Download (1) Give brief definitions or unique descriptions of the following terms:

Biochemistry 2000
Sample Questions 4
RNA, Lipids, Membranes
(1) Give brief definitions or unique descriptions of the following terms:
(a) exon
(b) glycosidic bond
(c) poly A tail
(d) open complex (transcription)
(e) holoenzyme
(f) anticodon
(g) trans fatty acid
(h) Fluid Mosaic Model
(i) Antisense strand
(j) Codon
(k) Unsaturated fatty acid
(l) Ganglioside
(m) Micelle
(2) The following figure indicates the typical steps associated with the production
of eucaryotic mRNA.
(a) Fill in the blanks describing each of the steps in the production of
mRNA.
(b) Identify each of the lettered regions of the figure.
(c) Identify the regions labelled 'i' and 'ii'.
(3) What are the four distinct steps in bacterial transcription?
(4) How does bacterial RNA Polymerase locate the promoter?
(5) Bacterial transcription is error prone. Explain why this is not always a
problem.
(6) Fill in the blanks in the following statements.
(a) RNA is synthesized in the ____________ direction and the template
DNA strand is read in the ___________ direction.
(b) Bacterial transcription rates are directly proportional to the
_____________ of RNA polymerase for the ______________.
(c) The two most common mechanisms for transcription termination are
____________________ and ____________________.
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Biochemistry 2000
Sample Questions 4
RNA, Lipids, Membranes
(7) What is the “Wobble Hypothesis”?
(8) List at least four features of the genetic code.
(9) What are the reactions required to generate an aminoacyl tRNA?
(10) What identifies the site at which bacterial translation is initiated?
(11) Consider the following mRNA sequence:
5'-GAGAAUAACAAUGCAAACAUUU ...
(a) What is the sequence of the corresponding “coding” strand?
(b) What is the primary sequence of the translation product?
(12) Identify each of the tRNA binding sites on the ribosome and the type of tRNA
present in the site.
(13) Describe each of the steps of the translation elongation cycle.
(14) Draw a stick diagram for each of the following:
(a) an 18:1n-9 fatty acid
(b) any triacylglycerol
(c) any glycerophospholipid
(d) uridyl-2',5'-guanosine
(15) In biological membranes, how does the ratio of saturated/unsaturated fatty
acid tails change as a function of temperature?
(16) Below you can find the sequencing of the DNA coding strand.
CAGGCTTGACACTTTATGCTTCCGGCTCGTATAATTTCTCCATATTGTGA
GCCGTTTGTAAGGAGGTGATCATGGTTCGCTATACCTAAGTATGATAGCG
Shine-Dalgarno sequence
CGCATGCTGCGCGCTTTTTTTTTGCATAGACT
a. Identify in this sequence the promoter and termination signal for
transcription as well as the start and stop codon for translation.
b. What is the amino acid sequence of the encoded protein? (Use the table
provided at the end of the questions)
(17) What is the function of the  factor for RNA-Polymerase?
(18) Write the reaction occuring during transcription initiation when the first
phosphodiesterbond is formed between adenosine and uridine. Draw the
structures as a stick diagram.
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Biochemistry 2000
Sample Questions 4
RNA, Lipids, Membranes
(19)
Translation
a. What is the functional difference between mRNA and tRNA?
b. Give the sequence of one codon matching the following anticodons and
state which amino acid is attached to the tRNA. (Use the table of the
genetic code provided in the lecture notes.)
anticodon
3’ – C G U – 5’
3’ – A A G – 5’
c. The mRNA specifying the a chain of human hemoglobin contains the base
sequence ...UCCAAAUACCGUUAAGCUGGA..... The C-terminal
tetrapeptide of the normal 'A' chain, which is specified by part of this
sequence, is ...-Ser-Lys-Tyr-Arg. In a mutant hemoglobin called
hemoglobin “Constant Spring”, the corresponding region of the 'A'-chain
has the sequence ...-Ser-Lys-Tyr-Arg-Gln-Ala-Gly-...
Specify the mutation on the mRNA level which causes hemoglobin
“Constant Spring”. (Again use the genetic code table.)
(20)
Draw the structures of the following lipids:
a. a triacylglycerol containing the following fatty acids: stearic acid (18:0),
palmitoleic acid (16:1n-7), and linoleic acid (18:2n-6).
b. Beewax consisting of palmitic acid (16:0) and a 30-carbon saturated
alcohol.
c. Phosphatidylethanolamine (a glycerophospholipid) with your choice of
fatty acids.
d. A ceramide, the simplest shingolipid.
(21)
Identify each as a fatty acid, steroid, triacylglycerol, glycerophospholipid
or sphingolipid:
1. cholesterol
2. glycerol, 2 fatty acids, phosphate, and choline
3. glyceryl tristearate
4. sphingosine, fatty acid, phosphate, and choline
(22)
Which of the following biomolecules are found in cell membranes?
a. cholesterol
b. triacylglycerol
c. carbohydrates
d. proteins
e. waxes
f. glycerophospholipids
g. sphingolipids
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Biochemistry 2000
Sample Questions 4
RNA, Lipids, Membranes
(23) Imagine the planet Gibo in a far-off galaxy where the life forms have a
similar appearance to those on earth. However, there is one huge difference:
heptane on Gibo has the role played by water on earth.
If the molecules that form cell membranes are the same as those on earth,
what is the major difference in the construction of cell membranes on Gibo
compared with earth?
(24) Membrane compositions of fish and other cold-blooded animals change
when their environmental temperature is lowered. The unsaturated fatty acid
content of the lipids in the cell membranes increases when the organism
becomes adapted to the lower temperature.
What is the purpose of this increase?
(25) Identify in the diagram the following components of the cell membrane:
Integral and peripheral membrane protein, carbohydrates, lipid bilayer, polar
heads of lipids, and nonpolar tails of fatty acids.
(26) The biochemists Eugene Kennedy and James Rothman have investigated
the biosynthesis of membrane lipids. They gave growing bacteria a short
pulse of radioactive phosphate which enters the cell so as to label the newly
synthesized phospholipids. Immediately afterwards, they added a membraneimpermeable TNBS label to the bacteria that combines with phopholipids.
Analysis of the resulting membrane showed that no TNBS-labelled
phospholipid was radioactively labelled.
a. Based on this experiment, where do you conclude are the phospholipids
synthesized?
b. Next, they repeated the experiment but allowed 3 min to elapse between
the pulse of radioactive phosphate and the TNBS addition. In this case,
about half of the TNBS-labelled phospholipid is also radioactive. How can
you explain this result?
4
Biochemistry 2000
Sample Questions 4
RNA, Lipids, Membranes
Answers:
(1)
(a) – The coding sequence that is retained in mature mRNA and directs
eucaryotic protein synthesis.
(b) – The covalent bond between the C1' of D-ribose and the N1(or N9) atom
of the nucleic acid base.
(c) – A stretch of 10s to 100s of adenylic acid nucleotide added to the 3'
terminii of eucaryotic mRNA.
(d) – The transcription initiation complex in which the DNA strands of the
bacterial promoter (-10 - +4) are single stranded.
(e) – The complete or active enzyme including all subunits.
(f) – The ribonucleotide triplet of tRNA that is complementary to the mRNA
codon.
(g) – An unsaturated fatty acid with a trans conformation about the double
bond.
(h) – Biological membranes are dynamic structures in which the lipids and
proteins are free to move in 2D.
(i) - Antisense strand – the antisense strand of DNA is complementary to the
RNA generated from this DNA (because it is the template strand during
transcription).
(j) - A codon is a base triplet in an mRNA that encodes one amino acid as
specified in the genetic code.
(k) An unsaturated fatty acid is a long chain carboxylic acid with one or more
double bonds in the hydrocarbon tail.
(l) A ganglioside is a complex shpingolipid consisting of a ceramide and 3 or
more sugars.
(m) A micelle is a globular aggregate of single-tail lipids in water where the
hydrocarbon tails are out of contact with water.
(2)
(a) – Transcription; Processing or Capping; Splicing
(b) – A, C, E, G, I , K, M, O are exons
B, D, F, H, J, L, N are introns
(c) – 'i' is the 5' cap (7-methyl guanosine)
'ii' is the 3' polyA tail
5
Biochemistry 2000
Sample Questions 4
RNA, Lipids, Membranes
(3) – The steps in bacterial transcription are
1. Template binding
2. Initiation
3. Elongation
4. Termination.
(4) – The RNA polymerase holoenzyme 70) binds dsDNA and 'scans'
along the dsDNA searching for a promoter. When a promoter is located, the
holoenzyme binds tightly to the promoter. Once transcription has initiated the
70 subunit is released.
(5) – Transcription errors are not necessarily a problem as (1) multiple transcripts
are produced from a single gene, (2) redundancy in the genetic code means
the resulting codon may encode the same amino acid residue and (3) many
mutations in protein sequence do affect function. In the case of eucaryotic
genes, mutations in introns may not appear in the mature mRNA.
(6) –
a–
5' to 3'
3' to 5'
b–
affinity
promoter
c–
r dependent
r independent
(7) – There are many more codons than tRNA molecules. Some tRNAs can
recognize more than one codon that differ in the third position of the codon by
forming non Watson-Crick base pairs.
(8) – Features of the genetic code include
(1) a codon or base triplet specifies an amino acid residue,
(2) base triplets are read in a particular frame,
(3) an amino acid can be specified by more than one codon (code is
degenerate)
(4) related codons specify amino acids with similar physiochemical properties
(code is non-random)
(5) virtually identical in all organisms (code is universal)
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Biochemistry 2000
Sample Questions 4
RNA, Lipids, Membranes
(9) – Amino Acid + ATP →
Aminoacyl adenylate + PPi
(Reaction 1)
Aminoacyl adenylate + tRNA →
Aminoacyl tRNA + Adenosine-5'-monophosphate
(Reaction 2)
(10) – Translation initiation depends upon an AUG codon and the presence of a
Shine-Dalgarno sequence a few nucleotides upstream that base pairs with
16S rRNA.
(11) – a – The coding sequence is identical to the mRNA sequence
5'-GAGAATAACAATGCAAACATTT ...
b – Translation starts at the AUG codon and produces: Met–Gln–Thr–Phe(12) – A site
P site
E site
contains aminoacyl tRNA
contains peptidyl tRNA
contains tRNA (deacylated tRNA)
(13) – The translation elongation cycle involves three steps:
(i) aminoacyl tRNA binding to the A site of the ribosome (catalyzed by EF-Tu)
(ii) peptide bond formation between P site tRNA and the A site tRNA
(iii) translocation of the A site tRNA to the P site and the P site tRNA to the E site
(catalyzed by EF-G)
In order for the cycle to continue, EF-Tu is converted from the GDP to the
GTP form by EF-Ts
7
Biochemistry 2000
(14) –
a–
Sample Questions 4
b–
RNA, Lipids, Membranes
c- Phosphatidic acid (x=H)
d–
(15) – Unsaturated fatty acids decrease the temperature of the order-disorder
transition. Consequently, the ratio of unsaturated to saturated fatty acids
increases as the temperature is lowered.
(16) a. Promoter, Termination signal, start and stop codon:
CAGGCTTGACACTTTATGCTTCCGGCTCGTATAATTTCTCCATATTGTGA
----------------------Promoter----------------------------------------GCCGTTTGTAAGGAGGTGATCATGGTTCGCTATACCTAAGTATGATAGCG
Shine-Dalgarno sequence Start codon
Stop codon
CGCATGCTGCGCGCTTTTTTTTTGCATAGACT
-------------Termination signal-----------b. Encoded amino acid sequence: Met-Val-Arg-Tyr-Thr-(Stop)
8
Biochemistry 2000
Sample Questions 4
RNA, Lipids, Membranes
(17) The  factor helps the RNA-Polymerase to find the promoter. Holoenzyme
(RNA-Polymerase including  factor) binds weakly to non-promoter DNA such
that it can move along DNA in search of the promoter. Holoenzyme binds
tightly only to the promoter. After transcription initiation, the  factor
dissociates and the resulting core enzyme (RNA-Polymerase without  factor)
binds tightly and continues with transcription elongation.
(18)
pppA + pppU  pppApU + PPi
H
N
2
O
N
N
N
H
O
O
O
O
O
O
O
P
O
P
N
O
P
O
O
P
N
N
O
P
O
P
O
H
O
O
H
O
O
O
O
O
O
O
+
O
H
H
O
H
O
O
H
H
N
2
N
N
O
O
O
O
P
N
O
P
N
O
P
O
H
O
O
O
O
O
O
O
O
P
O
P
H
O
N
H
O
H
+
O
O
O
O
P
O
O
N
O
O
H
O
O
H
(19) a. The function of the mRNA is to encode the sequence for a protein by
being a copy of the genetic information (DNA), while the tRNA has the
function to decode the mRNA by functioning as an adapter, i.e. the anticodon
of the tRNA base-pair with the codon of the mRNA and carries at its other end
the amino acid encoded by this codon.
b. anticodon
3’ – C G U – 5’
3’ – A A G – 5’
codon
5’ – G C A – 3’
5’ – U U C – 3’
amino acid
Ala
Phe
c. In the sequence CCAAAUACCGUUAAGCUGGA the U of the stop codon
following the Arg codon is changed to a C which generates a Gln codon.
The C is indicated in bold in the mutant sequence:
CCAAAUACCGUCAAGCUGGA
9
Biochemistry 2000
Sample Questions 4
(20) a.
O
O
O
O
O
O
b.
O
O
c.
CH2-CH2-NH3+
d.
H
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RNA, Lipids, Membranes
Biochemistry 2000
Sample Questions 4
RNA, Lipids, Membranes
(21)
a. cholesterol = steroid
b. glycerol, 2 fatty acids, phosphate, and choline = glycerophospholipid
c. glyceryl tristearate = triacylglycerol
d. sphingosine, fatty acid, phosphate, and choline = Sphingolipid
(22)
cholesterol, carbohydrates, proteins, glycerophospholipids and
sphingolipids are found in biological membranes.
(23)
If the same molecules form cell membranes, then these are lipids
(phosphoglycerolipids, sphingolipids) with a polar head and a nonpolar
tail. The polar heads (hydrophilic) are not soluble in heptane, but the
nonpolar tails are. Therefore the tails will point outwards, and the polar
heads will form the inner core of the lipid bilayer. That is the opposite
orientation compared to the lipid bilayers on earth.
(24)
Increasing the unsaturated fatty acid contents will create chains that
cannot pack as well within the membrane due to the kink within the
chains. Therefore, the unsaturated fatty acids have lower melting points
than saturated fatty acids. The loose packing means that the cell
membranes remain fluid and do not become too rigid even when the
temperature is decreasing.
(25)
A) Carbohydrates (attached to
proteins or as glycosphingolipids)
B) Lipid bilayer
C) Nonpolar tails of fatty acids
D) Polar heads of lipids
E) Integral and peripheral membrane
protein
(26)
a. The added TNBS remains outside the cells and labels only the outer
lipid layer of the bilayer in the cell membrane since it is membrane
impermeable. Consequently, the newly synthesized phospholipids can not
be in this outer lipid layer, but must be in the inner lipid layer of the
membrane. This indicates that newly made phospholipids are synthesized
on the cytoplasmic face of the membrane.
b. The newly synthesized, radioactive phospholipids must reach the outer
layer of the membrane to become also labeled with TNBS. Obviously, this
can occur in only 3 min and requires a flip-flop (transverse diffusion) of the
lipids. Usually, a flip-flop is extremely slow (in contrast to lateral diffusion).
The only possibility to explain the fast flip flop observed here is to assume
active transport of the phospholipids. (Indeed, this flip-flop is catalyzed by
enzymes known as flipases which are passive transporters.)
11