GRE Molecular & Cell Biology Questions 170, 171, 172

Today question is from GRE Biochemistry, Cell and Molecular Biology practice test.
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A1. From Fig. 1&2 we can see that protein synthesis continues for some time after inhibitor was added. That is caused by the fact that even when inhibitor is added and significant fraction of RNA Polymerase activity is halted, there is still a lot of "idle" or not yet used products of RNA Pol in the cell. This backup amount of RNAs is available for protein synthesis for short time. Answer is A.

A2. From Fig. 1 we can approximate rate of synthesis after inhibitor was added, and compare it to non-inhibited case. TO do that, let's just calculate derivative after inhibition, or slope of the lines. Take vertical distance (change in Counts Per minute, CPM), divide by change in time.

No inhibition: 6000-2000 / 20 hrs ~ 4000CPM/20min=200CPM/hr
With inhibition: 3000-2000 / 20 hrs ~ 50CPM/hr

After inhibition, rate goes down about 4x, thus we can estimate that inhibitor blocks about 75% of activity. Answer is B.

A3. In this question experiment was performed as following: proteins from nuclear extract were purified on a column. Peak were collected and used as a source for RNA polymerization. Since radioactive thymine was used, RNA polymerization activity of each fraction (peak) was measured afterwards.

In HeLa cells, there are 3 RNA polymerases:
RNA Pol I: synthesizes a pre-rRNA 45S, molecular weight 590 kDa
RNAPol II: synthesizes precursors of mRNAs and most snRNA and microRNAs, MW 550 kDa
RNA Pol III: synthesizes tRNAs, rRNA 5S and other small RNAs found in the nucleus and cytosol, MW 0.7MDa

Question is about type of the RNA Pol that is concentrated in first peak. Interestingly, Fig 3 gives much more information that asked in question, possibly setting up test-takers for a distraction. To answer, I had to Google RNA Pol chromatography, and it seems that peak 1 is mainly RNA Pol I.
 

 But even more widely known is that nucleolus is a site where ribosomal RNA (rRNA) is primarily produced. Answer is A.

Sample GRE Q55

Turns out this blog is still up and running :-) so here is a new one: Sample GRE biochem test, question 55 with P+ of 31% (from 2016 edition) [*P+ is percentage of test takers who gave correct answer.]

An amino acid transporter protein is responsible for the transport of a specific amino acid across a membrane. The KI values of several competitive inhibitors of the amino acid transporter are shown above. Based on these data, which of the following is most likely the amino acid transported by this protein?

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Correct answer is E And here is why. First, let's unwrap some of the language. "The inhibitor constant, Ki, is an indication of how potent an inhibitor is; it is the concentration required to produce half maximum inhibition." (here) So, the higher the Ki, more inhibitor concentration is necessary to prevent our transporter from moving amino-acid X through the membrane. It is also mentioned in the question that inhibitors are competitive. That means, to some degree, that inhibitors are similar in structure to that of the amino-acid X. Most similar inhibitor will then have lowest Ki, is est will occupy transporter's "slot" very nicely preventing amino-acid X from binding. In other words, inhibitor with lowest Ki will have most similar structure. Lowest Ki belongs to competitive inhibitor #3 (calledethyl benzene, by the way). It might be useful to notice second-best inhibitor as well, #5 (isoamyl alcohol). Both of these compounds are relatively not charged. Now let's look at structures of all possible answers:

(A)

(B)

(C)

(D)

(E)

As you can see, only tyrosine (E) carries benzyl group and also has less charge than other compounds. hence the answer is E

#15 RNA stem-loop stability

104. Which of the following mRNA molecules would form the most stable stem-loop structure?
(A) 5'...GGCUU......UUCGG...3'
(B) 5'...GGCUU......AAGCC...3'
(C) 5'...GGCUU......GGCUU...3'
(D) 5'...GGCUU......CCGAA...3'
(E) 5'...AAGCC......AAGCC...3'

Answer
First of all, stem-loops are formed by so-called inverted repeats. Is est, one strand of loop is complement to the other:
5'-...GGGCCC... (small loop here)
3'-...CCCGGG...
If this sequence is linearized now, we will get
5'-...GGGCCC......GGGCCC...-3' strand
Stem is that paired part of looped RNA.
Any mismatches (when, e.g. G is opposed by A) are called bulges.

So, hairpin (synonym of stem-loop) stability is defined by number of mismatches and stability of each base pair in the stem. That one arise from the fact that A have 2 hydrogen bonds with U (or T in DNA) and G have 3 hydrogen bonds with C, so GC pair is much more stable.

From first fact we can eliminate answer choices A, C, D and E, since neither A binds to C, nor U to U or A to U (just compare two nucleotides on the inner edges of uncovered sequences). Lets check answer B to be sure.
5'-...GGCUU...(loop)
3'-...CCGAA...
Should be stable!

This question is from easiest ones, so should not take a lot of time, which we are short of.

Yes, and I am back, will try to complete this blog during preparation.

#12 ER lumen protein

GRE-ST question number 54 is about ER proteins and functions.

The KDEL sequence, found on luminal proteins of the ER, is responsible for
(A) translocation of proteins into the ER lumen
(B) insertion of proteins into the membrane of the ER
(C) quality control in the ER
(D) recognition by signal peptidase of the signal sequence
(E) retrieval of ER luminal proteins from the Golgi

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This question is just seems to be hard. Just google and you'll probably find this wikipedia article on ER, where KDEL function is described clearly.
Point is that there are two types of proteins in ER lumen: those which should be transported further (to Goldgi) and those which work inside ER (e.g. linking oligosaccharides to proteins). So there should be some anchor for that "stable" proteins and it links to KDEL sequence on the end of those proteins. And because they cannot be fixed any way (or it would be very, very energetically unfavourable) they are transported back from Golgi to ER by specific KDEL-recetors.
So right answer is E.

#14 Protein secretion

GRE-ST question #98.

All of the following processes occur in the pathway leading to regulated protein secretion in animal cells EXCEPT
(A) formation of transport vesicles from the rough endoplasmic reticulum
(B) an increase in the concentration of cytosolic calcium ions prior to secretion
(C) synthesis of an amino-terminal signal sequence
(D) phosphorylation of a mannose residue in a glycoprotein
(E) trimming of N-linked oligosaccharides

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That is pretty simple. Question can be solved by just following common protein secretion pathway. That starts by transcription DNA's gene into RNA. Then that RNA — called matrix RNA, mRNA — caught by ribosome — which is actually RNA too, but ribosomal thus rRNA — and with assistance of different translation factors protein production begins (that is called translation mRNA into protein). Why secretion proteins are selected ones? That is because they have special signal at the beginning of polypeptide chain on N-terminal (which is actually some sequence at the beginning of mRNA, of course) which is caught by special cytoplasmic protein — Signal Recognition Particle or SRP. When SRP bind to that signal sequence translation stops and ribosome with mRNA and bound SRP flows to endoplasmic reticulum, where SRP-receptors are located. They bind SRP and passes signal through the lipid bilayer of ER. Then translation continues and protein get into ER lumen. After that happened, protein undergo different saccharides modifications. Pathway of Asn modification clearly described in Alberts book, includes trimming of N-linked oligosaccharides. Main steps of protein movement are: secretion in vesicle from ER to Goldgi apparatus and same movement through different compartments of Goldgi apparatus. After that vesicle with protein inside fuse into cell's membrane thus secreting protein into extracellular space.
So, I suppose that no calcium used in this pathway except signal transduction to muscle cells. So the answer is B.

Questionnaire #2

Several GRE-ST questions linked together.

Questions 118-121 refer to the following cellular processes.
(A) Phagocytosis
(B) Exocytosis
(C) Endocytosis
(D) Transcytosis
(E) Apoptosis

118. Movement of plasma membrane receptors from the basolateral surface to the apical surface of polarized epithelial cells
119. Up-regulation of glucose transporters at the plasma membrane
120. Selective retrieval of cell-surface proteins for recycling or degradation
121. Neurotransmitter release

Questions 124-126 refer to the following protein-modifying reagents.
(A) Chymotrypsin
(B) Cyanogen bromide
(C) Iodoacetamide
(D) Phenylglyoxal
(E) Pyridoxal 5′-phosphate

124. Forms a Schiff-base linkage with the epsilon-amino group of lysine residues
125. Specifically cleaves polypeptides on the carboxyl side of methionine residues
126. Generally used as a sulfhydryl-modifying reagent

Questions 127-129 refer to the following cell structures.
(A) Thylakoid membrane
(B) Nuclear lamina
(C) Eubacterial cell wall
(D) Plant cell wall
(E) Endoplasmic reticulum

127. Its assembly is inhibited by penicillin.
128. It is formed from polymeric fibrils composed of cellulose cross-linked by pectin and hemicellulose.
129. It is the site of dolichol phosphate function.

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It is important here to notice that one answer can be right for several questions.
Now lets try to describe each process announced. So phagocytosis is process of "eating" external organells by the cell. It is quite close to endocytosis, when – in contrast – molecules are being absorbed. Exocytosis is opposite process – secretion of molecules into external space. It can be hormones, antibodies and so on. Transcytosis is interesting process of transporting molecules from apical (absorbing) space of polarized cells to basolateral space, which happens, for example, when antibodies are transported through baby rat's gut. Main idea is that molecules are absorbed into internal vesicles (as during endocytosis) and then after several steps containers' content is exposed as during exocytosis. No target molecules are left in cell's plasma. Interesting is that cell can regulate exposure of some proteins in the cell membrane using transcytosis. During latent phase (e.g. while there is no great need in glucose) transporters are stored incorporated in vesicles inside the cell. But in case of emergency (or if cell is stimulated, by insulin in our case) that vesicles fuse into plasma membrane and transporters expose into extracellular space and begin to work. Last one is apoptosis which is self-destroying of the cell. That happens if crucial malfunction appear or cell's mission was fulfilled. So the answer are like that. I suppose, answer D is suitable for both two first questions.
118 D. 119 D. 120 C. 121 B.

Chymotripsin is protein, enzyme actually, of so-called class of proteases. It cleaves specific polypeptide regions near serine, so it is called serine protease.
Cyanogen bromide is simple small molecule CNBr and, as written in wikipedia, is used to immobilize proteins by gluing them to agarose gel during chromatography. But more important is that CNBr cleaves polypeptide chains at C-terminus of methionine.
Iodoacetamide is substance that can bind covalently with cysteine to prevent formation of disulfide bonds. So it can be called sulfhydryl modifying reagent.
Phenylglyoxal modifies arginine.
Pyridoxal 5′-phosphate (or PLP) is active form of vitamin B6. As written in wikipedia (again, yeah) it forms Schiff-base linkage the ε-amino group of a specific lysine group of the aminotransferase enzyme.
124 E. 125 B. 126 C.

This questions uncovers very specific properties of very important and well-known objects. So I will not discuss all of them, but just give right answers and some interesting details if I find any.
All options are common in one: they are membranes or structures, that acts like membranes – support shape (cell walls and nuclear lamina) and protect internal content. But they have different structure, functions and behavior.
127 C. Notice that eubacteria is synonym for bacteria.
128 D. 129 E. Dolichol phosphate acts in formation of membrane-associated glycoproteins, so I suppose its site is on the ER membrane.

#13 Hydrogen bonds

Which of the following pairs of molecules could NOT hydrogen bond with each other?

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Answer can be easily found in MBoC in few first chapters (page 57 :). Main idea about hydrogen bonds is that it is an interaction between two partial charges: d+ and d- or partial charge d+ with net charge on atom. So if there is shift of electrical density on some atom from hydrogen and same situation happened with atom on another molecule (actually this can be one molecule and hydrogen bond will occur because of its flexibility) hydrogen bond will establish. Note that its strength is about 20 times less than covalent bond's.
Right answer thus is D because there is no shift of electron density to the ring. In other cases there is possibility exists for hydrogen bonds to occur: between H-O- and O=H2 (A), H-OH and O= (B), -NH and O- (C), -OH and O= (E).

#10 Membrane is mosaic two-dimensional fluid (isn't it?)

GRE-ST question #109 – I reaaaally looove membranes, yeah?

All of the following statements about the fluid mosaic model of biological membranes are true EXCEPT:
(A) Lipid molecules in the membrane readily undergo lateral diffusion.
(B) Lipid molecules in the membrane readily undergo transverse (flip-flop) diffusion.
(C) Integral membrane proteins can undergo lateral diffusion.
(D) The saturated hydrocarbon chains of lipid molecules in the membrane undergo carbon-carbon bond rotation.
(E) The transition temperature of a membrane is sensitive to the composition of the lipid molecules in the membrane.

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That is rather easy question. To gt right answer, lets consider options one by one.
First, word "readily" was quite unclear, before I looked it up in the dictionary. It means "without hesitation or reluctance", i.e. without doubt, but in our case – rapidly, fast. So A is obviously true, because later (in plane) diffusion is primary characteristic of bilayer.
B is first candidate for right answer, because it is known, that one phohpolipid flip-flop by itself just once a month. But there are special proteins called phospholipid translocators. As wrote in MBoC, they "catalyze the rapid flip-flop of phospholipids from one monolayer to the other".
C is right too, because lots of proteins, e.g. channels and receptors, migrate in plane of bilayer rapidly.
E is true too. There two factors can be selected: saturation and length of phospholipids' tails and cholesterol amount in the bilayer. First two properties shift viscosity curve of membrane, while second smooth it as drawn on the picture below. Transition temperature is – in common sense – temperature when material undergoes phase transition. If tails are saturated (i.e. all carbon-carbon bonds are not double) and long, then it becomes easier to pack such bilayer tightly, thus temperature of freezing shifts to higher value. Contrary, if chains are unsaturated and short, their mobility is lower, thus freezing point will be shifted to lower temperatures. But cholesterol is thing that improve mobility of phospholipids at lower temperatures, as well as keep rigid structure at higher values.

Now, answer D. It is known, that tails of saturated hydrocarbons are very, vary mobile stuff. But they can't get far away from phospholipid core, hence they rotate around some links. Unsaturated phospholipids have double bonds in their tails. That double bonds are actually like two sticks, that connect atoms, so rotation around double bonds is impossible.
So right answer, as we excluded any other, is B and rapid flip-flop, even in presence of phospholipid translocators, doesn't happen.

#8 Membrane carrier vs. channel proteins

GRE-ST #26 question about difference between carrier and channel proteins. I just discovered that I don't even kind of idea of what membrane carrier protein is.

Membrane carrier proteins differ from membrane channel proteins by which of the following characteristics?
(A) Carrier proteins are glycoproteins, while channel proteins are lipoproteins.
(B) Carrier proteins transport molecules down their electrochemical gradient, while channel proteins transport molecules against their electrochemical gradient.
(C) Carrier proteins can mediate active transport, while channel proteins cannot.
(D) Carrier proteins do not bind to the material transported, while channel proteins do.
(E) Carrier proteins are synthesized on free cytoplasmic ribosomes, while channel proteins are synthesized on ribosomes bound to the endoplasmic reticulum.

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The best answer is provided in MBoC, of course. It is pretty short and uncovered in chapter 11, paragraph "There are two main classes of membrane transport proteins: carriers and channels". Briefly, carriers catch molecule of interest and push it inside or outside of compartment or cell by change in conformation, while channels organize – actually – a channel (often, filled with water or solution) that creates favorable conditions for transporting molecules of interest in- or outside.

Problem of transport through membrane is in its impermeability for charged molecules. That is why transport proteins are crucially important for cell. Transport differ, in common, into two groups: active and passive. It is transport against or down electrochemical gradient of molecules.
What is gradient? This topic is widely covered in calculus, but can be described simply with analogy of hill. If you put piece of rock on side of hill, rock will ran down to decrease its potential energy. Thus rock will roll down potential energy gradient. Same thing happens in the cell. If there are lots of molecules out of the cell and little inside, they will attempt to penetrate membrane. This process is spontaneous and do not need any energy, because it is actually hidden in solution and molecules themself, as energy for rolling is actually potential energy.
Another story with active transport. There we, obviously, need to take energy to drag molecule against its concentration gradient as we need energy to roll rock back on the hill.
Electrochemical gradient referred to two types of gradients. First one is gradient of electric charge. Because particles discussed now are charged, electric forces are involved. It takes some energy to pull positive charge into positively charged solute. We discussed chemical, or concentration, gradient above, it is about forces that drive molecules to occupy the larges volume. When speaking about electrochemical gradient, we are speaking actually about some mixture of those forces.

Now lets continue our talk about transport through membrane.
Important point here is that channels can't participate in active transport, i.e. force molecules against their electrochemical gradient, it was made possible by carrier proteins. As I mentioned, channel proteins create pore with channel, that act as defense from hydrophobic forces from phospholipid tails.

But what is the answer? That is difference in mechanism of transport. Using our brief discussion it is easy to identify false answers and explain their fallibility. Some trouble can be caused by answer A, because it is not clear are those proteins glycosilated or bound by any part to lipids. All – carriers and channel – proteins are membrane proteins, thus are synthesized on ER ribosomes. The difference between carriers and channels are, actually, in their names. As road tunnels don't ever touch cars, but ferries do, carriers closely interact and fix molecules on their surface, while channels just opens free road for rolling down gradient, so the right answer is D.

#9 Protein translocation in the ER

GRE-ST #39 question continues membrane proteins-related series of posts.

The common pathway of entry into the endoplasmic reticulum (ER) of secretory, lysosomal, and plasma membrane proteins is best explained by which of the following?
(A) Binding of their mRNAs to a special class of ribosomes attached to the ER
(B) Addition of a common sorting signal to each type of protein after completion of synthesis
(C) Addition of oligosaccharides to all three types of proteins
(D) Presence of a signal sequence that targets each type of protein to the ER during synthesis
(E) Presence of a zinc finger-binding domain in these three types of proteins

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ER plays important role in proteins' conformation maturation, transportation and translocation, which means their distribution in membrane bilayer.
Proteins from ER move out of the cell, into cell membrane and into cell's organelles membranes (e.g. lysosomes). Some of those proteins are expressed into cytoplasm after maturation (which includes proper folding and binding of oligosaccharides).
There are no special ribosomes, that are bound constantly to ER membrane and wait for mRNA to translate into protein. After gene (DNA) transcription of gene, mRNA is expressed into cytoplasm where ribosomes catch them. Actually, there are several binding sites on one mRNA so several ribosomes produce proteins simultaneously on one strand.
Main idea of that process is called "signal hypothesis", which says that there should be some signal that provide binding ribosome to ER and producing protein into ER, but not into cytoplasm. That signal is in first few amino acids of newly synthesized protein. After being exposed, this signal is recognized by special molecule – Signal Recognition Particle (SRP) which move ribosome with mRNA to SRP binding receptor on membrane of ER. This is very excited process which are uncovered in the MBoC clearly. Also prof. Schekman's lectures are very usefull.
Right answer is D. Gain more information in sources mentioned, because this is very interesting process, indeed.

#7 Bacteria and mammalian protein in vitro synthesis

GRE-ST question #29.

When bacteria produce mammalian proteins,
cDNA is used rather than genomic DNA. Which of the following is the best explanation?
(A) It is easier to clone cDNA than genomic DNA of comparable size.
(B) It is easier to clone RNA than DNA.
(C) It is not possible to clone the entire coding region of the gene.
(D) Most eukaryotic genes have introns that cannot be removed in bacteria.
(E) Most eukaryotic gene promoters do not function in bacteria.

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cDNA – complementary DNA, synthesized from mature mRNA (matrix RNA, protein-coding RNA, which is translated by ribosomes).
Intron – non-coding DNA region, that is transcribed into precursor RNA, but removed during splicing while RNA maturing.
Promoters – DNA regions, that signals begin of protein-coding sequences.

This question is uncovered by Alberts et al. in a tiny piece of text about cDNA libraries. Shortly, they remind that bacteria or yeast can't delete non-sense sequences (cos' their DNA have none of those) which are removed during splicing in mammalian. Hence right answer is D.

#2 Sweetened salad dressing

Ok, this one is from GRE Subject test (question number 16) and rather "chemical".

If sucrose and monosodium glutamate (MSG) are added to vinegar and oil salad dressing and shaken, the mixture will eventually separate into two phases of different density and polarity. Where will most of the sucrose and the MSG de located following phase separation?
(A) Both will concentrate in the vinegar
(B) Both will concentrate in the oil
(C) Both will concentrate at the interface
(D) Sucrose will concentrate in the oil and MSG – in the vinegar
(E) Sucrose will concentrate in the vinegar and MSG – in the oil

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Lets think a bit. The key word in the question for me is "density" and also I can't understand what "polarity" means and what role does it play.
So we will use density approach.
If you google a bit, you'll find – as I did – that oil density is about 0.9 g/ml and sucrose density is 1.5 g/cm3 (=1.5 g/ml) when MSG density is about twice less (lets say 0.7g/ml). So oil will be on the top, above vinegar. Sucrose will  precipitate (so it will be in vinegar). The question is where MSG will get to. Its approximate density is less than oil's so it will be on the top of the mixture.
So my answer is E. I have no idea how to get another way to find answer, maybe it is just knowledge test.