows the CRISPR system in a bacterium to distinguish its own genome from that of invading viruses. When Cas9 is directed to make a double-strand break in a gene, the break is generally repaired by nonhomologous end joining (not shown), which introduces local sequence errors that can disrupt gene function. A more precise mutation can be made as shown here, where the double-strand break is repaired by homologous recombination with an altered gene provided by the experimenter. By using a mutant form of Cas9 that can no longer cleave DNA, Cas9 can be used to activate a normally dormant gene or turn off an actively expressed gene

The synthesis of cDNA

Total mRNA is extracted from a particular tissue, and the enzyme reverse transcriptase is used to produce DNA copies of the mRNA molecules. A short oligonucleotide complementary to the poly-A tail at the 3′ end of the mRNA is first hybridized to the RNA to act as a primer for the reverse transcriptase, which then copies the RNA into a complementary DNA chain, thereby forming a DNA-RNA hybrid helix. Treating the DNA-RNA hybrid with a specialized ribonuclease (RNase H) that attacks only the RNA produces nicks and gaps in the RNA strand. DNA polymerase then copies the remaining single-stranded cDNA into double-stranded cDNA. Because DNA polymerase can synthesize through the bound RNA molecules, the RNA fragment that is base-paired to the 3′ end of the first DNA strand usually acts as the primer for the second strand synthesis, as shown. Any remaining RNA is eventually degraded during subsequent cloning steps. As a result, the nucleotide sequences at the extreme 5′ ends of the original mRNA molecules are often absent from cDNA libraries. mRNAs are reverse transcribed into DNA to to make the library. In this way, only transcribed regions are represented in the library.

Transgenic Plants Are Important for Agriculture

Transgenic plants can be made using recombinant DNA techniques optimized for plants ex: A disc is cut out of a leaf and incubated in a culture of Agrobacterium that carries a recombinant plasmid with both a selectable marker and a desired genetically engineered gene. The wounded plant cells at the edge of the disc release substances that attract the bacteria, which inject their DNA into the plant cells. Only those plant cells that take up the appropriate DNA and express the selectable marker gene survive and proliferate and form a callus. The manipulation of growth factors supplied to the callus induces it to form shoots, which subsequently root and grow into adult plants carrying the engineered gene.

vesicular transport

Transport vesicles bud from one compartment (donor) and fuse with another topologically equivalent (target) compartment. In the process, a subset of soluble components (red dots) are transferred from lumen to lumen. Note that membrane is also transferred and that the original orientation of both proteins and lipids in the donor compartment membrane is preserved in the target compartment membrane. Thus, membrane proteins retain their asymmetrical orientation, with the same domains always facing the cytosol.

_____ Electrophoresis Provides Greater Protein Separation

Two-dimensional Gel

Epitope tagging for the purification of proteins

Using standard genetic-engineering techniques, a short amino acid sequence can be added to a protein of interest. If the tag is an antigenic determinant, or epitope, it can be targeted by an appropriate antibody, which can be used to purify the protein by immunoprecipitation or affinity chromatography.

The Electron Microscope Resolves the Fine Structure of the Cell

Wavelength of an electron could be as small as .004 nm, much smaller than that of visible light! Resolution is limited by the "lenses" used to focus the electron beam The "lenses" in an electron microscope are *magnetic coils* (not glass)

The isolation of purified rough and smooth microsomes from the ER

When an intact cell or tissue is homogenized, many of its membrane-enclosed compartments form small sealed vesicles called microsomes. When this mixture of vesicles is sedimented to equilibrium through a gradient of sucrose, the two types of microsomes separate from each other on the basis of their different densities. Note that the smooth fraction contains non-ER-derived material. Thin section electron micrographs of the purified smooth and rough microsome fractions show an abundance of ribosome-studded vesicles in the rough microsome fraction that originated from the rough ER. These are not seen in the smooth microsome fraction, which primarily contains ribosome-free vesicles originating from the smooth ER, Golgi cisternae, and other organelles.

Protein Structure Can Be Determined Using ______. For this technique, the protein must be ____.

X-ray Diffraction crystallized

Living Cells Are Seen Clearly in

a Phase-Contrast or a Differential-Interference-Contrast Microscope

gene

a functional unit of inheritance, usually corresponding to the segment of DNA coding for a single protein

null mutation

a loss of function mutation that completely abolishes the activity of the gene

cholesterol

a sterol polar head group, rigid planar steroid ring structure, and nonpolar hydrocarbon tail cholesterol is found in animals; many other organisms use other sterols in the same way

Individual Proteins Can Be Fluorescently Tagged in Living Cells by

adding the coding sequence for a fluorescent protein Multiple Proteins Can Be Fluorescently Tagged with different fluorescent proteins

phosphatidylserine, phosphatidylethanolamine, and phosphatidylcholine

all derived from glycerol (has phosphate) all except phosphatidylserine are electrically neutral

genome

all of an organism's DNA sequences

Phospholipids Spontaneously Form

bilayers

alleles

alternative forms of a gene

translocation

breaks off a segment from one chromosome and attaches it to another

4 types of light microscopy

bright field- light transmitted straight through the specimen phase contrast - phase alterations of light transmitted through the specimen are translated into brightness changes differential interference contrast - highlights edges where there is a steep change of refractive index dark field - specimen is lit from the side and only the scattered light is seen

proteins can be separated by

column chromatography

cells can be separated into their component fractions via

centrifugation

Staining is used to increase

contrast

fluorescence microscopy second barrier filter

cuts out unwanted fluorescent signals, passing the specific green fluorescein emission between 520 and 560 nm

Largest intracellular compartment

cytosol

The longer the protein spends going through this cycle, the greater the chance it gets marked for

degradation

deletion

deletes a segment of a chromosome

sphingomyelin

derived from sphingosine, a sphingolipid, which has no phosphate

Sequence Variants Can Aid the Search for Mutations Associated with Disease

differences in SNPs (single nucleotide polymorphisms) in healthy and affected individuals of a particular disease can help target genes that affect the risk of developing a common disease

dominant negative mutation

dominant acting mutation that blocks gene activity, causing a loss of function phenotype even in the presence of a normal copy of the gene occurs when the mutant gene product interferes with the function of the normal gene product

The import receptor proteins have several FG binding sites, which can competitively bind to the FG repeats, allowing the cargo/receptor complex to "melt" its way through the Nuclear Pore Complex. This process can go in ___ direction

either

loss of function mutation

either reduces or abolishes the activity of a gene most common class of mutations usually recessive organism can usually function normally as long as one normal copy is retained

Biomolecular Condensates may Form and Disassemble in Response to Need

ex: Phosphorylation regulates the formation and dissolution of condensates during signaling. When a receptor at the plasma membrane is engaged by its ligand, its cytosolic tail and associated proteins become phosphorylated. This modification, along with surrounding amino acids, forms a specific binding site for various cytosolic and membrane proteins, many of which are multivalent. The multivalent proteins interact with each other to drive the formation of a condensate that has distinctive signaling properties. When the key sites become dephosphorylated, the condensate disassembles and signaling stops.

Screens for temperature sensitive mutations allow identification of essential genes.

ex: Screening for temperature-sensitive bacterial or yeast mutants

Nuclear Localization Signals Direct Proteins to the Nucleus

ex: T antigen without lysine won't get to nucleus

Mutant screens can be used to identify genes involved in a specific process

ex: flower formation

Different cell types have different lipid compositions

ex: liver has a lot for detoxifying purposes

The nucleolus is a condensate that has functionaly distinct regions

fibrillarin, nucelophosmin

Enzymes called _____, move selective phospholipids unidirectionally from one side of the bilayer to the other. *This process requires ATP*, concentrates specific membrane lipids on one side of the membrane, and so are considered active transporters.

flippases

Removal of the terminal glucose by a _____ releases the protein from calnexin, so it is free to move on from the ER

glucosidase

Oil immersion increases resolution Because oil

has a higher refractive index than air

numerical aperature

n sin θ function of a lens' light collecting ability for dry leneses, it cannot be more than 1 for oil immersion lenses, iit can be as high as 1.4 the higher the numerical aperture, the greater the resolution and the brighter the image

Cells derived from cancerous tissue are often easier to ____ (or may be that way already)

immortalize

gain of function mutation

increases the activity of the gene or makes it active in inappropriate circumstances usually dominant

phosphatidylserine

net negative charge

inversion

inverts a segment of a chromosome

types of chromatography

ion exchange, gel filtration, affinity

Biomolecular Condensates Create Biochemical Factories by

keeping multiple steps of a biochemical pathway in close proximity ex: the pyrenoid of the chloroplasts contains a condensate of Rubisco that exploits locally generated CO2.

Some β Barrels Form

large channels

fluorescence microscopy first barrier filter

lets through only blue light with a wavelength between 450 and 490 nm

Different fluorescent markers have various excitation and emission wavelengths The excitation wavelength is always ___ than the emission wavelength

lower

point mutation

maps to a single site in the genome, corresponding to a single nucleotide pair or a very small part of a single gene

cone shaped phospholipids form, cylinder shaped phospholipids form

micelles, bilayers

PCR can ___ the end of Specific Gene Sequences for easier Cloning

modify ex: DNA cloning by Gibson assembly. It is often useful to insert a DNA fragment in a circular bacterial plasmid. In Gibson assembly, a plasmid is cleaved at a specific site with a restriction nuclease. In parallel, the DNA sequence to be cloned is amplified by PCR of a cDNA or other source. In addition to sequences complementary to the ends of the DNA to be amplified, the PCR primers include 15-40 nucleotides of sequence that matches the sequence on either side of the cut site in the linearized plasmid. The PCR product and plasmid are treated with a 5′ exonuclease, which partially digests the DNA from the 5′ end, resulting in single-strand 3′ overhangs. These single-strand overhangs hybridize with their complementary sequence, neatly inserting the PCR product in the plasmid. DNA polymerase is then added to fill in any gaps in the sequence, and DNA ligase seals the nicks to provide the fully assembled plasmid.

conventional light microscope

objective lens - collects a cone of light rays to create an image condenser lens - focuses a cone of light rays onto each point of the specimen resolution - the resolving power of the microscope depends on the width of the cone of illumination and therefore on both the condenser and the objective lens.

Calnexin binds to incompletely folded proteins if they have

one terminal glucose

Peroxisomal precursor vesicles that bud from the ER contain a few

peroxisomal proteins

Four major phospholipids in mammalian plasma membranes

phosphatidylETHANOLAMINE phosphatidylSERINE phosphatidylCHOLINE sphingoMYELIN

Cholesterol has different effects on fluidity depending on

temperature

types of mutations

point, inversion, deletion, translocation

Multiple ribosomes can bind to one mRNA, forming a

polyribosome

conditional mutation

produces phenotypic effect only under certain conditions permissive conditon - not expressed ex: temp sensitive mutation, seen in high temps, but cold temps are permissive conditions

fluorescence microscopy beam splitting mirror

reflects light below 510 nm but transmits light above 510 nm

Most cell cultures obtained from animal tissues eventually die. They undergo _______, meaning that after some number of cell divisions, they stop dividing and die. Cells that do not have this problem are called ____

replicative cell senescence immortal

A Signal-Recognition Particle (SRP) binds to a ____ so that the signal sequence-binding pocket is near the growing polypeptide chain

ribosome binds to the large subunit specifically SRP binds to the large ribosomal subunit so that its signal sequence-binding pocket is positioned near the growing polypeptide chain exit site. Its translational pause domain is positioned at the interface between the ribosomal subunits, where it interferes with elongation factor binding

STR

short tandem repeat

Detergents can be used for ________ functional membrane protein systems.

solubilizing, purifying, and reconstituting ex: reconstituting a functional ion pump in a small vesicle for study

types of mass spectrometry

standard (MS) and tandem (MS/MS)

Cholesterol helps to maintain ____ of the plasma membrane

structural integrity and regulate fluidity

suppressor mutation

suppresses the phenotypic effect of another mutation, so that the double mutant seems normal intragenic suppressor mutation lies within the gene affected by the first mutation extragenic suppressor mutation lies in a second gene--often one whose product interacts directly with the product of the first

Some cells can be "immortalized" by engineering them to continuously produce

telomerase, which maintains the length of telomeres.

locus

the site of the gene in the genome

genotype

the specific set of alleles forming the genome of the individual

Scanning Electron Microscopy (SEM)

the specimen is scanned by a beam of electrons brought to a focus on the specimen by the electromagnetic coils that act as lenses The detector measures the quantity of electrons scattered or emitted as the beam bombards each successive point on the surface of the specimen and records the intensity of successive points in an image built up on a screen The SEM creates striking images of three-dimensional objects with great depth of focus and a resolution between 0.5 nm and 10 nm depending on the kind of instrument

phenotype

the visible character of the individual

For electron microscopy, proteins can be tagged with an antibody that is fastened to ____, which are easily visible

tiny metal particles (ex: Immunogold electron microscopy) Heavy Metals Can Provide Additional Contrast

cells can be isolated from ___ and grown in ____

tissues, culture

There are Multiple routes to the inner and outer membranes As with localization to other destinations, the necessary targeting information is encoded in the protein as a signal sequence. In the case of mitochondria and chloroplasts, a protein may require ___ signal sequences to get to the correct location.

two

Despite Their Fluidity, Lipid Bilayers Can Form Domains of Different Compositions

under some conditions, the phospholipids can spontaneously organize into domains, or lipid rafts note that because of their composition, raft domains are thought to have an increased membrane thickness

types of sedimentation (separation of cells)

velocity & equilibrium

In Most Transmembrane Proteins, the Polypeptide Chain Crosses the Lipid Bilayer in an ___ Conformation

α-Helical

purifying proteins

•Cells Can Be Separated into Their Component Fractions •Proteins Can Be Separated by Chromatography •Immunoprecipitation Is a Rapid Affinity Purification Method •Genetically Engineered Tags Provide an Easy Way to Purify Proteins •Purified Cell-free Systems Are Required for the Precise Dissection of Molecular Functions

Studying gene function and expression

•Classical Genetic Screens Identify Random Mutants with Specific Abnormalities •Mutations Can Cause Loss or Gain of Protein Function •Complementation Tests Reveal Whether Two Mutations Are in the Same Gene or Different Genes •Gene Products Can Be Ordered in Pathways by Epistasis Analysis •Mutations Responsible for a Phenotype Can Be Identified Through DNA Analysis Rapid and Cheap DNA Sequencing Has Revolutionized Human Genetic Studies

The compartmentalization of cells

•Construction of Most Organelles Requires Information in the Organelle Itself For example, if the ER is removed, the cell is unable to make a new ER

Misfolded Proteins in the ER Activate an Unfolded Protein Response

•If misfolded proteins accumulate in the ER, the cell increases production of several proteins, including those involved with: - Retrotranslocation out of the ER - Protein degradation - ER chaperones

Studying gene function and expression

•Linked Blocks of Polymorphisms Have Been Passed Down from Our Ancestors •Sequence Variants Can Aid the Search for Mutations Associated with Disease •Genomics Is Accelerating the Discovery of Rare Mutations That Predispose Us to Serious Disease •The Cellular Functions of a Known Gene Can Be Studied with Genome Engineering Animals and Plants Can Be Genetically Altered

Analyzing proteins

•Proteins Can Be Separated by SDS Polyacrylamide-Gel Electrophoresis •Two-dimensional Gel Electrophoresis Provides Greater Protein Separation •Specific Proteins Can Be Detected by Blotting with Antibodies •Mass Spectrometry Provides a Highly Sensitive Method for Identifying Unknown Proteins

Analyzing and manipulating DNA

•Restriction Nucleases Cut Large DNA Molecules into Specific Fragments •Gel Electrophoresis Separates DNA Molecules of Different Sizes •Purified DNA Molecules Can Be Specifically Labeled with Radioisotopes or Chemical Markers in Vitro •Genes Can Be Cloned Using Bacteria •An Entire Genome Can Be Represented in a DNA Library •Hybridization Provides a Powerful but Simple Way to Detect Specific Nucleotide Sequences •Genes Can Be Cloned in Vitro Using PCR •PCR Is Also Used for Diagnostic and Forensic Applications •PCR and Synthetic DNA Are Ideal Sources of Specific Gene Sequences for Cloning •DNA Cloning Allows Any Protein to Be Produced in Large Amounts

Looking at cells and molecules in the electron microscope

•The Electron Microscope Resolves the Fine Structure of the Cell •Biological Specimens Require Special Preparation for Electron Microscopy •Heavy Metals Can Provide Additional Contrast •Images of Surfaces Can Be Obtained by Scanning Electron Microscopy