Proteins Flashcards
1
Q
What three things do you need to fully describe a protein?
A
- the structure of the protein. 2. The reaction that is catalysed. 3. The mechanism of action.
2
Q
What isomer of amino acids are all proteins made up from?
A
L.
3
Q
What type of side chains are hydrophobic?
A
Non charged.
4
Q
Amino acids are either hydrophobic or hydrophilic. True or False?
A
False. They can have hydrophobic and hydrophilic parts. For example the oh group on tyrosine is hydrophilic whereas the benzene ring is hydrophobic.
5
Q
What two amino acids are carboxylic?
A
Aspartate and glutamate.
6
Q
What pH is histidine rarely found at?
A
Above 14. Then it becomes a amidizole ring with a net negative charge.
7
Q
What charge is histidine most commonly found at?
A
Neutral. This is between pH 6 and pH14.
8
Q
Why are peptide bonds rigid?
A
Electron localisation.
9
Q
The bottom right hand corner of a Ramachandran plot is disallowed for all amino acids. True or false?
A
False. Glycine is allowed.
10
Q
Beta strands are helical. True or false?
A
True.
11
Q
How many residues are there per turn in a beta sheet?
A
2.
12
Q
How many residues are there per turn in an alpha helix?
A
3.6.
13
Q
What is formed in a protein when the same phi-psi angles are found in succession?
A
The proteins secondary stucture- alpha helices and beta sheets.
14
Q
What does this define? ‘The arrangement of all atoms in the subunit, arrangement of the alpha-helices and beta sheets, side chains an any additional co factors.’
A
The tertiary structure.
15
Q
What is the definition of a proteins structure motifs?
A
Arrangement of a few helices and/or strands that occur often in different structures.
16
Q
Where is the helix-turn-helix often found?
A
DNA binding proteins.
17
Q
What is the definition of a domain?
A
Distinct sub division of a protein.
18
Q
What super-secondary structure is found in the TIM barrel?
A
Beta-alpha-beta unit.
19
Q
What are the two types of oligomers are found in quaternary structures?
A
Homo-oligomers and hetero-oligomers.
20
Q
What are dimers, timers and 24ers all examples of?
A
Homo-oligomers.
21
Q
What is ferritin an example of?
A
24mer.
22
Q
What is the definition of a hetero-oligomer?
A
Where copies of different chains assemble.
23
Q
What is the main technique used to see the structure of a protein?
A
X ray diffraction.
24
Q
Why can you see an electron density map of a proteins structure via x-ray crystallography?
A
The x-rays interact with the electrons.
25
Apart from X ray diffraction from protein crystals what are the other two methods to determine the structure of a protein?
NMR spectroscopy and high resolution cryo-electron microscopy.
26
What percentage of protein structure have been determined by x-ray crystallography?
90%.
27
What is being described here? Crystals of the highly purified target molecules are grown and exposed to X-rays to give diffraction patterns.
X ray crystallography.
28
What is the definition of resolution?
Level of detail that can be seen in a given map.
29
What does 1A equal in nm?
0.1
30
How much resolution do you need to see two bonded carbon atoms?
1.5A (the distance between two carbon atoms.)
31
How many A's are used in a low resolution map?
6.
32
How many A's are used in a medium density resolution map?
3.
33
What can you see in a low resolution map?
Sausages of helical density but no detail of side chains or atomic interactions.
34
What can you see in a medium density electron map?
Turns of a helix and side chains of blobs but not individual atoms.
35
How many A's can you see in a high resolution map?
1.6.
36
What can you see on a high resolution map?
Holes in aromatic rings and can almost see individual atoms.
37
What is the energy of interaction between atoms also known as?
Enthalpy.
38
Why are there few ionic interactions in a protein?
Because there are few charged amino acids.
39
What do ionic interactions depend on?
Distance.
40
Where are ionic interactions strongest?
In the centre.
41
Where are charged groups mainly found in the protein and why?
On the surface so they can interact with water.
42
What is the dielectric constant in the centre of a protein?
4.
43
What is the dielectric constant in water?
80.
44
Are ionic bonds or hydrogen bonds highly directional?
Hydrogen.
45
What do hydrogen bonds bind to on the exterior of a protein?
H20, ligands and the surface of other proteins.
46
What do hydrogen bonds depend on?
Distance and angle.
47
What do disulphide bonds do in hostile environments?
Add stability.
48
Why do you not find disulphide bonds in the interior of a protein?
There is reducing conditions.
49
What is the difference between delta H in a folded and unfolded protein?
0.
50
In a folded protein there are numerous VDW and H bonding interactions. What interactions are present in an unfolded protein?
The Hydrogen groups form hydrogen bonds with water and the rest of the chain makes VDW with water.
51
What is the key to protein folding?
Entropy.
52
What entropy change of protein folding is important?
The entropy of the water molecules.
53
Water molecules form cage like structures around unfolded proteins. What are these called?
Clathrates.
54
How are hydrophilic and hydrophobic residues arranged in a beta sheet?
They alternate.
55
How are the hydrophobic groups arranged in an alpha helix?
On the inside face.
56
For a novel protein is it quicker to predict the structure by using hydrophobic residues or by determining them experimentally?
Determining them experimentally.
57
What do groups of hydrophobic residues on the surface of a protein result in?
Sticky patches that can interact with hydrophobic ligands/ patches on other proteins.
58
How do basic amino acids interact with phosphate groups?
Ionic interactions.
59
What gives strands and helices specific shapes?
Hydrogen bonds.
60
What do enzymes allow?
Regulation.
61
How many reactions can each enzyme catalyse?
Generally only one.
62
How much do enzymes enhance the reaction rate by?
10^8 10^12
63
Are intermediates or transition states short lived?
Transition states.
64
What three things do enzymes do to molecules?
1. Bring substrates close together. 2. Allow them to be at optimal orientation. 3. Stabilize transition states and intermediates.
65
What do most enzyme reactions involve?
Hydrogen ion transfer.
66
What is the definition of acid-base catalysis?
When a proton is transferred going to or from the transition state.
67
What's the difference between a base and a nucleophile?
The bond formed when a nucleophile donates an electron is usually something other H.
68
What does RNase cleave?
Single stranded ribonucleic acids.
69
What type of enzyme is RNase?
A digestive enzyme.
70
Where is RNase produced?
The pancreas.
71
Where does RNase act?
The lower intestine.
72
RNase was worked on extensively in the 1980's because it was abundant. Where was it abundant?
Beef pancreas.
73
What is RNase S?
A clipped form of RNase.
74
Where in RNase do you clip to make RNase S?
Between residues 20 and 21.
75
Is RNAse active?
Yes.
76
Why was RNase S used in early mutagenesis experiments?
As the first 20 polypeptides could be replaced.
77
What type of base will RNase cut after?
Pyrimidines (U and C).
78
What bond does RNase cleave?
P-O5' bond.
79
What isotope of oxygen is the solution used for the RNase reaction enriched with?
O18.
80
What key catalytic intermediate is found in the RNase reaction which can unusually be isolated and characterised ?
2'-3' cyclic nucleotide.
81
The 2'-3' catalytic intermediate splits the RNase reaction into two steps. What are these steps?
1. The formation of the 2'-3' cyclic compound. 2. Cleavage.
82
The RNase reaction has two steps. What step is easier to study and revealed the pH dependance of the reaction?
The second step (the cleavage step).
83
What did the pH profile or Vmax show with the RNase reaction?
Two intersecting curves- this showed that two groups of a pka of roughly 7 were present. These groups were suspected to be histidine.
84
What is the principle behind chemical modification?
Reactive compounds chemically modify key groups in the enzyme.
85
One chemical modifier used to try and determine the structure of RNase was iodoacetate. What group did was this wrongly thought to chemically modify?
The SH groups or cysteine.
86
What chemical modifier was used to try and determine the structure of RNase?
Iodoacetate.
87
What method showed that cysteine was not an essential amino acid in RNase?
Cysteine.
88
What is special about the histidine residues in RNase?
They are unusually nucleophillic.
89
What are the key residues in the active site of RNase?
His12 and His119.
90
Why is only one His residue modified at a time in RNase?
As they are close together.
91
What do the two his residues act as in RNase?
One as an acid and one as a base.
92
The histidine residues in the active site of RNase are always hyper reactive. True or false?
False. They are only hyper reactive when the protein is folded.
93
Where was it determined that the modifying agent should lie in RNase?
Between the two histidine residues.
94
RNase was the forth protein to have its structure determined. When was it determined?
1967.
95
How was the structure of RNase determined?
X ray crystallography.
96
What does the polypeptide chain of RNase fold into?
A 3-stranded V-shaped anti-parallel beta sheet.
97
What is the polypeptide chain of RNase cross linked by?
4 disulphide bridges.
98
What shape is the active site in RNase?
A deep cleft.
99
Where is the catalytic residue His12 found in RNase?
The N terminal.
100
What is the role of Lys41 in the specificity pocket of RNase?
Stabilises the -ve phosphate group in the intermediate.
101
What two other basic residues asset with binding in RNase?
Lys and Arg.
102
What residue in the specificity pocket of RNase makes VDW contacts with the RNA base?
Phe 120.
103
What two residues in the RNase are involved in H bonding?
Ser123 and Thr45.
104
Why can the purines not bind to RNase?
The pocket is too small.
105
Different hydrogen bonding occurs with RNase and U and RNase and C. Why this is possible?
Ser and The can both be donors and acceptors.
106
Dinucleotides and longer molecules were cleaved by RNase when they entered the specificity site, meaning you can not visualise how the two interact. What useful non- cleavable analogue was used to allow visitation of this interaction?
UpCH2A.
107
What are the three active site residues in RNase?
Lys 41, His 12, His 119.
108
What are the three specificity pocket residues in RNase?
Phe 120, Thr 45, Ser 123.
109
What type of catalysis happens in the RNase enzyme?
General acid base.
110
What his residues acts as the acid in the first half of the RNase reaction?
His 119.
111
What residue acts as a base in the RNase reaction?
His 12.
112
How does the second half of the RNase reaction work?
The system is attack by water and the whole thing is reversed.
113
How many classes of RNase are there roughly?
100.
114
Most classes of RNase are related to RNase A. True or false?
False.
115
What is the function of RNase H?
Cleaves RNase in RNA/DNA duplexs.
116
What is the function of RNase L?
Destroys all RNA in the cell (innate anti-viral and apoptosis.)
117
What is function of RNase P?
Riboenzyme involved in processing tRNAs.
118
What is angiogein?
A homologue of ribonuclease A.
119
How similar is angiogein to ribonuclease A?
33%
120
What does angiogenin do?
Promotes development of blood vessels in healthy cells and in tumours.
121
How is it though that angiogenin works?
Translocated to the nucleus of endothelial cells where it cleaves tRNA and RNA molecules involved in signalling.
122
What is special about the binding inhibitor angiogenin?
It is one of the tightest binding inhibitors known.
123
What product is made after RNase cleavage?
5' OH and 3' P.
124
Where is the key oxygen on a deoxyribose ring?
3.
125
RNA has an extra hydroxyl group attached to the ribose ring. What position is this in?
2.
126
What carbon of the sugar ring is the phosphate group attached to?
5.
127
What distances are hydrogen bonds usually?
2.8A-3.2A.
128
Sometimes you can get hydrogen bonds shorter than the average hydrogen bond (you can never get them larger.) How short can these bonds be?
2.4A.
129
Due to repulsion hydrogen bonds are fairly linear. How much variation is there on this?
20 degrees or 30 CHECK
130
Are purines or pyrimidines larger?
Purines.
131
How many carbons are in the purine rings?
5/6.
132
How many carbons are in the pyramidines rings?
6.
133
What is different between U and T?
Thymine has an extra methyl group.
134
C, U and T are all pyrimidines with the same hydrogen bonding potential. True or false?
False, T/U have a different hydrogen potential to C.
135
By knowing the covalent structure of the bases we also know the 3D structure. True or false?
False.
136
The 3D structure of each nucleotide is determined by __ conformational angles.
7. The value of these three angles= the 3D structure.
137
What was the main source of evidence for Watson and Cricks DNA structure?
X-ray fibre diffraction.
138
What 5 methods were used to determine the structure of DNA?
1. Electron microscopy and light scattering. 2. Chargaff's rules... ratio of bases. 3. X-ray fibre diffraction. 4. Titration of DNA. 5. Model-building studies.
139
How big is the diameter of DNA?
20A. Most proteins are roughly 50A.
140
Why can you not see DNA without a microscope?
Visible lights wavelength is about 500A. You need 1.5A.
141
X rays are the ideal wavelength to visualise DNA however they can't be used. Why?
There is no suitable lens to view them with meaning the diffraction pattern will not be seen.
142
What are used as lenses in an electron microscope?
Magnets.
143
In the 1950's electron microscopes were not very good meaning that could not show the overall molecule shape. What could they show?
The overall molecule shape, which was a long thin molecule with a diameter of 20nm.
144
What other method showed that DNA was the same structure as electron microscopes had previously done?
Light scattering- some light hits the protein/ nucleic acid and is scattered.
145
What did Chargaff discover?
A=T and C=G.
146
G/C +A/T =?
1.
147
Although diffraction patterns from X-ray fibre diffraction can not be seen with lenses they can be seen with ______ \_\_\_\_\_.
Photographic film.
148
What is the X Ray passed though in X ray fibre diffraction ?
1000s of aligned DNA molecules pulled through a fibre.
149
What does X ray diffraction results show?
A regular structure.
150
X ray diffraction patterns showed repeating distances of 3.4A. Why was this relevent?
3.4A is the thickness of an aromatic ring. (Stack of pennies.)
151
In the 1950's Franklin and Wilson placed DNA fibres in controlled humidity chambers. What two humidities did they use?
Less than 75% and 92%.
152
What form of DNA is found in 92% humidity?
B.
153
Type A DNA is found in
A.
154
Did Franklin or Watson/ Crick concentrate on B form DNA (
Watson and Crick.
155
What pattern was showed in the B type DNA?
Double diamond with 2 fold symmtery- this showed a helix.
156
All forms of DNA are clockwise (right handed). True or false?
False. Z form is anti-clockwise.
157
Is A or B DNA shorter and fatter?
A.
158
How are the base pairs arranged in B strand DNA?
Perpendicular.
159
What angle are the base pairs at in A DNA?
20 degrees.
160
How many bp to As does Type A DNA have?
11bps in 28A.
161
How many base pairs to As does type B DNA have?
10bps in 34A.
162
How are the bases arranged in A and B DNA?
They are stacked.
163
What type of DNA isn't neatly stacked and is 'hollow'?
A.
164
What way does A DNA turn?
Towards.
165
What way does B DNA turn?
Away.
166
Does changing from type A to type B DNA require bond breaking?
No.
167
Is the A or B form of DNA rare?
B.
168
What can cause the DNA sequence to be slightly twisted?
The sequence of proteins.
169
Most DNA in the cell is free. True or false?
False.
170
What RNA was used to determine is structure?
RNA from reovirus.
171
What is the only type of RNA you get?
A.
172
Why can you not get B form RNA?
RNAs hydroxyl group can not fit into the B structure.
173
Why can't RNAs hydroxyl group fit in the B structure?
Steric clashes with the next base.
174
A DNA can form DNA/RNA complex. What is it hence involved in?
Transcription.
175
B DNA can not form a DNA/RNA complex as it can only pair with a B strand. What is B DNA hence involved in?
Replication.
176
How many types of DNA were used in fibre diffraction?
All of them.
177
What is single crystal diffraction also called?
Crystallography.
178
What two words can describe DNA crystallography?
Detailed and simple.
179
The downside of crystallography is that you can not mathematically interpret the data. True or false?
False. You can.
180
What is used instead of a lense in x-ray crystallography?
A computer.
181
Is there ambiguity in X ray crystallography?
No.
182
What was the problem with X ray crystallography in the 1970's?
It was not poogen
183
When was the 'Z form' of DNA found?
1979.
184
What sequence was found in Z DNA?
GCGCGC- repeated purines/ pyrimidines.
185
What shape is the backbone for Z DNA?
Zig Zag.
186
How long is Z DNA?
Very short.
187
How manu bp/ A is there in Z DNA?
12bp and 45A.
188
Where was Z DNA found in 1999?
Human editing enzyme.
189
What was found in 2003 with Z DNA?
DNA/RNA binding proteins, some found in tumour response and viral pathogenicity.
190
Although unclear, what may regions of Z DNA be involved in?
Unwinding/ supercoiling.
191
Has Z DNA with a top half of A or B been found?
B.
192
B DNA with a bottom of Z has been found. What is found at this junction?
AT base pair.
193
Who proposed tRNA?
Crick.
194
How many types of tRNA are there?
More than 20.
195
tRNAs are quite big and easy to purify. True or false?
True.
196
What was the tRNA base structure worked out in in 1965?
Yeast analine.
197
Why was it thought that tRNA might base pair with itself?
Complementary parts of the sequence.
198
tRNA is made out of 4 B DNA 'arms'. True or false?
False. It is made out of 4 B DNA arms.
199
What arm is found on the left side of tRNA?
D.
200
What arm is found on the right side of tRNA?
A.
201
Where do amino acids attach to in tRNA?
Tha aa arm.
202
Where do anticodons attach to in tRNA?
The ac arm.
203
What percentage of bp in tRNA pair in a Watson/Crick manner?
55%.
204
How does the ribosome bind to the tRNA?
Through the T loop in the top left corner (third corner.)
205
Unusual base triplets are found in tRNA, what do this add?
Extra stability.
206
95% if the aromatic bases in tRNA are stacked on top of each other?
That base stacking is a major force of stability in tRNA.
207
What causes the bases to stack in A/B DNA and in tRNA?
The edges of the bases are rich in N and O, allowing hydrogen bonds to form. This means they want to form hydrogen bonds with water/ each other.
208
What part of bases is hydrophobic and what does this cause?
IThe face/ the aromatic rings. These then stack on top of each other to avoid water, resulting in stacking.
209
In X ray fibre diffraction what does big spacing on the pattern correlate to?
Small spacing in the molecule.
210
What group in DNA can always be titrated?
Phosphates.
211
What pH do the phosphates in DNA titrate at?
2.
212
What pH do the bases titrate at?
It varies depending on the base.
213
You can titrate the bases in DNA easily?
False. They can only be titrated in extreme circumstances as they are not exposed.
214
Watson and Crick used model building studies to determine the structure of DNA. What did they use to make these?
Known information on stereochemistry of phosphates, sugars and the bases.
215
What were the four key features of Watson and Crick's model of DNA?
1. Helical stacking. 2. Base stacking. 3. Two chains. 4.Regular sugar phosphate backbone.
216
What evidence did Watson and Crick use to support helical stacking?
Diffraction pattern.
217
What evidence did Watson and Crick use to support base stacking?
3.4A repeats in diffraction pattern.
218
What evidence did Watson and Crick use to support two chains?
Density measurements and x ray patterns.
219
What evidence did Watson and Crick use to support the regular- sugar phosphate backbone?
The backbone was shown to be the same in DNA from different species. It also did not depend on base composition.
220
Why did Watson and Crick think the bases were hydrogen bonded to each other?
As titration experiments showed they were buried from water.
221
Watson and Crick determined that DNA contained hydrogen bonds through titration experiments. How did they work out that they were between the two chains?
As hydrogen bonds are linear and meaning they can not be present between the same chain.
222
Watson and Crick determined that the hydrogen bonds in DNA had to be between two different chains. What other restriction was there?
The x ray diffraction patterns had shown the the base pair composition could not be affected by the base pairs.
223
Why did Watson and Crick conclude that the base pairs had to be Pu:PY?
As Purines are larger than pyrimidines meaning that if the bases were paired as Pu:Pu and Py:Py the distances would not be the same (Pu:Pu larger) and due to the random incorporation of the bases the backbone would not be universal.
224
AT and GC have almost identical geometry provided the chains run in opposite directions. True or False?
False. It is identical.
225
What did thermal denaturation of DNA provide information on and why?
The base pairs due to the fact that GC has 3 hydrogen bonds which correlates to a higher melting point.
226
Why was the determination of different size DNA grooves important?
As this provided vital information later on to determine protein/drug DNA interaction.
227
How long is a turn in DNA?
34A.
228
How long is a repeat unit in DNA?
3.4A.
229
What is the role of the base pairing in DNA?
Carries genetic information.
230
What is the role of the sugar-phosphate backbone in DNA?
Stability.
231
What is the lysozyme enzyme involved in?
The first line of defence against bacterial attack.
232
Where are lysozyme found in humans?
Bodily secretions.
233
Lysozyme are small enzymes. How many amino acids are they made up of?
129.
234
How many SS bridges are there in lysozyme?
4.
235
Bacteriophages have lysozymes related to human lysozymes in both chemistry and structure . True or false?
False, their chemistry is the same but the structure is different.
236
Lysosomes are part of what huge group of enzymes?
Glycosidase enzymes.
237
What is the main role of glycosides enzymes?
They hydrolyse sugars.
238
What type of bacteria have a thick peptidoglycan cell wall?
Gram positive.
239
What two alternating sugars are peptidoglycan molecules made up of?
NAM and NAG.
240
From what sugar does peptidoglycan cross link?
NAM.
241
Where does the lyzosome cleave the peptidoglycan?
Between NAM and NAG where they aren't too many cross links.
242
What two unusual amino acids are found in the peptidoglycan cross link?
D-ala and D-glu.
243
What does the side chain of D-Glu bind to with an unusual link in peptidoglycan?
Main chain of lys.
244
What does the side chain of lys bind to with an unusual linkage in peptidoglycan?
Main chain of Gly.
245
What enzymes synthesis peptidoglycan cross links and are also targets of penicillin and related antibiotics?
D-Ala-D-Ala transpeptidase.
246
What linkage does the lysozyme cleave?
NAM B(1-4) NAG linkage in peptidoglycan.
247
When the NAM NAG linkage is cleaved where does the O molecule go?
To NAG. H20 then turns this and the exposed group on NAM into two H20 molecules.
248
How long is a typical peptidoglycan sugar chain?
25-35 disaccharide units.
249
How many domains do lysozymes have?
2 separated by a deep cleft.
250
What does the left domain of the lysozyme contain?
A small Beta sheets made mainly of hydrophobic residues.
251
What does the right domain of the lysozyme contain?
A hydrophobic core surrounded by a short alpha helices.
252
What substrate was observed binding to the top half of the cleft/active site of the lysozyme?
triNAG.
253
What did the rate of hydrolysis of NAG show?
That the active site will bind 6 sugars.
254
What sites did triNAG bind to?
ABC.
255
Why could NAM only be found in sites B, D and F?
Because it is bigger.
256
What is hexaNAG cleaved into?
(NAG)4 and (NAG)2.
257
What pattern of interactions was found in the lysozyme active site?
H bonds to O and N on the edges of the sugar rings and hydrophobic/ VDW interactions on the faces of the sugars.
258
What sites is the NAM/NAG cleavage site found between?
D and E.
259
What are the only two amino acids in the D/E region of the enzyme that can accept/donate protons meaning they are the catalytic groups?
Asp52 and Glu35.
260
Glu35 and Asp52 both have carboxylic acid functional groups, however at pH 6 they are not both found as carboxylate ions. Why?
Glu is in a hydrophobic microenvironment which makes it protonated (uncharged).
261
What method showed that Glu35 is found in its protonated state in the lysozyme?
Neutron diffraction.
262
What intermediate was proposed in the now wrong Phillips mechanism?
Carbonium intermediate.
263
What was the role of Asp 52 in Phillips mechanism?
Stabilises the distorted carbonic ion intermediate in the D site.
264
What was the main reason that the Phillips mechanism was disregarded?
Carbonium ion is high energy and isn't a likely intermediate.
265
What bond was involved in the second proposed mechanism of the lysozyme?
enzyme-glycosol covalent bond.
266
What acts as a nucleophile in the second proposed mechanism for the lysozyme?
Asp52.
267
Nucleophilic attack by _____ forms covalent ___________ intermediate. ____ (\_\_\_\_\_) donates a proton and E-F diffuses away, making the first product. Attack by \_\_\_\_\_\_. OH added to C1 of D and a proton to \_\_\_\_\_. A-B-C-D is the second product.
Asp52, glycosyl-enzyme, Glu35 buried, water, Glu35.
268
What type of evolution are lysozymes thought to have followed and why?
Convergent evolution as they have no sequence similarity but all their mechanisms use two carbonyl groups.
269
Name 5 types of glycosidases.
1. Lysozymes. 2. Lactases. 3. Amylases. 4. Cellulases. 5. Neuraminidases.
270
What do cellulases do?
Break down cellulose to glucose.
271
What is the purpose of neuramindases?
Used by viruses and bacteria to penetrate cell walls in a process called pathogenesis.
272
Glu 35 is protonated in the lysozyme. What can this also be known as?
Glutamic acid.
273
What are the three steps of the lysosome mechanisms?
1. Nucleophilic attack by ASP52 to create a covalent glycosyl-enzyme intermediate. 2. Glu35 donates a proton. 3. Water attacks.
274
What are 6 examples of DNA BP?
1. Proteins that regulate gene expression. 2. DNA packaging proteins. 3. Restriction enzymes. 4. Polymerases. 5. Repair proteins. 6. Unwinding proteins.
275
Why are DNA binding proteins often positive?
As the DNA backbone is negative due to the phosphates.
276
What fits easily into the major groove?
The edges of bases (specific sequences can be detected easily).
277
Can the edges of bases be seen by the minor groove?
Yes.
278
DNA is always found in is low energy A and B forms. True or false?
False. It can bend away from the structure and some sequences are more likely to do this then others.
279
When were the first DNABP established?
1980's.
280
Do DNABP react with the phosphate group specifically?
No.
281
What two bases are involved in specific binding with adenine in the major groove?
Asn and Gln.
282
What other reactions are involved with DNABP?
Non polar interactions, hydrophobic interactions, non hydrogen bonding contacts.
283
After hydrogen bonds between bases have been taken into consideration how many other options of Hydrogen bonds can be made in the major groove?
3.
284
After hydrogen bonds between bases have been taken into consideration how many other options of Hydrogen bonds can be made in the minor groove?
2.
285
What is the pattern of donors and acceptors between A and T in the major groove?
ADA.
286
What is the pattern of donors and acceptors between A and T in the minor groove?
AA.
287
What is the pattern of donors and acceptors between G and C in the major groove?
AAD.
288
What is the pattern of donors and acceptors between G and C in the minor groove?
DA.
289
What base has a methyl group that proteins/ residues?
Thymine.
290
What fits neatly in the major groove into the B DNA?
Alpha helix
291
What amino acid is in the turn in the helix turn?
Glycine as it fits well into the B groove of DNA.
292
What is the zinc finger model made up off?
2 beta strands and 1 alpha helix.
293
How are the zinc finger motifs usually arranged?
In strings.
294
Why are zinc fingers not normally found on their own?
They don't give much specificity.
295
The tilt of the helix relative the the groove of DNA doesn't change. True or false?
False.
296
What amino acids are present in the zinc finger model?
2 cysteines on the beta strand and 2 histidines.
297
When is the basic leucine zipper motif used?
In non DNAbp polymerisation.
298
How are the helices arranged in the leucine zipper motif?
At the same height.
299
The basic leucine zipper motif is one continuous helix. True or false?
False, it can be one continuous helix but it can also double back on itself in a loop.
300
How are B ribbons formed?
By two beta strands.
301
Where does the beta ribbon fit into and what does it do?
It fits into the major groove of DNA and the side chains of the strand interact with the edges of the bases.
302
What type of contacts can be made by a b strand and the minor groove of DNA in packaging?
Non specific contacts.
303
How may degrees does DNA rotate onto itself?
180.
304
Where is the symmetry in DNA?
Through and between every base pair (approximately, ignore the base pairs).
305
What is the only DNA sequence that has true 2 fold?
Palindromic.
306
Where are palindromic sequences often found?
Binding sites.
307
What do most prokaryotic repressors and activators recognise?
Palindromic DNA sequences.
308
When is the basic leucine zipper used?
Non DNABP in a polymerisation/ dimerisation fashion.
309
The basic leucine zipper motif is always a continuous helix. True or false?
False. It can be double back on itself in a loop.
310
When were ribozymes discovered?
1983.
311
What are ribozymes made from?
RNA.
312
Where was the first example of a ribozyme found?
In the protozoan Tetrahymena.
313
What part of the mRNA makes up the ribozyme?
Intron.
314
What are the three main points regarding the ribozymes structure?
1. The majority of base pairs form WC pairs in A-form double helices. 2. Almost all bases are stacked, even if they aren't paired. 3. Base triplets and metal ions can link bits o the structure together.
315
What forms the 3D structure in the ribozymes?
H bonds.
316
What drives the folding of the ribozymes?
Base stacking of hydrophobic surfaces.
317
What is the probable ancestor of the spliceosome?
Self splicing intron.
318
What are the catalytic triads in the group 2 ribozymes?
CGC or AGC.
319
What do the catalytic triads (CGC and AGC) bind to in the group 2 ribozymes?
2 Mg2+ ions.
320
What two things do RNA enzymes need to do/have?
1. They need to specifically bind substrates. 2. Have groups with unusual activity that can catalyse reactions.
321
How do RNA enzymes bind their substrates?
They have unpaired bases which can bind complementary sequences in DNA and RNA.
322
Why do ribozymes have much less scope for reactivity than proteins do?
They only have four bases.
323
Ribozymes are only made up of 4 bases so they are less chemically reactive than protein enzymes. What two methods can increase their reactivity?
1. Bound metal ions can give unusual reactivity. 2. Nucleobases in unusual 3D environments can give unusual chemical properties.
324
What are ribozymes thought to be the remainder of?
RNA world.
325
Why is it thought that most cofactors have a nucleotide in their structure?
They may have originally been cofactors to ribozymes.
326
What is the definition of a riboswitch?
mRNA segments that can fold up and bind small target molecules and affect the transcription of the mRNA.
327
What do riboswitches demonstrate?
That RNA can regulate gene synthesis.
328
What do riboswitches show in regards to RNA?
That they can bind small molecules.
329
When could riboswithes be descended from?
RNA world (pre protein regulatory machinery.
330
What can riboswitches be?
Potential antibiotic targets.
331
Riboswitches are mainly found in bacteria but they have recently been found in some eukaryotes. What are these?
Plants and fungi.
332
How many Svedborg units is the prokaryotic ribosome made out of?
70s. 50s large 30s small
333
What is the overall size of the prokaryotic ribosome in megadaltons?
2.7 megadaltons.
334
What is the overall size of the small prokaryotic ribosome subunit in megadaltons?
1.0 mega daltons.
335
What is the overall size of the large prokaryotic ribosome subunit in megadaltons
1.7 mega daltons.
336
Both subunits in the prokaryotic ribosome are made up of two pieces of RNA. True or false?
False. The small subunit only has one.
337
How many proteins are in the large subunit of the prokaryotic ribosome?
34.
338
How many proteins are in the small subunit of the prokaryotic ribosome?
21.
339
What main method was used to determine the structure of the ribosome?
Neutron scattering.
340
In the 1980's Ada Yonath tried to crystallise ribosomes from many bacterial species, including T thnermophlillus. What were her results?
She obtained poor quality crystals which resulted in weak diffraction patterns (spots were too close to be distinguised between.)
341
In the 1990s technological advances helped with the determination of the structure of the ribosome. What are 5 examples of these improvements?
1. Synchrotron radiation ( 1000x stronger than conventional sources.) 2. Improved detector technology ( increases diffraction intensities) 3. Cryocooling (prevents radiation damage). 4. Improvements in computer power ( Improved date processing.) 5. EM structures present.
342
What happened in 1999 in regards to the ribosomes structure?
Low resolution gave the overview of the structure. 6A gave the structure of both subunits. The structure of the complete ribosome and the tRNAS were done at 8A.
343
At a low resolution what was determined about the structure of the ribosome?
The overall shape of the protein/ RNA maintain but no detail of side chains or bases.
344
When did the detailed structure of the small and large ribosomal subunits become available?
August 2000.
345
Were all components of the DNA and the proteins determined in the August 2000 model of the ribosome?
Yes.
346
What is the purpose of the large ribosomal subunit?
Catalyses peptide bond formation.
347
How many domains are in the larger piece of RNA in the large subunit?
6
348
What is thought to form the core of the ribosome?
Complex 3D jigsaws of RNA.
349
A few proteins are found close to the active site of the ribosome. True or false?
False, no proteins are close to the active site. the N and C tails of some proteins however are.
350
What is the core of the large subunit made from?
Tightly packed mass of RNA helices.
351
What stabilises interactions between RNA domains?
Ribosomal proteins on the outside of the domains.
352
What is the active site for protein synthesis in the ribosome made form?
100% RNA.
353
What can the ribosome also be called?
A ribozyme.
354
How was the active site of the ribosome located?
Soaking crystals with known inhibitors.
355
The proposed catalytic mechanism for the active site of the ribosome is complex. What does it involve?
A crucial adenine in an unusual 3D environment very close to a phosphate group. This unusual environment results in abnormal protonation.
356
What is the peptide exit tunnel is bounded by?
RNA domains.
357
What is the small ribosomal subunit responsible for?
Decoding genetic information during translation.
358
What does the small subunit bind?
mRNA and tRNA (to read the mRNA).
359
How many proteins/ nucleotides is the small subunit made from?
20/ 1518.
360
All 20 proteins in the small subunit have been identified. What proportion of the nucleotides have been identified?
96%.
361
What is the decoding centre in the small subunit made from?
100% RNA.
362
What are the three key parts of the ribosome?
1. Decoding centre 2. Peptide tunnel 3. Peptidyl active site.
363
The eukaryotic ribosome is larger than the prokaryotic ribosome. What is its size?
4200 KDA.
364
Is translation more complex in eukaryotes?
Yes.
365
The ribosomes of both prokaryotes and eukaryotes are very closely related. True or false?
False. They are only closely related in the active site.
366
Several classes of antibiotics are directed against the bacterial ribosome. What two antibiotics are directed at the small subunit?
Tetracycline. Streptomycin.
367
Several classes of antibiotics are directed against the bacterial ribosome. What two antibiotics are directed to the large subunit?
Chloramphenicol. Erythromycin.
368
Do AB bind to the RNA or the protein in the ribosome?
RNA.
369
What is tetracycline used for?
Respiratory tract infections.
370
What is chlorampheniocal used for?
Bacterial meningitis.
371
What do palindromic base sequences allow?
2-fold protein to interact symmetrically with bases as well as the backbone.
372
What are the three main DNA binding proteins in bacteria?
1. CAP. 2. CRO. 3. Lambda repressor.
373
The three main DNABP in bacteria are CAP, CRO and the lambda repressor. These all have very different structures but are similar in two ways. What are these ways?
1. All dimers. 2. All have a helix-turn-helix motif.
374
What is the purpose of CAMP in the CAP/DNA complex?
It switches on DNA binding.
375
In the CAP/DNA complex the recognition helixes bind to the major grooves in the DNA. What happens to the DNA molecule?
It bends around the cap complex to maximise interactions.
376
When is the trp repressor active?
When trp can act as the co repressor.
377
At low trp concentrations the repressor is not active. How much mRNA is hence transcribed to make trp?
5 pieces corresponding to 5 enzymes.
378
When trp is bound to the trp repressor how far apart are the helices?
34A. (Can bind)
379
When trp is not bound to the repressor how far apart are the helices?
26A. (Can not bind).
380
Endonuclease EcoR1 and DNAP1 (Klenow fragment) both cleave DNA. Which one is a dimer?
EcoR1.
381
Endonuclease EcoR1 and DNAP1 (Klenow fragment) both cleave DNA. Which one is a monomer?
DNAP1 klenow fragment.
382
Endonuclease EcoR1 and DNAP1 (Klenow fragment) both cleave DNA. Which one only cleaves palindromic sequences?
EcoR1.
383
Endonuclease EcoR1 and DNAP1 (Klenow fragment) both cleave DNA. Which one cleaves all sequences?
DNAP1 klenow fragment.
384
Endonuclease EcoR1 and DNAP1 (Klenow fragment) both cleave DNA. Which one is described to 'embrace DNA'?
EcoR1.
385
Endonuclease EcoR1 and DNAP1 (Klenow fragment) both cleave DNA. Which one is described as a ' large circular cleft that wraps around the DNA like a hand.'
DNAP1 klenow fragment.
386
Structure of the p53 core domain complexed with DNA revealed critical contacts. What were these (3)?
1. Zn2+ ion stabilising binding loops (p53) 2. Strand loop helix motif of P53 fits into B DNA major groove. 3. Arg248 contacts backbone in minor groove.
387
How often is the Arg 248 residue of P53 mutated in cancer patients?
10% of cancer causing mutations.
388
What do the 6 most common P53 mutations normally affect?
Non specific DNA binding.
389
What are the 6 most common P53 mutations resulting in 40% of the p53 derived tumours?
1. 2 arginines- sugar phosphate backbone. 2. 3 arginines- H bond within the protein structure and stabilise protein conformation. 3. Gly245 stabilises structure of the loop in the minor groove.
390
60% of P53 tumours have mutations of residues close to the protein-DNA interface. What two things does this cause?
1. Disruption of sequence specific interactions. 2. Proteins no longer recognise correct DNA target sequence.
391
What shape is p53?
A tetramer.
392
Some anticancer drugs work by blocking DNA replication. How?
Disrupt DNA-protein interactions and prevent transcription.
393
What three types of cancer drug are there?
1. Intercalating. 2. Major groove binding. 3. Minor groove binding.
394
What type of drug is actinomycin?
Intercalating.
395
What type of drug is cisplatin?
Major groove binding.
396
What type of drug is neutrospin?
Minor groove biding.
397
