1
Q
What are the macromolecules of translation?
A
- ribosomes (polypeptides, >50) and rRNA (3-4)
- amino acid activating enzymes (20)
- tRNA molecules (40-60)
- soluble proteins (translation factors) involved in polypeptide chain initiation, elongation, & termination
2
Q
What are ribosomes composed of?
A
- proteins and several different rRNAs
- a large and a small subunit that assemble
3
Q
What is the key role of a ribosome?
A
= protein synthesis, formation of peptide bonds b/n amino acids
4
Q
Describe the prokaryotic ribosome
A
- 50S subunit
- 30S subunit
- complete prokaryotic ribosome is 70S
5
Q
Describe the eukaryotic ribosome
A
- 60S subunit
- 40S subunit
- complete eukaryotic risbosome is 80S
6
Q
What does the S in the subunits stand for?
A
Svedberg units
= a measure of how the ribosomal complex sediments in a gradient during centrifugation
- densities are not additive due to the way the complete ribosome sediments
7
Q
Describe tRNAs
A
- adapters b/n amino acids and codons in mRNA
- the first anticodon position of tRNA base pairs with 3rd codon position of mRNA
8
Q
Where do the amino acids attach to the tRNA?
A
- via the 3’-ACC-5’ sequence at the 3’ end
9
Q
Key facts about the ribonucleotides in tRNA?
A
- A, G, C, and U
- often modified post-transcriptionally by cellular enzymes adding -CH3 or H
10
Q
How many tRNA synthetase do cells contain per amino acid?
A
- at least one
11
Q
What do tRNA synthetases do?
A
- catalyze formation of aminoacyl tRNAs
12
Q
What joins an amino acid to their respective tRNA?
A
amino acyl tRNA synthetase, contain an active site, a pocket for the amino acid to bind and a pocket for the tRNA to bind
- help form the tRNA-amino acid bond (high energy!) with help of ATP to AMP conversion (ATP hydrolysis)
13
Q
How is aa joined with tRNA?
A
with a covalent bond that is high energy, this results in the aminoacyl tRNA = charged tRNA
14
Q
What molecule does the formation of amino-acyl tRNA synthetase require?
A
ATP - needs to be hydrolyzed to help from the high energy bind
15
Q
Describe the reaction that results in the formation of the aminoacyl tRNA?
A
- first step: aa reacts with ATP
- then: aminoacyl-AMP and PPi are produced
- second step: amino acids transferred t tRNA and AMP is released
16
Q
Steps of translation in both Eukaryotes and Prokaryotes?
A
- very similar in both
Initiation
Elongation
Termination
17
Q
Describe the initiation complex that forms during initiation of translation in Prokaryotes
A
composed of mRNA, large and small ribosomal subunits, the initiation factors (IF1-3) and GTP (guanosine triphosphate)
18
Q
How does the initiation complex form in Prokaryotes?
A
- IF-3 binds to small subunit to prevent large subunit from binding and allows small one to bind to mRNA
- tRNA charged with N-formylmethionine forms a complex with IF-2 and GTP
- all initiation factors dissociate from the complex and GTP is hydrolized to GDP
- large subunit joints to create 70S initiation complex
19
Q
Where is the Shine Dalgarno sequence located in initiation?
A
- mRNA
- 16S rRNA, a component of 30S small ribosomal subunit contains the complement
20
Q
Where does pairing between Shine-Dalgarno and rRNA complement position the ribosome?
A
- near the AUG start codon
21
Q
At the end of initiation, where is the ribosome and where is the first tRNA in Prokaryotes?
A
- the ribosome is assembled on the mRAN
- the first tRNA is attached to the initiation codon
22
Q
What is the Shine-Dalgarno sequence?
A
5’-AGGAGG-3’
also called the consensus sequence
23
Q
What tRNA is specific to the initiation codon?
A
= the initiation codon is 5’-AUG-3’
therefore, Methionine, so Fmet-tRNA (IAC)
- the methionine is modified with formyl group (hence fMet)
24
Q
Where do you see fMet?
A
only in prokaryotes!
25
What are the three distinct regions in a ribosome after it is completely assembled?
- Aminoacyl site (A)
- Peptidyl site (P)
- Exit site (E)
26
What happens at the A site?
The aminoacyl site accepts newly charged aminoacyl tRNAs specific for the codon at that position
27
What happens at the P site?
the peptidyl site is the position where the peptide bond forms b/n amino acid on tRNA on that site and the newest one at the A site
28
What happens at the E site?
The exit site is where the tRNA that has had its amino acid removed, following peptide bond formation, exits the ribosome
29
Describe the amino acid on the initiator tRNA in Eukaryotes
- it is not formylated, it is just MET (not fMET)
30
Where does the initiation complex form in Eukaryotes for translation?
- at the 5'-terminus of the mRNA (the 7-methylguanosine cap on the 5' end) and there is no Shine-Dalgarno sequence in eukaryotes!
- then ribosome scans inward from there
31
Which sequence influences the efficiency of which AUG in vicinity of 5' 7-MG cap is used to start translation in eukaryotes?
= KOZAK sequence
- 5'-GCC (A or G) CC AUG G-3'
- the AUG start codon is imbedded within the Kozak sequence
- spacing of the nucleotides is important but some difference can occur but need A or G two nt before AUG and a G after!
32
What promotes translation initiation in eukaryotes?
- the poly A tail of the eukaryotic mRNA interacts with the mRNA 5' 7MG cap structure via cap binding protein complex (CBC) to promote translation initation
33
During elongation, where does the anticodon of an incoming tRNA bind?
- binds with the codon of the mRNA that is exposed at the A site
- occurs with the help of EF-Tu (Elongation factor Tu) and GTP
34
How does bind formation occur during chain elongation?
- peptide bond is formed by peptidyl transferase
- the dipeptide is attached to the tRNA in the A site
- tRNA in the P site is now devoid of its amino acid
- therefore bond formation occurs b/n the NH2 group of the amino acid attached to the tRNA in the A site and the -COO group of aa attached to tRNA in P site via peptidyl transferase activity of the ribosome
35
How does the ribosome move on to the next codon after the peptidyl bond forms?
- with the help of EF-G and GTP
- at this point the tRNA that was originally in the P site is now devoid of its aa and is ejected from the ribosomal E site and newly charged aminoacyl tRNA specific to unbound codon in A site binds
36
What is the role of GTP in elongation?
= provides energy
37
When does elongation end?
= when one of the stop codons is encountered
| - specifically, when a chain-termination codon (stop codon) enters the A site of the ribosome
38
What are the stop codons?
UAA
UAG
UGA
39
What happens when a stop codon is encountered?
a release factor binds to the A site
| - essentially there is no tRNA molecule that can bind to the stop codons and that is why they stop elongation
40
What does release factor 1 recognize?
UAG and UAA stop codons
41
What does release factor 2 recognize?
UAA and UGA stop codons
42
What do RF-1 and RF-2 do?
- both release factors alter the activity of peptidyl transferase, which results in a water molecule being added to the carboxyl terminus of the nascent polypeptide releasing it and thereby ending elongation/translation
43
How is the ribosome dismantled?
- RF-3 and GTP help in dismantling
44
Which researchers won the Nobel Prize in Chemistry in 2009 for their discovery of ribosome structure?
- Ramakrishnan, Steitz, and Yonath
45
How does Chloromycetin inhibit bacterial protein synthesis?
- stops formation of peptide bonds
46
How does Erythromycin inhibit bacterial protein synthesis?
- stops translocation of mRNA along ribosome
47
How does Neomycin inhibit bacterial protein synthesis?
- stops interaction between tRNA and mRNA
48
How does Streptomycin inhibit bacterial protein synthesis?
- stops initiation of translation
49
How does Tetracycline inhibit bacterial protein synthesis?
- stops binding of tRNA to ribosome
50
How does Paromomycin inhibit bacterial protein synthesis?
- stops the validation of mRNA-tRNA match
51
What is a nonsense mutation
- formation of premature stop codon that prevents the synthesis of full-length protein
52
What are some quality control mechanisms in the cell
- mechanisms that ensure the quality of mRNA and mechanisms that ensure the quality of proteins
53
How is the quality of mRNA ensured?
- by degrading mRNAs with errors that can create problems in translation (like nonsense mutations or if they stall ribosomes or have unusual secondary structures or if damaged by chemicals)
54
What are chaperones and how do they help in quality control of proteins?
= they assist in the proper folding of newly-synthesized proteins
- chaperones are often associated with the ribosome during translation
55
What is a mechanism to control the quality of proteins other than chaperones?
= modifying amino acids post-translation by phosphorylation, methylation or acetylation, ubiquitination
- can modify protein function and activity
56
What causes the disease Xeroderma Pigmentosum (XP)?
a mutation of one or more genes in the ncleotide excision DNA repair pathway
- results in inability to repair sunlight induced UV light lesions in DNA in skin cells and patients often develop skin cancer
57
What may occur when a damage in DNA is not corrected?
= inherited change in genetic information = a mutation
58
What is a mutation?
= heritable change in the sequence of an organisms genetic material
- mutation may alter the phenotype of the organism
- the process by which genetic change occurs
59
What is a mutant?
= organism that carries one or more mutations in its genetic material
60
What is the connection b/n evolution and mutation?
- Natural selection preserves the combinations best adapted to the existing environment = evolution
61
How does recombination during meiosis play into variability?
= recombination during meiosis rearranges genetic variability between homologous chromosomes into new gene combinations
62
What is somatic tissue?
- tissue that divides mitotically
63
What is germinal tissue?
- tissue that divides through meiosis and produces gametes
64
Can somatic mutations be transferred to progeny?
No, only germinal mutations can be transferred to progeny following meiosis and sexual reproduction
65
What are the types of gene mutations?
- somatic mutations (in somatic cells, will only occur in descendants of that cell and not be transmitted to progeny)
- germinal mutations (occur in germ-line cells and transmit through gametes to progeny)
66
What is the origin of many varieties of fruit and vegetables?
= somatic mutations
- original mutation occurred in fruit trees and those were somatic mosaics
- vegetative propagation allows fro somatic mutation to be perpetuated
- some somatic mutations can lead to human diseases like cancer
67
What is the importance of bacterial and phage mutants?
- very useful in genetic studies and have helped establish the disciplines of genetics and molecular biology as well as the biotechnology industry
68
What are the types of point mutations?
- base substitutions
- frameshift mutations
- tautomeric shifts
= all of them occur at localized sites in DNA
69
What is an example of a dynamic mutation?
- expanding nucleotide repeats
| = the nucleotide repeat copy number can expand or contract dramatically in each cell
70
What is a gross chromosomal rearrangement?
= a change in chromosome number or structure
71
What are the types of base substitutions?
- transition
| - transversion
72
What happens during a transition?
- a pyrimidine is replaced with another pyrimidine or a purine is replaced with a purine
73
What happens during a transversion?
- a pyrimidine is replaced with a purine or vice versa
74
How many different base substitutions exist?
12
75
What is a frameshift mutation?
= an insertion or deletion of one or two base pairs that alter the reading frame of the gene distal to the site of mutation, i.e. dramatic change after the frameshift
- can be any number of bases except a multople of 3
- even a single C:G insertion (C on one strand, G on other) causes a shift in frame and after the insertion a change in amino acids = mutant polypeptide occurs
76
What is tautomeric shift?
= movement of H atoms from one position in a purine or pyrimidine base to another
- it is rare but can occur spontaneously during DNA replication and alter DNA base pairing
- can cause some spontaneous mutations
- results in keto and enol (O vs OH) or amino and imino form (NH2 vs NH)
77
Which are the normal tautomeric forms of bases?
= keto (O) and amino (NH2)
78
What is often a consequence of a frameshift mutation?
= a non-functional protein
79
How can tautomeric shifts cause spontaneous mutations?
- when the shifts occur during DNA replication
- they alter DNA base pairing, when bases are in rare enol or imino states, they can form A-C and G-T base pairing through hydrogen atoms shifting and allowing for bonds
80
What are transition mutations?
= they result from tautomeric shifts during DNA replication
- if G undergoes tautomeric shift to rare enol form, it prefers to base pair with T, so GT pair forms, at the next round of replication the base T pairs with A to form a mutant AT base pair in the new strand
- therefore, only get the transition from CS to AT
- also means there is only ever 1 mutant cell among the 4 produced by two rounds of DNA replication
81
Describe expanding nucleotide repeats
- expansion of triplet repeats like CAG is cause of numerous human diseases when in coding regions of eukaryotic genes
- the mechanism of expansion involves DNA replication
- it is termed dynamic b/c the repeat copy number is in flux with each round of replication
82
Through what mechanism does an expanding nucleotide occur?
- when there is a run of CAG repeat the polymerase can pause and slide backwards and repeat the copy
- can cause hairpins to form in the newly synthesized strand since base pairing is possible which causes part of the strand to be replicated twice thereby further increasing the number of repeats
83
What is another way of classifying mutations?
- looking at their effect on the phenotype of an organism
84
What is a forward mutation?
= genetic alteration that changes the wild-type phenotype to mutant
85
What is a reverse mutation?
= genetic alteration that changes the mutated site back to normal, thereby reversing the mutant back to the wild-type phenotype
86
What can forward and reverse mutations affect?
= protein function
87
What are gene mutations that are classified by their effect on gene or protein function
- missense mutation
- nonsense mutation
- silent mutation
88
What is a missense mutation?
= base substitution that results in an amino acid change in the protein
89
What is a nonsense mutation?
= base substitution that changes a sense codon to one of the three nonsense stop codons (UAG, UGA, UAA)
- premature termination of translation
90
What is a silent mutation?
= base substitution at the 3rd codon position that changes the codon to one still specifying the same amino acid, no change in amino acid sequence
91
What is a neutral mutation?
= missense mutation in which aa is changed to one of a similar chemical type (i.e. glycine to alanine)
- little to no effect on protein function
92
What is a loss-of-function mutation?
= result of mutations that cause complete or partial loss of normal protein function (example cystic fibrosis results form loss of function in C gene)
93
What is a gain-of-function mutation?
= result of a mutation that causes the cell to produce a protein or gene product whose function is not normally present
94
What is a conditional mutation?
= expressed only under certain conditions, i.e. temperature-sensitive mutation)
95
What is a lethal mutation?
= results in premature cell death
96
What is a suppressor mutation?
= it is a second site mutation that hides or suppresses the effect of the first mutation
- suppressor mutations can be within the same gene (intragenic)
- for instance if missense mutation alters single codon and second mutation at different site in same gene restores original aa
97
What is an intergenic suppressor?
= supressor mutation present within a different gene but still has the ability to suppress or revere the mutagenic effect of the first muation
- example: base substitution results in stop codon and nonfunctional protein
- in a different gene an incorrect base results in an anticodon on tRNA that can pair with stop codon and translation can continue past the stop codon, so there is then an additional aa but it is more likely to be functional protein
98
What are key facts about mutation frequencies?
- occur at low frequency but can be increased markedly by treating cells with mutagens that damage DNA
99
What is the mutation rate in eukaryotes?
- varies from 10^-4 to 10^-7 per gene per generation
| - very low spontaneous mutation rates
100
What is the mutation rate for bacteria and bacteriophages?
- varies from 10^-8 to 10^-10 per nucleotide pair per generation
101
What is the frequency of mutation in bacteria like after treatment with mutagens?
- can increase f by >1% per gene
102
What is an induced mutation?
= mutation that occurs as a result of exposure to external factors such as environmental chemicals and radiation
103
What is a spontaneous mutation?
= one that results from internal factors under normal circumstances and just occurs randomly as part of biochemical pathways and processes
104
What constitutes the largest source of damage to DNA?
INTERNAL FACTORS that are generated by normal metabolic processes
- meaning: DNA is usually damaged under normal physiological conditions inside the cell by water (hydrolysis), oxygen (thru oxidation) and alkylating agents (through alkylation)
105
What causes damage to DNA under normal physiological conditions, i.e. which endogenous chemical rxns?
water, oxygen, alkylating agents
106
What is an estimate of errors made during DNA replication per year?
- conservative estimate = 6x10^14
| or 60 billion mistakes due to replication in a typical 50 minutes lecture
107
What is endogenous DNA damage?
= internal, i.e. result of normal biochemical processes
- produces thousands of lesions every day
- antibody formation in developing immune system also causes 10^11 breaks that normally occur (and are repaired) each day
108
What is exogenous DNA damage?
= external, i.e. environment
- the only type of exogenous DNA damage really worth mentioning is that due to peak hour sunlight
- damages skin cells and generates lesions in DNA in pyrimidine dimers
109
Why is DNA damage not that much of an issue as numbers would suggest?
repair systems
110
What specifically causes spontaneous DNA damage?
- DNA replication errors
- DNA replication pausing (polymerase pauses)
- endogenous chemical reactions
111
What are examples of DNA replication errors that result in spontaneous DNA damage?
- tautomeric shifts = movement of H atoms from one position to another on purine or pyrimidine ring (result: non-standard bp leading to transition mutations)
- strand slippage during replication = in repeated sequences, but also misalignments during recombination - can result in addition or omission of nt
- wobble-induced base mispairing
112
What is wobble induced base mispairing?
flexibility in base-pairing (wobble) can result in non-standard G-T and C-A base pairs which lead to transition mutations
113
Describe replication pausing
= when at a DNA nick (generated by endogenous ROS or enzymes like topoisomerase) replication stalls
- results in unusual DNA structure or bulky lesion that generates broken DNA like DNA double-stranded break
114
What is a DSB?
DNA double stranded break
| = lethal or mutagenic lesion unless properly repaired by recombination mechanism
115
What are endogenous chemical reactions that lead to spontaneous DNA damage?
1. Depurination
2. Deamination
3. Oxidation
4. Alkylation
116
How does depurination lead to spontaneous DNA damage?
= spontaneous loss of purine base from nucleotide through hydrolysis of glycosidic bond (approx. 10000 cell/day, so 10,000 purines lost)
(pyrimidines only about 500 per cell per loss)
- when a single stranded DNA contains an AP (apurinic/apyrimidinic) site, it can be replaced with an A or C and this generates a transition or transversion mutation (A or C will be on new strand)
117
What is deamination?
- can lead to spontaneous DNA damage
- spontaneous loss of -NH2 group on DNA bases, causes transition mutation
- can result in Cytosine eventually becoming uracil or 5-Methylcytosine eventually being replaced by Thymine
118
What is oxidation in the process of spontaneous DNA damage?
= endogenous reactive oxygen species (ROS) damage DNA and can produce oxidized bases such as 8-oxoG, which mispairs with C or A to produce a transversion mutation (GC to TA)
119
What is alkylation and how does it lead to spontaneous DNA damage?
= endogenous alkylating agents (S-adenosyl methionine or SAM) can add methyl groups to DNA bases and can lead to transition mutations (GC - AT)
- example of a base modified like this: O6-methyl guanine
120
What are the types of induced DNA damage?
- chemical agents
- radiation
(these are known mutagens)
- first discovered: Hermann Muller 1927 in fruit fly, first hint that gene is target of X rays
121
What are the two categories of chemicals that induce mutation?
- chemicals that are mutagenic to replicating AND nonreplicating DNA (alkylating agents and nitrous acid)
- chemicals mutagenic only to replicating DNA (like base analogs and acridine dyes)
122
What are alkylating agents?
= mutagens that react w DNA bases and add methyl or ethyl groups and can directly/indirectly induce transitions, transversions, frameshift mutations, and chromosomal aberrations (there are natural agents as well, like SAM)
- examples: mustard gas, EMS, EES
123
How does nitrous acid induce DNA damage?
= deaminating agent, removes amino (NH2) groups from DNA bases, A, C, and G and causes transition mutations
- nitrous acid: HNO2
124
Describe the molecular mechanism of the transitions caused by nitrous acid
- Adenine reacts w HNO2 to give Hypoxanthine which can bond with cytosine: AT to GC
- Cytosine with HNO2 gives Uracil, which bonds with Adenine and results in CG to TA
- Guanine reacts with HNO2 to give Xanthine and this can still bond with cytosine, therefore not mutagenic!
125
How does Hydroxylamine lead to DNA damage?
= hydroxylating agent
- hydroxylates the amino (-NH2) group of cytosine causing the modified base to pair with adenine after replication = leads to transition mutation
- chem structure: NH2OH
- C:G -> OHC:A -> T:A therefore CG to TA transition mutation
126
What do base analogs do?
- affect replicating DNA
- two common base analogs are 5-bromouracil (5-BU resembles T) and 2-aminopurine (2-AP resembles A or G)
- incorporated into DNA during replication and when rare tautomers arise will eventually cause transition mutations
- if cell exposed to 5-BU during replication it will be incorporated into DNA instead of T an bp with A, but then if 5-BU goes from being keto to enol it will base pair with guanine, therefore
- can also cause TA to CG and AT to GC but takes 3 replications
127
How do the acridines cause DNA damage?
- intercalation of an acridine dye causes frameshift mutations during DNA replication
- examples: Proflavin, Ehtidium bromide, and acridine organge
- cause insertions or deletions of one or two base pairs altering the reading frame of gene distal to the site of mutation
- essentially these molecules can insert between the DNA bases and stretch out the helix, then during replication the polymerase then inserts or deletes a nt
128
How does gel electrophoresis work?
- can use acridines since they reflect UV light, allows to look at DNA during electrophoresis
129
How does radiation cause mutations?
- UV light induces mutations through EXCITATION
- X-rays (even shorter wavelength than UV) cause mutations thru IONIZATION
- damaging effects of radiation are inversely proportional to wavelength: the longer wavelengths are less damaging than shorter
130
What are the longest rays on the electromagnetic spectrum?
longest: radio waves
| then microwaves, then infrared, followed by visible light
131
What are the shortest rays on the electromagnetic spectrum?
= shortest cosmic rays
visible light < UV < X rays < Gamma rays < cosmic rays
shortest wavelengths have highest energy
132
What exactly happens during ultraviolet irradiation?
= UV ligh causes cross-linking of adjacent thymines, which results in thymine dimers that block DNA replication (T=T on same strand) b/c DNA polymerase cannot replicate through the dimer
- can cause DNA breaks = broken chromosome = lethal to cell
- can also occur b/n cytosines (i.e. pyrimidines)
- the ringed pyrimidines absorb UV light and this excites the molecule causing a rearrangement of bonds
133
Where is UV irradiation a major concern and how can it manifest?
- in sunlight and tanning beds (tanning beds major cancer threat, as lethal as cigarettes)
- causes skin cancer
- peak sunlight: exposed human skin can acquire about 4500 thymine dimers and UV induced photoproducts per hour
134
What does ionizing radiation do to DNA?
- caused by X-rays, can cause DNA breaks and other types of damages and results in a change in chromosome structure
- damage: nicks and double-stranded breaks, faulty repair by recombination can then cause rearrangements like deletions, duplications, inversions, and translocations
- first experiment to prove this: Muller with his Drosophila
- i.e. ionization is much more powerful than radiation b/c it disrupts DNA at atomic level
135
What is the rate of mutation in the cell like?
- DNA constantly exposed to endogenous (mostly) and exogenous agents that cause DNA damage
- HOWEVER, rate of mutation in the cell is low
- this is due to repair mechanisms (nobel prize to Lindahl, Modrich, and Sancar for DNA repair)
MBG 2040
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