Module 9 Flashcards
1
Q
- are the monomeric units that make up the nucleic acids DNA (Deoxyribonucleic acid) and RNA (Ribonucleic acid)
A
Nucleotides
2
Q
- are responsible for the storage and passage of the information needed for the production of proteins
A
Nucleic acids
3
Q
Each nucleotide consists of __
A
a pentose sugar, a nitrogenous base, and a phosphate group
4
Q
Result from linking one or more phosphates with a nucleoside onto the 5’ end of the molecule through esterification
A
Nucleotides
5
Q
A __ is a 5-carbon sugar in a pentose ring form
A
pentose sugar
6
Q
__ contains ribose, which has a hydroxyl group in both the 2’ and 3’ positions (prime refers to the carbon of the sugar)
A
RNA
7
Q
__ has only a single hydroxyl group in the 3’ position
A
DNA
8
Q
A __ is attached by a glycosidic bond to the 1’ carbon of the nucleotide’s sugar
A
nitrogenous base
9
Q
__ consist of linked 5-membered and 6-membered rings (Adenine and Guanine, A and G) which can be found in DNA or RNA
A
Purines
10
Q
__ consist of 6-membered rings Cytosine, Thymine, and Uracil (C, T, and U)
A
Pyrimidines
11
Q
(pyrimidines)
__ is found in DNA or RNA, T is found in DNA, while U is found in RNA
A
Cytosine
12
Q
- Planar, aromatic, and heterocyclic
- Derived from purine or pyrimidine
- Numbering of bases is “unprimed”
A
Nitrogenous Bases
13
Q
The sugar derivatives
__ participate in sugar interconversions and in the biosynthesis of starch and glycogen
A
UDP-glucose and UDP-galactose
14
Q
nucleoside-lipid derivatives such as __ are intermediates in lipid biosynthesis.
A
CDP-acylglycerol
15
Q
The __ serve
as the second messengers in hormonally regulated events, and GTP and GDP play key roles in the cascade of events that characterize signal transduction pathways.
A
cyclic nucleotides cAMP and cGMP
16
Q
__ are nitrogen-containing heterocycles, cyclic structures that contain, in addition to carbon, other (hetero) atoms such as nitrogen.
A
Purines and pyrimidines
17
Q
Pyrimidine vs Purine
A
Note that the SMALLER
PYRIMIDINE MOLECULE has the longer name and the LARGER PURINE MOLECULE the shorter name, and that their six-atom rings are numbered in opposite directions.
18
Q
__ are weak bases (pKa values 3-4), although the proton present at low pH is associated, not as one might expect with the exocyclic amino group, but with a ring nitrogen, typically N1 of adenine, N7 of guanine, and N3 of cytosine.
A
Purines or pyrimidines with an ´NH2 group
19
Q
The sugar in ribonucleosides is D-ribose, and in deoxyribonucleosides is
2-deoxy-D-ribose. Both sugars are linked to the heterocycle by a __, almost always to the N-1 of a pyrimidine
or to N-9 of a purine
A
B-N-glycosidic bond
20
Q
__ are nucleosides with a phosphoryl group esterified to a hydroxyl group of the sugar.
A
Mononucleotides
21
Q
Additional phosphoryl groups, ligated by __ to the phosphoryl group of a mononucleotide, form nucleoside diphosphates and triphosphates.
A
acid anhydride bonds
22
Q
Steric hindrance by the heterocycle dictates that there is no freedom
of rotation about the β-N-glycosidic bond of nucleosides or nucleotides. Both therefore exist as noninterconvertible __. While both syn and anti
conformers occur in nature, the anti conformers predominate.
A
syn or anti conformers
23
Q
The conjugated double bonds of __ absorb ultraviolet light.
A
purine and pyrimidine derivatives
24
Q
__ serves as an allosteric regulator and as an
energy source for protein synthesis.
A
GTP
25
__ serves as a second messenger in response to nitric oxide (NO)
during relaxation of smooth muscle
cGMP
26
__ forms the urinary glucuronide conjugates of bilirubin and of many drugs, including aspirin.
UDP-glucuronic acid
27
__ participates
| in biosynthesis of phosphoglycerides, sphingomyelin, and other substituted sphingosines
CTP
28
__ have two acid anhydride bonds and one ester bond.
Nucleotide triphosphates
29
The purine analog __, used in treatment of hyperuricemia and gout, inhibits purine biosynthesis and xanthine oxidase activity.
allopurinol
30
__ is used in chemotherapy of cancer
Cytarabine
31
__ which is catabolized to 6-mercaptopurine, is employed during organ transplantation to suppress immunologic
rejection
azathioprine
32
The 5′-phosphoryl group of a mononucleotide can esterify a second hydroxyl group, forming a __. Most commonly, this second hydroxyl group is the 3′-OH of the pentose of a second nucleotide.
phosphodiester
33
This forms a __ in which the pentose moieties are linked by a 3′,5′-phosphodiester bond to form the “backbone” of RNA and DNA.
dinucleotide
34
Posttranslational modification of preformed polynucleotides can generate additional structures such as __, a nucleoside in which D-ribose is linked to C-5 of uracil by a carbon-to-carbon bond rather than by the usual β-Nglycosidic
bond.
pseudouridine
35
- contains a single phosphate group, which is a strong acid
- can be attached through the oxygen of a hydroxyl at either the 5’ or 3’ position of the sugar
- It is more commonly attached to the 5’ position.
phosphate
36
A __ is the term for a sugar and a base
nucleoside
37
From __ can be attached to nucleosides to from nucleoside mono, di, or triphosphates
1 to 3 phosphates
38
A __ can also be called a nucleotide
nucleoside monophosphate
39
(Naming Conventions)
| Nucleosides
Purine nucleosides end in “-sine”
- Adenosine, Guanosine
Pyrimidine nucleosides end in “-dine”
- Thymidine, Cytidine, Uridine
40
(Naming Conventions)
| Nucleotides
Start with the nucleoside name from above and add “mono-”, “di-”, or “triphosphate”
- Adenosine Monophosphate, Cytidine Triphosphate, Deoxythymidine Diphosphate
41
__ are linked together by phosphodiester bonds between the 3’ hydroxyl on the sugar of one nucleotide through a phosphate molecule to the 5’ hydroxyl on the sugar of another nucleotide
Nucleotides
42
Nucleotide bonds are broken by __
phophodiesterases
43
Adenine nucleotides are components of 3 major coenzymes:
NAD+, FAD, COENZYME A
44
__ are activated precursors in nucleic acid synthesis.
Nucleoside triphosphates
45
(PURINE NUCLEOTIDE SYNTHESIS)
| First purine derivative formed is __
Inosine Mono-phosphate (IMP)
46
SOURCES OF ATOMS IN PURINE BIOSYNTHESIS
```
N1 - Aspartate
C2, C8 - 10-Formyl-THF
N3, N9 - Glutamine
C6 - CO2
C4, C5, N7 - Glycine
```
47
The activated sugar used is __
- is generated by the action of PRPP synthetase and requires energy in the form of ATP
- This reaction releases AMP. Therefore, 2 high energy phosphate equivalents are consumed during the reaction.
- First committed step of purine synthesis (Regulated)
5-phosphoribosyl-1-pyrophosphate, PRPP
48
5-phosphoribosyl-1-pyrophosphate, PRPP is inhibited by __
ADP and GDP
49
The major site of purine synthesis is in the __.
liver
50
Synthesis of the purine nucleotides begins with PRPP and leads to the first fully formed nucleotide, __.
inosine 5'-monophosphate (IMP)
51
The synthesis of IMP requires __
five moles of ATP, two moles of glutamine, one mole of glycine, one mole of CO2, one mole of aspartate and two moles of formate
52
The formyl moieties are carried on tetrahydrofolate (THF) in the form of __.
N5,N10-methenyl-THF and N10-formyl-THF
53
(SYNTHESIS OF AMP OR GMP)
__ represents a branch point for purine biosynthesis, because it can be converted into either AMP or GMP through two distinct reaction pathways.
IMP
54
The pathway leading to AMP requires energy in the form of GTP; that leading to GMP requires energy in the form of __
ATP
55
The accumulation of excess __ will lead to accelerated AMP synthesis from IMP instead, at the expense of GMP synthesis.
GTP
56
the conversion of IMP to GMP requires ATP, the accumulation of excess __ leads to accelerated synthesis of GMP over that of AMP.
ATP
57
The synthesis of PRPP by PRPP synthetase is feed-back inhibited by __ (predominantly AMP and GMP).
purine-5'-nucleotides
58
The amidotransferase reaction (2nd step) catalyzed by __ is also feed-back inhibited allosterically by binding ATP, ADP and AMP at one inhibitory site and GTP, GDP and GMP at another.
PRPP glutamyl amidotransferase
59
__ is regulated in the branch pathways from IMP to AMP and GMP. The accumulation of excess AMP leads to accelerated synthesis of GMP, and excess GMP leads to accelerated synthesis of AMP.
purine biosynthesis
60
Catabolism of the purine nucleotides leads ultimately to the production of __ which is insoluble and is excreted in the urine as sodium urate crystals.
uric acid
61
The synthesis of nucleotides from the purine bases and purine nucleosides takes place in a series of steps known as the __.
salvage pathways
62
The free purine bases, adenine, guanine, and hypoxanthine, can be reconverted to their corresponding nucleotides by __.
phosphoribosylation
63
Two key transferase enzymes are involved in the salvage of purines:
1. adenosine phosphoribosyltransferase (APRT)
| 2. hypoxanthine-guanine phosphoribosyltransferase (HGPRT)
64
A critically important enzyme of purine salvage in rapidly dividing cells is __ which catalyzes the deamination of adenosine to inosine.
adenosine deaminase (ADA)
65
Deficiency in adenosine deaminase (ADA) results in the disorder called __
severe combined immunodeficiency, SCID
66
__ can also contribute to the salvage of the bases through a reversal of the catabolism pathways. However, this pathway is less significant than those catalyzed by the phosphoribosyltransferases.
Purine nucleotide phosphorylases (PNPs)
67
- separate kinase for each nucleotide
Nucleoside Monophosphate kinases
68
- single enzyme with broad specificity
Nucleoside Diphosphate kinases
69
One genetic disorder of pyrimidine catabolism,
| __, is due to total or partial deficiency of the enzyme dihydropyrimidine dehydrogenase.
β-hydroxybutyric aciduria
70
This disorder
of pyrimidine catabolism, also known as __, is also a disorder of β-amino acid biosynthesis, since the formation of β-alanine and of β-aminoisobutyrate is
impaired.
combined uraciluria-
| thyminuria
71
The three processes that contribute to purine nucleotide biosynthesis are, in order of decreasing importance.
1. Synthesis from amphibolic intermediates (synthesis de novo).
2. Phosphoribosylation of purines.
3. Phosphorylation of purine nucleosides.
72
__ has a low level of PRPP glutamyl amidotransferase, and hence depends in
part on exogenous purines.
Human brain tissue
73
__ cannot synthesize 5-phosphoribosylamine
and, therefore, also utilize exogenous
purines to form nucleotides.
Erythrocytes and polymorphonuclear
| leukocytes
74
The rate of PRPP
synthesis depends on the availability of ribose 5-phosphate and on the activity of __, an enzyme whose activity is feedback inhibited
by AMP, ADP, GMP, and GDP
PRPP synthase
75
__ also inhibit hypoxanthine-guanine phosphoribosyltransferase,
which converts hypoxanthine and guanine to IMP and GMP, and GMP feedback inhibits PRPP glutamyl amidotransferase
AMP and GMP
76
Reduction of the 2′-hydroxyl of purine and pyrimidine ribonucleotides,
catalyzed by the complex that includes __, provides the deoxyribonucleoside diphosphates (dNDPs) needed for both the synthesis and repair of DNA
ribonucleotide
| reductase
77
Five of the first six enzyme activities of pyrimidine biosynthesis
reside on __.
multifunctional polypeptides
78
For further pyrimidine synthesis to occur, dihydrofolate must be reduced
back to tetrahydrofolate. This reduction, catalyzed by dihydrofolate
reductase, is inhibited by __
methotrexate
79
- are alternate substrates for orotate phosphoribosyltransferase.
Both drugs are phosphoribosylated, and allopurinol is converted
to a nucleotide in which the ribosyl phosphate is
attached to N1 of the pyrimidine ring.
Allopurinol and 5-fluorouracil
80
Humans convert adenosine and guanosine to __. Adenosine is first converted to inosine by adenosine
deaminase.
uric acid
81
In mammals other than higher primates, __,converts uric acid to the watersoluble
product allantoin. However, since humans lack __, the end product of purine catabolism in humans is uric acid.
uricase
82
__ is a condition that results from the precipitation of urate (uric acid) as monosodium urate (MSU) or calcium pyrophosphate dihydrate (CPPD) crystals in the synovial fluid of the joints, leading to severe inflammation and arthritis.
Gout
83
Most forms of gout are the result of excess purine production or to a partial deficiency in the salvage enzyme, __
Hypoxanthine-guanine phosphorybosyl transferase (HGPRT)
84
increased activity of PRPP synthetase leads to excess PRPP leading to increased purine nucleotide production that can increase the rate of purine degradation and subsequently increase __
uric acid synthesis
85
Responsible for re-forming IMP and GMP from hypoxanthine and guanine
Hypoxanthine-guanine phosphorybosyl transferase (HGPRT) and the Salvage Pathway
86
Most forms of gout can be treated by administering the antimetabolite __ .
This compound is a structural analog of hypoxanthine that strongly inhibits xanthine oxidase.
allopurinol
87
- results from the loss of a functional HGPRT gene.
- is inherited as a sex-linked trait, with the HGPRT gene on the X chromosome (Xq26-q27.2).
- Patients with this defect exhibit not only severe symptoms of gout but also a severe malfunction of the nervous system.
- In the most serious cases, patients resort to self-mutilation.
Lesch-Nyhan syndrome
88
- is most often (90%) caused by a deficiency in the enzyme adenosine deaminase (ADA).
- This is the enzyme responsible for converting adenosine to inosine in the catabolism of the purines
SCID (Severe Combined Immunodeficiency Disease)
89
- selectively leads to a destruction of B and T lymphocytes, the cells that mount immune responses.
- deoxyadenosine is phosphorylated to yield levels of dATP that are 50-fold higher than normal
ADA deficiency
90
A less severe immunodeficiency results when there is a lack of __, another purine-degradative enzyme.
purine nucleoside phosphorylase (PNP)
91
- One of the many glycogen storage diseases __ also leads to excessive uric acid production.
- results from a deficiency in glucose 6-phosphatase activity.
- The increased availability of glucose-6-phosphate increases the rate of flux through the pentose phosphate pathway, yielding an elevation in the level of ribose-5-phosphate and consequently PRPP
- The increases in PRPP then result in excess purine biosynthesis.
von Gierke disease
92
3 different enzyme defects can lead to gout:
- PRPP synthetas elevated
- HGPRT deficiency
- glucose-6-phosphatase deficiency
93
APRT lacking
Renal lithiasis
94
PNP lacking
Immunodeficiency
95
Xanthine oxidase absent
Xanthinuria
96
(Pyrimidine Nucleotide Biosynthesis)
The first completed base is derived from __
1 mole of glutamine, one mole of ATP and one mole of CO2 (which form carbamoyl phosphate) and one mole of aspartate.
97
The carbamoyl phosphate used for pyrimidine nucleotide synthesis is derived from __, within the cytosol
glutamine and bicarbonate
98
Carbamoyl phosphate is then condensed with aspartate in a reaction catalyzed by the rate limiting enzyme of pyrimidine nucleotide biosynthesis, __.
aspartate transcarbamoylase (ATCase)
99
UMP Synthesis Overview
- 2 ATPs needed: both used in first step
* One transfers phosphate, the other is hydrolyzed to ADP and Pi
- 2 condensation reactions: form carbamoyl aspartate and OMP
100
Pyrimidine ring is synthesized from __
Carbamoyl phosphate and Aspartate
101
Carbamoyl-P for pyrimidine synthesis:
a. Formed in cytosol
b. Formed by a cytosolic form of the carbamoyl-P-synthetase II
c. Uses Glutamine as N-donor
d. Pyrimidine are attached to PRPP after synthesis
102
(DIFFERENCES IN PURINE AND PYRIMIDINE BIOSYNTHESIS) 1
1. The ring structure is assembled as a free base, not built upon PRPP.
* PRPP is added to the first fully formed pyrimidine base (orotic acid), forming orotate monophosphate (OMP), which is subsequently decarboxylated to UMP.
103
(DIFFERENCES IN PURINE AND PYRIMIDINE BIOSYNTHESIS) 2
2. There is no branch in the pyrimidine synthesis pathway. UMP is phosphorylated twice to yield UTP (ATP is the phosphate donor).
* The first phosphorylation is catalyzed by uridylate kinase and the second by ubiquitous nucleoside diphosphate kinase.
104
(DIFFERENCES IN PURINE AND PYRIMIDINE BIOSYNTHESIS) 3
3. UTP is aminated by the action of CTP synthase, generating CTP.
* The thymine nucleotides are in turn derived by de novo synthesis from dUMP or by salvage pathways from deoxyuridine or deoxythymidine.
105
- is a multifunctional enzyme that contains redox-active thiol groups for the transfer of electrons during the reduction reactions.
Ribonucleotide reductase (RR)
106
- is reduced in turn, by either thioredoxin or glutaredoxin.
- The ultimate source of the electrons is NADPH.
- The electrons are shuttled through a complex series of steps involving enzymes that regenerate the reduced forms of thioredoxin or glutaredoxin.
Ribonucleotide reductase (RR)
107
ATP ACTIVATES REDUCTION OF
- CDP
| - UDP
108
- induces GDP reduction
| - inhibits reduction of CDP. UDP
dTTP
109
- inhibits reduction of all nucleotides
dATP
110
- stimulates ADP reduction
| - inhibits CDP,UDP,GDP reduction
dGTP
111
(Synthesis of the Thymine Nucleotides)
| The de novo pathway to __ first requires the use of dUMP from the metabolism of either UDP or CDP.
dTTP synthesis
112
(Synthesis of the Thymine Nucleotides)
The dUMP is converted to dTMP by the action of __.
thymidylate synthase
113
(Synthesis of dTMP from dUMP)
The unique property of the action of __ is that the THF is converted to dihydrofolate (DHF), the only such reaction yielding DHF from THF.
thymidylate synthase
114
(Synthesis of dTMP from dUMP)
In order for the thymidylate synthase reaction to continue, THF must be regenerated from DHF. This is accomplished through the action of __.
dihydrofolate reductase (DHFR)
115
(Synthesis of dTMP from dUMP)
| THF is then converted to N5,N10-THF via the action of __.
serine hydroxymethyl transferase
116
__, unlike mammals, cannot use exogenous folate but must synthesize it from PABA. This pathway is thus essential for production of purines and nucleic acid synthesis in bacteria
Sulfonamide-susceptible organisms
117
__ is also useful as an antibacterial; it does not affect mammalian cells because it is about 50,000 times less efficient in inhibition of mammalian dihydrofolate reductase
Trimethoprim
118
The activity of __ (one of the various deoxyribonucleotide kinases) is unique in that it fluctuates with the cell cycle, rising to peak activity during the phase of DNA synthesis; it is inhibited by dTTP.
thymidine kinase
119
The regulation of pyrimidine synthesis occurs mainly at the first step which is catalyzed by __.
aspartate transcarbamoylase, ATCase
120
__ is inhibited by CTP, UDP, UTP, and dUTP; it is activated by ATP
ATCase
121
The role of glycine in ATCase regulation is to act as a competitive inhibitor of the __ binding site.
glutamine
122
Catabolism of the __ leads ultimately to β-alanine (when CMP and UMP are degraded) or β-aminoisobutyrate (when dTMP is degraded) and NH3 and CO2.
pyrimidine nucleotides
123
These deficiencies result in __ that causes retarded growth, and severe anemia caused by hypochromic erythrocytes and megaloblastic bone marrow.
- Leukopenia is also common in __
orotic aciduria
124
Orotic aciduria can be treated with __, which leads to increased UMP production via the action of nucleoside kinases.
uridine and/or cytidine
125
__ can also cause orotic aciduria because it can act as an alternate substrate and compete with orotic acid for degradation
Allopurinol
126
- Storage, transmission, and expression of genetic information
Nuclei Acids
127
- Lack nucleus
- Single chromosome
- Plasmids (nonchromosomal DNA)
Prokaryotes
128
- Nucleus
- Mitochodrion
- chloroplasts
Eukaryotes
129
The DNA Structure
- Deoxyribonucleoside monophosphate polymer
- 3’ → 5’ covalent phosphodiester bond
* Cleaved chemically (ONLY RNA cleaved by alkali)
* Enzymatically: deoxyribonucleases (DNAse) and ribonucleases (RNAse)
- Occur as double stranded (exception ssDNA viruses)
130
The DNA Structure (Prokaryotes vs Eukaryotes)
Prokaryotes: associated with nucleoid
Eukaryotes: nucleoproteins
131
- 2 chains coiled around the axis of symmetry
- Antiparallel chains/strands
- Deoxyribose-phosphate backbone: hydroPHILIC
- Base pairs: hydroPHOBIC
- “Twisted ladder”
- Proposed structure: 1953 by Watson and Crick
DNA
132
provide spatial access to DNA-binding proteins
Minor (narrow) and major (wide) grooves
133
__ intercalates into the minor groove
Dactinomycin (actinomycinD)
134
- In a dsDNA, amount of A is equal to T, amount of G is equal to C, amount of purines A and G = pyrimidines T and C
Chargaff Rule
135
Base Pairs are Complementary (1)
- The base of one strand is paired to the other
- Base pairs are perpendicular (900 ) to axis of symmetry
- A to a T (U), G to a C
- A to T (2 hydrogen bonds)
- G to C (3 hydrogen bonds)
- Strand separation: pH ionization and heat
136
Base Pairs are Complementary (2)
- NOTE: Phosphodiester bonds not broken down by pH and heat
- Denaturation: loss of helical DNA
* Measured at 260 nm absorbance
* ssDNA has higher relative absorbance
- Tm : melting temperature where there is loss of half of the DNA helical structure
137
AT DNA will have a higher peak of relative absorbance. __ will form at a lower temperature compared to high GC containing DNA
More single stranded DNA
138
The Structural Variants of the DS DNA
1. B Form
2. A Form
3. Z Form
139
- Right-handed helix with 10.4 nucleotide residues per 3600 turn
- most common, cross like
- Base planes perpendicular to axis
- Chromosomal DNA
B Form
140
- Formed by moderately dehydrating the B
- Right-handed helix
- 11 base pairs per turn
- Base pair plane are 20 away from perpendicularity to helical axis
- DNA-RNA hybrids and ds RNA
A Form
141
- Left handed
- 12 base pairs per turn
- Found in DNA with alternating purine and pyrimidine a poly GC
- B and Z helical transitions play a role in regulating gene expression
Z Form
142
Linear and Circular DNA
- Eukaryotic mitochondrial and chloroplastic DNA are closed and circular chromosomes
- Prokaryotes have single, ds, supercoiled, circular chromosome
143
- small, circular, extrachromosomal DNA molecules that undergo replication but may or may not be synchronized to chromosomal division. *Carry antibiotic resistance
Plasmids
144
DNA Replication
- Semiconservative
- Each parental strand incorporated into the duplex
- DNA replication is well-studied in Escherichia coli
- Eukaryotic DNA replication is complex
145
- parent strand will divide into two. One each will go to the daughter strands
Semiconservative
146
Prokaryotic DNA Replication (1)
- DNA replication starts at one origin of replication (a single, unique nucleotide sequence noted to be consensus)
* Exclusively at AT pairs (It will start at AT because it is where the hydrogen bonds are the weakest.)
* Eukaryotes have multiple origins of replication
- DNA polymerases use single strand DNA therefore the parental strands are melted
- Parental strand is unwound and separated
147
Prokaryotic DNA Replication (2)
- 2 replication forks are produced
- BIDIRECTIONAL movement forming a replication BUBBLE
- DNA strand-separation proteins (prepriming complex)
148
[DNA strand-separation proteins (prepriming complex)]
| - binds to the specific nucleotides at the origin of replication and melts AT-rich regions; ATP-dependent
Dna A protein
149
[DNA strand-separation proteins (prepriming complex)]
- bind to ssDNA near the replication fork; move into the neighboring double-stranded region and force strands apart; ATP-dependent NOTE: DnaB is E.coli’s principal helicase; must bind to DnaC
Helicase
150
[DNA strand-separation proteins (prepriming complex)]
- bind to the ssDNA brought about by the helicase; NOT ENZYMES; protect DNA from nucleases
SSB
151
A replication fork consists of four components that form in the following sequence:
(1) the DNA helicase unwinds a short segment of the parental duplex DNA;
(2) a primase initiates synthesis of an RNA molecule that is essential for priming DNA synthesis;
(3) the DNA polymerase initiates nascent, daughter-strand synthesis; and
(4) SSBs bind to ssDNA and prevent premature reannealing of ssDNA to dsDNA.
152
- acts on the lagging strand to unwind dsDNA in a 5′ to 3′ direction.
- associates with the primase to afford the latter proper access to the template.
helicase
153
* Positive supercoil or “super twists” interfere with unwinding
* Negative supercoil
* Topoisomerase – used to release the stress or tension brought about by the helicase
SUPERCOILING PROBLEM
154
– Nuclease and ligase properties
– Reuses energy stored from cleaving the phosphodiester bond to resealing the strand
– The intact DNA is passed through the break before it is resealed
– Relaxes negative supercoils in E.coli and both supercoils in eukaryotes
– energy efficient
Type I DNA topoisomerase: single strand break
155
– Causes a second stretch of DNA double helix to pass through the break
– ATP-requiring
– Needed for separation of interlocked DNA molecules following chromosomal replication
– DNA gyrase: unusual property to introduce negative supercoils into a relaxed circular DNA; neutralizes the positive supercoils during double helix opening
Type II DNA topoisomerase: double strand break
156
Etoposide, teniposide, and doxurubicin (anticancer agents) targets __.
human toposiomerase II
157
__ target DNA gyrase.
Quinolones
158
– Copied in the direction of the advancing replication fork
– 3’→ 5’
– Synthesize continuously
Leading strand
159
- Copied in the direction away from the replication fork
- Synthesize discontinuously
- Okazaki fragments (short fragments)
- 3’→ 5’
Lagging strand
160
DNA polymerase
- Read 3’→ 5’
| - Synthesize 5’ → 3’
161
RNA Primer Initiates Chain Elongation
- DNA pol cannot initiate synthesis of complementary strands on a ss template
- Require an RNA primer (short, double-stranded region of RNA-DNA hybrid)
- Hydroxyl group of RNA primer serves as first acceptor of a deoxynucleotide
- Primase (DnaG or RNA pol): synthesize short RNA sequences (10 nucleotides)
* Leading strand:1
* Lagging strand: multiple
162
RNA Primer Initiates Chain Elongation (2)
- Requires 5’ – ribonucleotide triphosphates
- Added via 3’ to 5’ phosphodiester bond
- Primosome: addition of primase to prepriming complex
163
__ is the initiator complex plus the helicase and the Single stranded binding protein (plus the primase = PRIMOSOME)
3’ complex
164
The mobile complex between helicase and primase has been called a __.
primosome
165
HOW THE SLIDING CLAMP FITS
The sliding clamp will be loaded by the clamp loader → magbubukas siya, you will need ATP → pag napasok na niya yung ssDNA, magfoform na siya ng dsDNA → irerelease niya yung ATP
→ hydrolyze the phosphate group
→ magbibind na ngayon yung clamp tapos dun na magbibind yung polymerase
166
- A number of different DNA polymerase molecules engage in DNA replication. These share three important properties: (1) chain elongation, (2) processivity, and (3) proofreading.
DNA POLYMERIZATION
167
DNA POLYMERIZATION
* DNA pol III uses the 3’ –OH of RNA primer as first acceptor
* Highly processive enzyme
* Remains bound to template and does not diffuse away
* Its β subunit forms a ring that encircles the template DNA thus serving as a sliding DNA clamp
* New strand grows from 5’ to 3’, antiparallel to parent
* 5-deoxynucleoside triphosphates as substrates
168
DNA POLYMERIZATION 2
* PPi is released when each new deoxynucleoside monophosphate is added to the growing chain
* DNA synthesis stops if a nucleotide is depleted
* DNA pol III has 3’ to 5’ proofreading (exonuclease)
* Checks to make certain the added nucleotide is complementary to base template
169
RNA Primer Excision and DNA Replacement
* DNA pol I has a 5’ to 3’ exonuclease activity besides 3’ to 5’ exonuclease and 5’ to 3’ polymerase activity
* DNA pol I locates the space or nick between the 3’-end of newly-synthesized DNA and 5’-end of adjacent RNA primer
170
RNA Primer Excision and DNA Replacement (2)
* Removes RNA primer via 5’ to 3’ exonuclease and puts deoxyribonucleotides via 5’ to 3’ direction and proofreads via 3’ to 5’
* 5’ to 3’ exonuclease activity removes one nucleotide that is properly base-paired and 1-10 nucleotides if mutated
171
- mediates the formation of the final phosphodiester bond between the 5’-phosphate group on the DNA chain synthesized by DNA pol III and the 3’-OH group on the chain made by DNA pol I
DNA ligase
172
High-Fidelity DNA Synthesis
High fidelity = konting error lang ang pwede niyang magawa
173
- Closely follows prokaryotic DNA synthesis
- Contains multiple origins of replication
- RNA primers removed by Rnase H and FEN1 rather than DNA pol I
Eukaryotic DNA Replication
174
• Contains primase
• Initiates DNA
synthesis
• Proofreading (-)
Pol α (alpha)
175
* Repair
| * Proofreading (-)
Pol β (beta)
176
• Replicates
mitochondrial DNA
• Proofreading (+)
• Location: mitochondria
Pol γ (gamma)
177
• Thought to elongate
Okazaki fragments
of the lagging strand
• Proofreading (+)
Pol δ (delta)
178
• Thought to elongate
the leading strand
• Proofreading (+)
Pol ε (epsilon)
179
- Complexes of noncoding DNA plus proteins located at the linear chromosomal ends
- Maintain the structural integrity
- Prevent nuclease attack
- Allow repair systems to distinguish a true end from a dsDNA break
Telomeres
180
Telomeres
- In humans, telomeric DNA contains several thousand tandem repeats of noncoding hexameric , AG3T2, base-paired to complementary region of Cs and As
- GT-rich strand is longer than its CA complement leaving ssDNA a few hundred nucleotides in length at 3’ end
- Single stranded region is thought to fold back on itself – forming a loop
181
- Following removal of RNA primer from the extreme 5’ end of the lagging strand, there is no way to fill in the remaining gap with DNA
- Telomeres shorten with each successive cell division
- Once telomeres shorten beyond some critical length, the cell is no longer able to divide
Telomere shortening
182
- Cancer cells, stem cells and germ cells, telomeres do not shorten and cells do not age
- This is due to the presence of the ribonucleoprotein telomerase
Telomere shortening
183
This complex contains a protein that acts as a
reverse transcriptase, and a short piece of RNA that acts as a
template. The CA-rich RNA template base-pairs with the GT-rich,
single-stranded 3'-end of telomeric DNA
Telomerase
184
- Translocates to the newly synthesized end
- GT-rich strand has been lengthened
- Add TTAGGG
- Primase can use it as a template to synthesize an RNA primer
- The RNA primer is extended by DNA pol and the primer removed
- Viewed as mitotic clocks
- Their length is inversely related to the number of times the cells have divided
- Aging and cancer
Telomerase
185
- Results from endogenous and exogenous causes
- Most DNA damage is repaired before DNA is replicated
- Mutagens are most effective in causing damage during the S phase when new DNA is being synthesized
DNA Damage
186
- Rate of mutations occurring from endogenous (internal cellular)
- Absence of environmental mutagens
- Caused by errors during DNA replication
- Spontaneous tautomeric shifts
- Spend very little time in their less stable forms so mutation from tautomeric shift is rare
Basal Mutation Rate
187
– there are changes in the base pair. It’s still the same molecule but may isang double bond na nareplace sa isang position pero pag binilang mo siya same lang. Shift in the double bonds can have an effect on the fidelity of the DNA, mutation may happen.
Tautomeric shift
188
– Outside influences: ionizing radiation (X-ray and radioactive radiation), UV radiation, hydrocarbons and oxidative free radicals
Exogenous Agents
189
DNA Repair Mechanisms
- Necessary because cells are continuously bombarded by environmental mutagens and basal mutations
- Most cases, cells use the undamaged strand of the DNA as template to correct the mutations
- When both strands are damaged, the cell resorts to use the sister chromatid or to an error-prone recovery mechanism
190
DNA Repair Mechanisms: General Scheme
recognition, removal, repair and religation
191
DNA Repair Mechanisms
1. Direct Repair
2. Mismatch Repair
3. Base Excision Repair
4. Nucleotide Excision Repair
5. Homologous Recombination
6. Nonhomologous End Joining
192
- Removes alkyl groups by a direct transfer to MGMT in a one-time reaction
- Repairs only one type of lesion
- NOT an enzyme
- May be the most efficient of all repair paths
- Cells must be able to continually manufacture more MGMT to perform the function
Direct Repair
193
- Deals with correcting the mismatches of normal bases that fail to maintain normal Watson-Crick base pairing
- Due to DNA pol mistakes during replication
- In eukaryotes, mismatch is accomplished by MSH2, MLH1, MSH6, PMS1, and PMS2 gebes
- Hereditary nonpolyposis colon cancer (HNPCC) at young age
Mismatch Repair
194
- Required to correct spontaneous depurination and deamination
- 10,000 purine bases lost per cell per day
- Spontaneous deamination of cytosine results in uracil
- Involves recognition of nucleotides that have lost the bases or have been modified
Base-Excision Repair
195
- Remove UV light-induced DNA damage
- UV light is nonionizing and cannot penetrate beyond the outer layer of the skin
- Form pyrimidine-pyrimidine dimers (cytosine and guanine)
- Necessary to recognize chemically induced bulky additions to DNA that distort the shape if DNA double helix and cause mutations
Nucleotide Excision Repair
196
- Carcinogens like benzopyrene in cigarette smoke
| - Xeroderma pigmentosum
Nucleotide Excision Repair
197
- Takes advantage of sequence information available from unaffected homologous chromosome
- BRCA1 and 2 proteins
- Breast CA, Fanconi’s anemia
Homologous Recombination
198
- Permits joining ends even if there is no sequence similarity between them
- Error-prone
Introduce mutations during repair
- Is important before the cell has replicated its DNA because there is no template available for repair by HR
Nonhomologous End Joining
199
Eukaryotic DNA Organization
- 46 linear chromosomes
- Paternal and maternal origins
- 20000 to 25000 genes
- MtDNA has 37 genes (all maternal)
200
Eukaryotic DNA Organization (2)
- DNA + proteins = chromatin
- Human haploid genome (23 chromosomes) = 3 x108 base pairs
- Total uncoiled DNA in a cell: 1 meter
- Uncoiled individual chromosome: 1.7 -8.5 cm in length
201
– loosely packaged and transcriptionally active
Euchromatin
202
__ is always condensed -- essentially inactive. It is found in the regions near the chromosomal centromere and at chromosomal ends (telomeres).
Constitutive heterochromatin
203
__ is at times condensed, but at other times it is actively transcribed – uncondensed and appears as euchromatin
Facultative heterochromatin
204
Genomic Organization
- Organized into unique and repetitive sequences
- Repeat Sequences: satellites and LINES and SINES
- Alpha satellite: 171 bp extending several million base pairs
- Minisatellite: 20-70 bp, few thousand base pairs
- Microsatellite: 2,3,4 bp anf few hundred in length
- Trinucleotide repeats: microsatellite sequences when expanded associated with a disease
205
* Less than 106 copies
* Short interspersed elements (SINES): 90-500 bp (100,000 copies)
* Long interspersed elements (LINES): 7,000 bp (20-50,000 copies)
* Wala siyang ineencode na any protein but sobra siyang nag repeat in a single sequence
SINES AND LINES
206
- Done for trisomy 21, to see the problems in the genes, translocations, deletions, mutations
- Giemsa stain is used. Fluorescent microscope is used to see the fluorescent antibodies binding to different chromosomes
Spectral Karyotyping
207
CLASSES OF RNA
1. Protein coding RNA – mRNA
2. Non-protein coding RNA
* Large – rRNA and lncRNA(long non-coding)
* Small – tRNA, snRNA, miRNA and siRNA
208
Types: ≥10^5 Different species
Abundance: 2%-5% of total
Stability: Unstable to very stable
Messenger (mRNA)
209
Types: 28S, 18S, 5.8S, 5S
Abundance: 80% of total Stability: Very stable
Ribosomal (rRNA)
210
SIMILARITIES BETWEEN DNA SYNTHESIS AND RNA SYNTHESIS
1. General steps of initiation, elongation, and termination with 5' to 3' polarity
2. Involves large, multi-component initiation complexes
3. Adherence to Watson-Crick base pairing rules
211
DIFFERENCE BETWEEN DNA SYNTHESIS AND RNA SYNTHESIS
1. Ribonucleotides are used in RNA synthesis rather than deoxyribonucleotides
2. U replaces T as the complementary base pair for A in RNA
3. Primer is not involved in RNA synthesis
4. Only a portion of the genome is transcribed or copied into RNA, whereas the entire genome must be copied during DNA replication
5. No proofreading function during RNA transcription.
212
Exons vs Introns
Exons - one that codes for protein
Introns - one that is a noncoding proteins
213
RNA is synthesized from a DNA template by an RNA polymerase
TRANSCRIPTION
214
Template Strand vs Coding Strand
Template strand – strand that is transcribed into an RNA molecule
Coding strand – the other DNA strand; sequence corresponds to the primary transcript which encodes the protein
215
Information on template strand is read out in __
3’ to 5’ direction
216
- specific site to which the RNA polymerase attaches; always located upstream from the transcription start site
* upstream nucleotide has a negative number
Promoter
217
- region of DNA that extends between the promoter and terminator; always located downstream from transcription start site
Transcription Unit
218
- Responsible for the polymerization of ribonucleotides into a sequence complementary to the template strand of the gene
DNA-DEPENDENT RNA POLYMERASE (RNAP)
219
- Exist as 400 kDa core complex consisting of:
* Two identical alpha subunits
* Similar but not identical B and B' subunits
* omega subunit
* sigma subunit
- B subunit binds Mg++ ions
(Core enzyme: omega, 2 alpha, B and B' subunits)
DNA-DEPENDENT RNA POLYMERASE (E coli)
220
[DNA-DEPENDENT RNA POLYMERASE (E coli)]
| __ enables the core enzyme to recognize and bind the promoter region to form the preinitiation complex (PIC)
Sigma factor/Sigma subunit
221
- Binding of RNAP holoenzyme (core + sigma factor) to template at promoter site
TEMPLATE BINDING
222
RNA Synthesis
1. Initiation
2. Elongation
3. Termination
223
(RNA Synthesis)
- First nucleotide (usually a purine) associates with initiation site on B subunit of enzyme
- RNAP catalyzes the coupling of the nucleotide to the second nucleotide
- RNAP undergoes a conformational change after RNA chain length reaches 10-20 and then able to move away from the promoter
INITIATION
224
(RNA Synthesis)
- Successive residues are added to 3’ terminus with sequence dictated by base pairing rules
- DNA unwinding must occur to provide access for appropriate base pairing; unwinding about 17 bp / polymerase molecule
ELONGATION
225
(RNA Synthesis)
- Termination signal recognized by rho factor (an ATP-dependent RNA-DNA helicase)
- RNA chain release; core enzyme separates from DNA template
TERMINATION
226
RNA POLYMERASE
- More than one RNA polymerase may transcribed the same template strand of a gene simultaneously
227
RNA polymerase continues to incorporate nucleotides +3 to ~+10, at which point the polymerase undergoes another conformational change and moves away from the promoter;
this reaction is termed __
promoter clearance
228
RECOGNITION OF INITIATION SITE (E coli)
- RNAP scans DNA sequence at rate of 103 bp/sec until it recognizes specific regions of DNA to which it binds with higher affinity (promoter)
229
The __ contains regions of highly conserved nucleotide sequence – located 35 and 10 bp upstream from the start site of transcription
promoter
230
- A T rich sequence
- Low melting temperature (lack G C)
- Eases dissociation between coding and non-coding strands so RNAP can have access to nucleotide sequence of immediately downstream coding strand
TATA BOX (PRIBNOW BOX)
231
- 40 nucleotide pairs in length
- Contains hyphenated or interrupted inverted repeat, followed by a series of AT base pairs *forms hairpin structure
- With aid of rho factor [an ATP-dependent RNA-stimulated helicase], RNAP stops and dissociates from DNA template and releases RNA transcript
TERMINATION SIGNAL in E coli
232
(Classification of RNA Polymerase)
major products: rRNA
sensitivity to alpha-amanitin: insensitive
RNA polymerase I
233
(Classification of RNA Polymerase)
major products: mRNA, miRNA
sensitivity to alpha-amanitin: high sensitivity
RNA polymerase II
234
(Classification of RNA Polymerase)
major products: tRNA, 5s RNA, snRNA
sensitivity to alpha-amanitin: intermediate sensitivity
RNA polymerase III
235
- Present in genes that lack TATA box; requires same transcription factors
INITIATOR (Inr) SEQUENCE/ DOWNSTREAM PROMOTER ELEMENT (DPE)
236
CLASS II GENE IN EUKARYOTES Requires:
RNA polymerase II, transcription factors A, B, D, E, F, H
237
- Binds to TATA box
- Only one of the factors capable of binding to specific sequences of DNA
- Originally considered to be a single protein
- Consist of TBP (TATA binding protein) and 8 TAFs (TBP associated factors)
TF IID
238
- Similar to bacterial sigma factor
TF IIF
239
- Has kinase activity
TF IIH
240
- Increases kinase activity of TF IIH
TF IIE
241
STEPS IN THE ASSEMBLY OF PREINITIATION COMPLEX
1. TBP binds to TATA box in the minor groove of DNA and causes an approximately 100 degree bend of DNA helix which facilitates interaction of TAFs
2. Binding of TFIIA, then TFIIB binds to TFIID/promoter complex
3. Pol II/TFIIF complex binds to the ternary complex of TFIIA/TFIIB/TFIID to the promoter
4. Complex attracts Pol II-TFIIF to promoter
5. Addition of TFIIE and TFIIH >> assembly of PREINITIATION COMPLEX (PIC)
242
- Sequences upstream from start site which determines frequency of transcription
- Binds protein factors, Sp1 and CTF
GC and CAAT boxes
243
- Increase or decrease rate of transcription initiation
- Can exert their effect when located hundreds or thousands of bases away from transcription units located on same chromosome
Enhancers /Silencers
244
- For steroids, T3, TRH, cAMP, prolactin, etc.
| - Acts as or in conjunction with enhancers or silencers
Hormone response element (HRE)
245
- Consists of 12 subunits
- Has a carboxy terminal repeat domain (CTD)
- Activated when phosphorylated on the Ser and Thr residues; inactive when dephosphorylated
EUKARYOTIC POLYMERASE II
246
- Contains a heptad repeat of Tyr-Ser-Pro-Thr-Ser-Pro-Ser
- Substrate for several kinases
- Binding site for proteins known as Srb (suppressor for Polymerase B) or mediator protein
carboxy terminal repeat domain (CTD)
247
THREE CLASSES OF TRANSCRIPTION FACTORS INVOLVED IN mRNA GENE TRANSCRIPTION
1. Basal Components
2. Coregulator
3. Activators
248
- RNA polymerase II, TBP, TFIIA, B, D, E, F, H
Basal components
249
- Bridging factor that communicate between the upstream activators, proteins associated with Pol II or the other components of TFIID
* TAFs, Mediator, Chromatin modifiers, Chromatin remodelers
Coregulators
250
- Binds to DNA and stimulate PIC formation or PIC function
| * SP1, ATF, CTF, AP1
Activators
251
TWO MODELS FOR THE ASSEMBLY OF THE PREINITIATION COMPLEX
1. Stepwise assembly
| 2. Recruitment
252
Sequential addition of components; takes place in DNA template
Stepwise assembly
253
Role of activators and coactivators may be solely to recruit preformed PIC to the promoter
Recruitment
254
- Transcribes tRNA and 5SRNA
| - Recognizes a promoter internal to the gene to be express (intragenic promoter)
RNA POLYMERASE III
255
- mRNA subjected to little modification and processing
| - Serve as translation templates even before their transcription has been completed
RNA PROCESSING (PROKARYOTES)
256
- Primary transcript undergoes extensive processing within the nucleus
* Capping
* Nucleolytic and ligation reactions
* Terminal addition
* Nucleoside modification
RNA PROCESSING (EUKARYOTES)
257
(PROCESSING OF mRNA)
Primary transcript which is processed to generate mRNA
hn RNA (heterogenous nuclear RNA)
258
(PROCESSING OF mRNA)
Sequence that represent the amino acid-coding portion
Exons
259
(PROCESSING OF mRNA)
Intervening sequences between the exons that are excised
Introns
260
PROCESSING OF mRNA
- Introns sequences are cleaved out and exons are spliced together
- Addition of 5’ cap and poly(A) tail at 3’ terminus
261
- Structure involved in converting the primary transcript to mRNA
- Consists of primary transcript and snRNAs– U1, U2, U4/U6, U5
SPLICEOSOME
262
(PROCESSING OF mRNA)
- 5’end of the intervening sequence is joined via 2’ – 5’ phosphodiester linkage to an __ 28-37 nucleotide upstream from the 3’ end of the intervening sequence
adenylate residue
263
Alternative RNA Processing
1. Use of alternative transcription start site
2. Use of alternative polyadenylation sites
3. Use of alternative splicing and processing
264
- Precursor tRNA reduced in size by a specific class of ribonucleases which recognize molecules capable of folding into functionally competent products
Modification of standard bases
- Attachment of CCA terminus at 3’end in the cytoplasm
PROCESSING OF tRNA
265
- Eukaryotic ribosome made up of:
60S subunit – 5S, 5.8S, 28S
40S subunit – 18S
- Transcribed from a single large precursor molecule (45S) which encodes an 18S, 5.8S and 28S rRNA
- Processed in nucleolus; undergoes methylation
PROCESSING OF rRNA
266
Inhibits gene expression by decreasing specific protein production
miRNA
267
(miRNA PROCESSING)
Primary transcript termed __; transcription unit either located independently in the genome or within intronic DNA of other genes
pri-miRNA
268
(miRNA PROCESSING)
Processed by __ maintaining its hairpin structure
Drosha-DGCR8 nuclease
269
(miRNA PROCESSING)
Transported through nuclear pore via action of __
exportin 5
270
- Once in the cytoplasm, further processed by Dicer nuclease-TRBP complex
- One of two strands loaded to RISC (RNA-induced silencing complex) composed of Argonaute proteins (Arg 1>>4)
- RISC complex promote mRNA degradation or inhibit translation
miRNA PROCESSING
271
- RNA molecules as with catalytic activity
- Generally involves transesterification reactions, splicing and endoribonuclease activities
- Plays central role in peptide bond formation
Ribozymes
272
- From the poisonous mushroom, Amanita phalloides
| - Forms a tight complex with RNA polymerase II and a looser one with RNAP III
alpha-Amanitin
273
- Inhibits transcription by binding to subunit of prokaryotic RNA polymerase
Rifamycin B and Rifampicin
274
- Intercalating agent
| - Tightly binds to duplex DNA, strongly inhibiting both transcription and replication
Actinomycin D
275
- Important in the understanding of protein synthesis and mutation
- Refers to the collection of codons that specifies amino acids
GENETIC CODE
276
– sequence of 3 nucleotides that codes for a specific amino acid
Codon
277
Occurs in the ribosome where the various classes of RNA interact
PROTEIN SYNTHESIS
278
– synthesize proteins that remains within the cell
Free ribosome
279
– membrane-bound polyribosome; synthesize integral membrane proteins and proteins to be exported
Rough Endoplasmic Reticulum
280
PROTEIN SYNTHESIS
| Requires the participation of the different RNAs
mRNA
| tRNA
281
contains the nucleotide sequence that translate to amino acid sequence of the protein
mRNA
282
recognizes specific nucleotide sequence as well as specific amino acids
tRNA
283
forms the structure of the ribosome where protein synthesis takes place
rRNA
284
- Specifies each of the 20 amino acids
- 4 different nucleotides (A, T, C, G)
- 3 nucleotides in a codon
- therefore, 43 = 64 possible combinations
* 64 specific codons
- 3 nonsense codons
- 61 amino acid codons
CODON
285
(BASIC CHARACTERISTICS OF THE GENETIC CODE)
– multiple codons must code for the same amino acids
Degenerate
286
(BASIC CHARACTERISTICS OF THE GENETIC CODE)
– for a given codon, only a single amino acid is indicated
Unambiguous
287
(BASIC CHARACTERISTICS OF THE GENETIC CODE)
- Reading of the genetic code during the process of protein synthesis does not involve any overlap of codons
Non-overlapping
288
(BASIC CHARACTERISTICS OF THE GENETIC CODE)
- Read in a continuing sequence of nucleotide triplets until a nonsense codon is reached
No punctuations
289
(BASIC CHARACTERISTICS OF THE GENETIC CODE)
- Same for all organisms
- Exception: 4 codons reads differently in the mitochondria and cytoplasm within the same cell (AUA, UGA, AGA, AGG)
Universal
290
At least one __ exist for each of the 20 amino acids
tRNA
291
(tRNA)
| – site of attachment of specific amino acid
Acceptor arm
292
(tRNA)
| – involved in binding of aminoacyl tRNA to ribosomal surface at site of protein synthesis
TYC
293
(tRNA)
| – for proper recognition of a given tRNA species by its proper aminoacyl-tRNA synthetase
D loop
294
(tRNA)
| – recognize codon in mRNA
Anticodon region
295
- Enzyme that attaches the amino acids to their specific tRNAs
- Capable of recognizing specific tRNA and specific amino acids
AMINOACYL tRNA SYNTHETASE
296
STEPS IN THE RECOGNITION AND ATTACHMENT OF AA TO tRNA
1. Aminoacyl tRNA synthetase binds to a specific amino acid forming an activated intermediate of aminoacyl-AMP-enzyme complex
2. Complex recognizes a specific tRNA to which it attaches the aminoacyl moiety at 3’OH adenosine terminus
297
- Base pairing between last nucleotide of codon and corresponding nucleotide of anticodon is not strict
- occurs allowing formation of hydrogen bonds on bases other than the standard ones
- E.g., 3 codons for glycine (GGU, GGC, GGA) can form base pair from one anticodon, CCI
WOBBLE
298
Change in the nucleotide sequence
MUTATION
299
(MUTATION: Single Base Substitution)
- Pyrimidine changed to another pyrimidine or purine changed to another purine
- Eg. A—>G; C—>T
Transition
300
(MUTATION: Single Base Substitution)
- Purine changed to a pyrimidine or pyrimidine changed to a purine
- Eg. A—>T, C—>G
Transversion
301
(EFFECTS OF BASE SUBSTITUTION)
- More likely if changed base in mRNA is the 3rd nucleotide of a codon
No detectable effect
302
(EFFECTS OF BASE SUBSTITUTION)
- Occurs when a different amino acid is incorporated at the corresponding site in protein molecule
- Might be acceptable, partially acceptable, or unacceptable depending on location of the amino acid in the specific protein
Missense effect
303
(EFFECTS OF BASE SUBSTITUTION)
- Results in premature termination of amino acid incorporation into peptide chain >> peptide fragment >> non-functional
Nonsense Effect
304
- Results from deletion or insertion of nucleotide in the gene thus generation an altered reading frame in mRNA >> results in garbled translation
FRAME SHIFT MUTATION
305
(Effects of Frameshift mutation)
If 3 or multiple of 3 nucleotides are missing/added >> on translation, protein will __
lack or have an added amino acid
306
(Effects of Frameshift mutation)
Insertion/deletion of 1, 2 or non-multiple of 3 >> reading frame will be distorted
a. Garbled amino acid sequences
b. Generation of nonsense codon
c. Reading through normal termination codon
307
- mRNA sequence is translated into sequence of amino acids of specified protein
- Message is read from 5’ to 3’ direction
PROTEIN SYNTHESIS
308
PROTEIN SYNTHESIS (Prokaryotes vs Eukaryotes)
Prokaryotes: translation can begin even before transcription is completed
Eukaryotes: primary transcript must first be process to generate mature mRNA before translation can begin
309
3 PHASES OF PROTEIN SYNTHESIS
1. Initiation
2. Elongation
3. Termination
310
PROTEIN SYNTHESIS: INITIATION
| a. Ribosomal dissociation
1. eIF-3 and eIF-1A binds to 40s subunit
| 2. Dissociation of the 80s ribosome to 40s and 60s
311
PROTEIN SYNTHESIS: INITIATION
b. Formation of 43s preinitiation complex
1. Binding of GTP by eIF-2
2. Binary complex binds to met-tRNA (first codon to be translated is usually AUG)
3. Ternary complex binds to 40s (with eIF-3 and eIF-1A) to form 43s preinitiation complex
312
PROTEIN SYNTHESIS: INITIATION
c. Formation of 48s initiation complex
1. eIF-4F binds to cap of mRNA
2. eIF-4B binds to mRNA and reduces the secondary structure of 5’ end
3. mRNA associates with 43s preinitiation complex with hydrolysis of ATP to form 48s initiation complex
4. Complex scans mRNA for a suitable initiation codon (AUG)
313
(Initiation in eukaryotic cells)
Precise initiation codon determined by __ that surrounds the AUG
Kozak consensus sequences
314
(Initiation in E. coli)
- A purine-rich sequence located 6-10 bases upstream of the AUG codon of the mRNA (5’-end)
- Complementary to the sequence near the 3’-end of the 16s rRNA of the 30s subunit
- Facilitates the binding and positioning of the mRNA on the 30s subunit
Shine-Dalgarno sequence
315
(PROTEIN SYNTHESIS: INITIATION)
D. Formation of 80s initiation complex
1. 60s binds to 48s initiation complex with hydrolysis of GTP (bound to eIF-2) by eIF-5
2. Release of initiation factors
3. Formation of 80s ribosome
316
- 2 sites in complete ribosome – A and P sites
- After the formation of 80s ribosome:
* met-tRNA at P site
* A site is free
INITIATION
317
– consist of subunits alpha, B, gamma
- eIF-2 alpha when phosphorylated binds tightly to and inactivates the GTP-GDP recycling protein eIF-2B >> prevents formation of 43s preinitiation complex >> blocks protein synthesis
- Kinases are activated when cell is under stress and energy expenditure is deleterious for the cell
eIF-2
318
(POINTS OF REGULATION IN INITIATION:eIF-4E)
– made up of 4E and 4G-4A complex
* 4E – binds to m7G cap at 5’ end (rate limiting step in translation)
* 4G – scaffolding protein; binds to eIF-3, 4A and 4B
eIF-4F
319
– binds to m7G cap at 5’ end (rate limiting step in translation)
4E
320
(POINTS OF REGULATION IN INITIATION:eIF-4E)
– links 4F to 40s ribosomal subunit
eiF-3
321
(POINTS OF REGULATION IN INITIATION:eIF-4E)
– ATPase and helicase complex; unwinds RNA
eIF-4B
322
LEVELS OF REGULATION OF 4E
1. 4E when phosphorylated by insulin and mitogenic growth factors binds to cap more avidly
2. 4E bound and inactivated by protein BP1, BP2 and BP3, preventing it from binding to 4G
* BP1 dissociates from 4E when BP1 is phosphorylated by insulin or other growth factors
323
Cyclic process on the ribosome in which one amino acid at a time is added to the nascent peptide chain
Elongation
324
(PROTEIN SYNTHESIS: ELONGATION)
a. Binding of aminoacyl-tRNA to A site
1. eEF-1A forms a complex with GTP and the entering aminoacyl-tRNA
2. The complex with the aminoacyl tRNA enters A site
3. GTP is hydrolyzed
4. eEF-1A, GDP and PO4- is released which will then be recycled to eEF-1A•GTP
325
(PROTEIN SYNTHESIS: ELONGATION)
b. Peptide bond formation
1. alpha-Amino group of new aminoacyl-tRNA in A site carries out a nucleophilic attack on esterified carboxyl group of peptidyl-tRNA occupying P site catalyzed by peptidyltransferase
2. Growing peptide chain is now attached to the tRNA in the A site
326
__ is a component of the 28s RNA of the 60s ribosomal subunit
Peptidyltransferase activity
327
(PROTEIN SYNTHESIS: ELONGATION)
| c. Translocation
1. Deacylated tRNA is attached by its anticodon to the P site at one end and by the open CCA tail to an E (exit) site
2. eEF-2 binds to displace the peptidyl tRNA from the A site to the P site; deacylated tRNA is on the E site from which it leaves the ribosome
3. eEF-2•GTP complex is hydrolyzed to eEF-2•GDP moving the mRNA forward by one codon and leaving the A site open for occupancy by another ternary complex of amino acyl tRNA-eEF1alpha•GTP and another cycle of elongation
328
- Energy requirement for the formation of one peptide bond = 4 high-energy phosphate bonds
* 2 ATP for charging of tRNA with aminoacyl moiety
* 1 GTP for entry of aminoacyl tRNA to A site
* 1 GTP for the translocation of peptidyl tRNA from A site to P site
ELONGATION
329
1. After multiple cycles of elongation with polymerization of amino acids into protein molecule, termination codon of mRNA appears in A site
2. Releasing factors recognize termination signal, with GTP and peptidyltransferase, they promote hydrolysis of bond between peptide and tRNA at the P site
3. tRNA and protein molecule are released from P site
4. 80s ribosome dissociates into 40s and 60s subunits
Protein Synthesis: TERMINATION
330
- Many ribosomes can translate the same mRNA molecule simultaneously – about 80 nucleotides apart
- A single mammalian ribosome is capable of synthesizing about 400 peptide bonds per minute
PROTEIN SYNTHESIS
331
- Many proteins are synthesized from mRNA as precursor molecules which are then modified to the active protein
- e.g. Insulin
POST-TRANSLATIONAL PROCESSING
332
- Structural analogue of tyrosyl-tRNA
- Binds to A site >> peptide chain formed between peptide and free NH2 group of puromycin >> premature termination of chain growth
- Unselective; inhibits both prokaryotic and eukaryotic protein synthesis
PUROMYCIN
333
- Binds to 60s ribosomal subunit inhibiting peptidyltransferase activity
- Blocks eukaryotic protein synthesis
CYCLOHEXIMIDE
334
- Catalytically inactivates eEf-2 by ADP-rybosylation
Diphtheria Toxin
335
- Poisonous plant protein that catalytically inactivates the eukaryotic large subunit
Ricin/Abrin
336
- inhibits the binding of aminoacyl tRNAs to the prokaryotic small subunit
Tetracycline
337
- Inhibits elongation in prokaryotes by binding to EFG•GDP in a way that prevents its dissociation from large subunits
Fusidic Acid
338
- Inhibits peptidyltransferase on the prokaryotic large subunit
Chlorampenicol
339
- Inhibits translocation by the prokaryotic large large subunit
Erythromycin
340
- Causes mRNA misreading and inhibits chain initiation in prokaryotes
Streptomycin
341
- block the bacterial translation by binding reversibly to the 30s subunit and distorting it in such a way that the anticodons of the charged tRNA cannot properly align with the codons of the mRNA
Tetracycline
342
- bind reversibly to the 50s subunit. they appear to inhibit elongation of the proteins by preventing peptidyltransferase from forming peptide bond between the amino acids
Macrolide (erythromycin, azithromycin, clarithromycin, dinithromycin)
