1
Q
What are codons?
A
Each three letter “word” in mRNA.
2
Q
What does each codon consist of?
A
Each codon consists of three consecutive bases that specify a single amino acid
3
Q
What direction do we read proteins?
A
5’ -> 3’
N terminus -> C terminus
4
Q
How many amino acids are there?
A
There are 20 different amino acids
5
Q
What do the 20 amino acids have in common?
A
The 20 common
amino acids that make up
proteins have similar
structures.
6
Q
What features does each amino acid have?
A
Each amino acid consists of a central carbon atom (C α) attached to:
(1) an amino group (NH3+),
(2) a carboxyl group (COO−), (3) a hydrogen atom (H), and
(4) a radical group, designated R.
7
Q
How are amino acids joined together?
A
Amino acids are joined together by peptide bonds. This forms in a dehydration reaction (lose H2O)
8
Q
What is primary structure?
A
Primary structure of a protein is its sequence of amino acids
9
Q
What is secondary structure?
A
Interactions between amino acids cause the primary structure to fold into a secondary structure, such as an alpha helix or beta sheet
10
Q
What is important for secondary structure?
A
R groups do not participate
11
Q
What is tertiary structure?
A
Secondary is folded even further into the tertiary
12
Q
What is quaternary structure?
A
Two or more polypeptide chains may associate to create a quaternary structure
13
Q
What do codons specifiy?
A
Three consecutive ribonucleotides (CODON) specifies one amino acid.
14
Q
What are the three nucleotides called in DNA vs. mRNA?
A
In DNA the 3 nucleotides are called as Triplet Code
In mRNA the 3 nucleotides are called as CODON
15
Q
Why did it need to be a triplet code?
A
- 20 amino acids
- 4 nucleic acid bases
- Number of possible codons = 4^n (n = # nucleotides per codon)
- 1 base not enough
- 2 bases not enough
- 3 bases → 64 possible codons [ENOUGH!]
16
Q
What do mRNA codons instruct the ribosome to do?
A
mRNA codons instruct the
ribosome to incorporate
specific amino acids into a
polypeptide – Translation
17
Q
What do all tRNAs have?
A
All tRNA’s from all organisms have a similar structure.
18
Q
What is the same for all tRNA molecules?
A
The amino acid attachment site is the same for ALL tRNA molecules.
-Sequence is always 5’-CCA-3
19
Q
What does the anticodon read and how?
A
The ANTICODON reads the information in a mRNA sequence by base pairing with a codon.
20
Q
How many codons are there?
A
There are a total of 64 codons
21
Q
How many sense codons are there?
A
There are 61 sense codons, they code for amino acids
22
Q
What does the 61 sense codons include?
A
Includes a start codon, AUG
23
Q
What does AUG initiate and what does it code for?
A
AUG initiates translation and codes for the amino acid methionine (MET)
24
Q
How many stop/nonsense codons are there?
A
There are 2 stop or nonsense codons
25
What are the three stop codons?
UAA, UAG, UGA
26
What do stop codons do and what do they code for?
They terminate translation and do NOT code for amino acids.
27
Are all codons exclusive to one amino acid?
No, there are multiple codons
per amino acid except
Methionine (Met) and
Tryptophan (Trp).
28
The genetic code is _________ but not ___________?
The Genetic Code is
Degenerate but not Ambiguous
29
What is the ratio of sense codons to amino acids?
61 sense codons, but only 20 amino acids!
30
What does degeneracy mean?
Some amino acids are specified by more than one codon - degeneracy.
31
What are synonymous codons?
Codons that specify the same amino acids are synonymous codons.
32
The genetic code is degenerate but not...?
The code is degenerate but NOT AMBIGUOUS!!!
33
What does it mean for the genetic code to not be ambiguous?
A codon NEVER specifies more than one amino acid.
34
What do most synonymous codons differ in?
Most synonymous differ in only the 3rd (3′) base, with the 1st and 2nd base being most important for distinguishing the amino acid.
35
What do mutations in the third position cause?
Mutations in the third position
rarely change amino acid
specification
36
Example of degeneracy with human and chimp?
Example., comparison of the coding region of human and chimp Leptin genes.
- Five differences between the sequences but only one (red arrow) of the five encode for a
different amino acid.
37
What are the two types of degeneracy?
Partial and complete
38
What is partial degeneracy?
Partial – changing the third base in a codon from a purine to a purine (G <-> A), or from a
pyrimidine to a pyrimidine (C <-> U).
39
What is complete degeneracy?
Complete – changing the third base in a codon to any of the four bases, e.g. GGU, GGC, GGA, or GGG, all code for
glycine.
40
What is degeneracy accommodated by?
Degeneracy of the genetic code is accommodated by:
1. Isoaccepting tRNA
2. Wobble effect
41
What is isoaccepting tRNA?
tRNAs bind (‘accept’) same
amino acid, but recognize different codons (by using
different anticodons).
42
What is the wobble effect?
allows same aa-tRNA to pair with more than one codon.
43
How many tRNAs and amino acids do we have?
30 different tRNAs, but only 20 amino acids!
44
Are all tRNAs used? How are they isoaccepting?
All tRNAs are used! So different tRNAs accept the same amino acid -> ISOACCEPTING.
45
Example of isoaccepting tRNA?
Example: tRNAs with anticodon 3’ AGG 5’ and 3’ AGU 5’
both accept serine.
46
How many anticodons and sense codons do we have? What is this solved by?
61 sense codons, but only 30 anticodons (30 tRNAs).
- solved by the wobble effect
47
Are all codons used? What does this mean?
All 61 codons are used! So the same anticodon base pairs with different codons.
48
Example of same anticodon pairing with codons?
Example: tRNA with anticodon 3’ AGG 5’ base pair with codons 5’ UCC 3’ and 5’ UCU 3’.
49
What does the wobble effect allow?
WOBBLE EFFECT allows the same anticodon to base pair with more than one codon.
50
Where does the wobble effect occur?
Wobble occurs between the 1st (5′) base of an anticodon of t-RNA (Wobble Position) and the 3rd (3′) base of a codon of mRNA.
51
How does a non-Watson and Crick base pair occur?
The 1st (5’) base of a anticodon is allowed to move slightly “wobble” from its
normal position to form a non-Watson & Crick base pair with the codon.
52
Wobble at the 5' end allows what?
Wobble at the 5' end of the anticodon allows variable nucleotide at the 3' end of
the codon, therefore more than one codon for an amino acid.
53
With an anticodon wobble position of G, what does it pair with?
A G wobble position pairs with C or U at the third position of the codon
54
With an anticodon wobble position of C and A, what does it base pair with?
A C or A anticodon wobble position will base pair with G or U respectively at the third position of the codon
55
With an anticodon wobble position of U, what does it base pair with?
A U anticodon wobble position base pairs with A or G at the 3rd position of the codon
56
What does an anticodon wobble position of I pair with?
An I will pair with either A, U or C at the third position of the codon
57
What is inosine?
Inosine is an intermediate in
the metabolism of purine.
58
Where is inosine found and what is it essential for?
Inosine is commonly found
in tRNAs and is essential for
proper translation of the
genetic code in wobble base
pairs.
59
Example of serine codons?
Serine is encoded by six codons, four are shown below. Differ only in the third
position of the codon.
- Has two tRNAs for 4 of the codons
60
How many amino acids, tRNAs and codons do we have?
20 amino acids
30 isoaccepting tRNAs
61 codons
61
What is a reading frame?
Reading frame refers to protein-coding region of the mRNA.
62
What does a reading frame specify?
Specifies a single protein starting and ending at internal sites within the mRNA.
63
What do all reading frames begin with?
All RFs begin with AUG, therefore all proteins begin with
Methionine (M).
64
How are codons read?
Codons consisting of three nucleotides are read 5’ to 3’.
65
Do codons overlap?
Codons are not overlapping, each base is part of only one
codon.
66
Do codons have gaps?
No gaps, each base in the coding region of an mRNA is
part of a codon.
67
How is the reading frame set?
Message is translated in a reading frame set by the
initiator codon, AUG.
68
What is an open reading frame?
An open reading frame is a portion of a RNA molecule that, when translated into
amino acids, contains no stop codons.
69
What does the presence of stop codons in reading frames 1-3 mean?
Presence of stop codons in 2 of the above reading frames means they are not open reading frames (ORFs). A long open reading frame is likely part of a gene.
70
How many reading frames are there for a sequence?
There are three! Only one is the correct open reading frame
71
When do you look for an AUG?
“Use the start codon to determine the reading frame”
“This is the full mRNA transcript”
“The transcription start site is in (italics and underlined) and the transcription termination site is (underlined)”
72
Possible question: 1) DNA sequence → mRNA transcript → amino acid sequence
Notice for the first type of question you need to orient yourself to the mRNA (5’→3’)
… be given template or non-template strand.
73
Possible question: DNA or RNA sequence … find an open reading frame (no stops), AUG not necessary
Notice for the second type of question we are looking for an ORF, so for DNA you may need to check both strands.
74
Possible question: Full mRNA transcript → aa sequence of gene “look for the start codon to determine the reading frame"
Notice for the second and third types of questions we are looking for an ORF, so we
automatically know that we are talking about the non-template strand, which is the
same as the mRNA strand.
75
What is a missense mutation?
The new codon encodes a different amino acid and there is a change in the amino acid sequence
76
What is a nonsense mutation?
The new codon is a stop codon, there is a premature termination of translation
77
What is a silent mutation?
The new Condon encodes the same amino acid, there is no change in amino acid sequence.
78
What do missense, nonsense, and silent mutations have in common?
They are all types of point mutations, where just one nucleotide changes
79
What are frameshift mutations?
These are insertions and deletions that shift the frame in which the codon sequence is read.
80
What are the two types of frameshift mutations?
They can be deletions or insertions
81
What is translation?
Translation is the process of converting genetic information stored in nucleic acid
sequences into proteins.
82
Sequences of mRNA are translated into what?
Sequences of mRNA are translated into unique sequence of amino acids in a polypeptide chain (linear order is preserved throughout!)
83
Where does translation take place?
Translation of an mRNA takes place on a RIBOSOME.
84
How does the ribosome read the mRNA?
The mRNA is read in the 5’ to 3’ direction.
85
Where are amino acids attached to the polypeptide?
Amino acids are attached to the carboxyl (C) terminus of the growing polypeptide chain; polypeptide is made in the amino (N) to carboxyl (C) direction.
86
The N-terminus is what?
The N-terminus is the one where the amino group is free
- the beginning
87
The C-terminus is what?
The C-terminus is the one where the carboxyl group is free
- the ending
88
Can multiple ribosomes translate at once?
Multiple ribosomes can
simultaneously translate the
same mRNA molecule –
polyribosome (or polysome)
89
What are the 6 requirements for prokaryotic translation?
* mRNA template
* tRNAs (transfer RNAs)
* Amino acids
* Ribosomes
* Many accessory proteins
* Energy provided by GTP hydrolysis
90
prokaryotic mRNA is..?
Prokaryotic mRNA: Polycystronic, i.e. multiple ORFs
91
What do tRNAs have in common?
All tRNA's from all organisms have a similar structure.
92
What do tRNAs serve as?
Serves as a link between the genetic code in mRNA and the amino-acid sequence of a protein.
93
What is the amino acid attachment site?
The amino acid attachment site is the same for ALL tRNA molecules.
- Sequence is always 5’-CCA-3’
94
How does the anticodon read the mRNA?
The ANTICODON reads the information in a mRNA sequence by base pairing with a codon
95
What is the direction of travel for the ribosome?
The ribosome reads mRNA from 5' to 3'
96
What 3 positions can hold tRNA?
The E, P and A sites
97
What is the A site?
This is where new tRNAs (charged with amino acids) can bind and deliver their amino acid
98
What is the ribosome consisting of?
RNA-protein complex: Ribonucleoprotein - made as RNA and protein
99
What does the prokaryotic ribosome have for RNA and sub-units
Prokaryote version has 3 RNA molecules and 2 sub-units
100
rRNA has what activity?
rRNA is the part of the ribosome that actually has enzymatic activity.
101
What are the 2 subunits?
50S and 30S that assemble to form 70S ribosome
102
What does the ribosome direct?
Directs the synthesis of
proteins.
103
Where does synthesis of proteins take place?
Synthesis takes place in the
cavity between subunits
104
Role of small subunit?
Small subunit holds mRNA
105
Where does the growing peptide exit?
Growing polypeptide exits
through the tunnel in the large
subunit.
106
How many amino acids added per second?
Adds 20 amino acids per
second.
107
3 binding sites of tRNA?
3 tRNA binding sites:
A = Aminoacyl binding site
P = Peptidyl binding site
E = Exit site
108
Where does the tRNA binding site span?
tRNA binding site spans large and small subunits, so tRNA anticodon loop can make contact with mRNA.
109
Small subunit holds..? Why?
Small subunit holds mRNA so only 1 codon at a time can be read by a tRNA.
110
What happens to the amino acid at the end of the tRNA?
Amino acid at end of tRNA makes contact with catalytic region of the large subunit that joins amino acids together to form peptide
bonds.
111
How is the arm of the tRNA positioned?
The arm of the tRNA is
positioned in such a way as to allow polypeptide chain to
exit through a tunnel in the back of the ribosome.
112
What is the growing peptide attached to?
The growing polypeptide is always attached to a tRNA.
113
What do many antibiotics attack?
Many antibiotics attack
translational apparatus
– e.g., binding to tRNA
sites, blocking exit
tunnel…
114
What kind of process is translation?
Four step process
115
What are the four steps of translation?
1. tRNA Charging
2. Initiation
3. Elongation
4. Termination and
peptide release
116
What is tRNA charging?
Attachment of an amino acid to the tRNA is referred to as tRNA charging.
117
What are all amino acids attached to?
ALL amino acids are attached to the adenine (A) nucleotide in the 3’ end acceptor stem.
118
What is the carboxyl group of the amino acid linked to?
The carboxyl group of an amino acid is linked to the 3’
hydroxyl (OH) group of the A nucleotide of the tRNA.
119
What is the R group?
R group = unique side chain specific to a particular amino acid.
- dictates which amino acid is being attached
120
What is energy needed for?
Energy required for binding of aa to tRNA supplied by ATP
121
What is a charged tRNA known as?
tRNA+aa = aminoacyl-tRNA
* e.g.: aminoacyl-tRNAAla has alanine bound to tRNA
* Could also write Ala-tRNAAla
122
What does aminoacyl-tRNA synthetase do?
Aminoacyl-tRNA synthetase correctly attaches an amino acid to the appropriate
tRNA.
123
How many aminoacyl-tRNAs are there?
20 different aminoacyl-tRNA
synthetase.
124
What does each aminoacyl-tRNA synthetase recognize?
Each aminoacyl-tRNA synthetase recognizes one amino acid and attaches it to the correct set of tRNA’s.
125
What is the anticodon not involved in?
The anticodon region of tRNA is not involved in helping the synthetase recognize amino acids, it works with the mRNA
126
What are the 2 key steps of translation initiation?
1. Assembly of ribosomal subunits at the translation start site.
2. Base pairing of initiator-tRNA anticodon with start site codon in the mRNA.
127
What are the 5 requirements for translation initiation?
1) mRNA
2) Small and large subunits
3) Initiator-tRNA
4) Initiation factors (IFs)
5) GTP
128
What's the role of IF3?
IF3 binds to the small subunit, preventing the large subunit fro binds, thus allowing the small subunit to attach to mRNA.
129
How does imitation in prokaryotes begin?
IF-3 binds to the small subunit, preventing the large subunit from binding.
130
What occurs when the large and small subunits are apart?
Small ribosome subunit bind to the mRNA.
131
What is the Shine-Dalgarno sequence what what does it bind to?
16S rRNA of the small ribosome subunit complementary base
pairs with the Shine-Dalgarno sequence
132
What does the initiator-tRNA base pair with?
Initiator-tRNA anticodon (3’
UAC 5’) base pairs with mRNA
initiation/start codon (5’ AUG
3’).
133
What is the initiator-tRNA charged with?
Initiator tRNA is charged with
fMet amino acid
(N-formylmethionine).
134
What is the role of IF3 protiens?
IF-3 proteins keep the small
and large ribosomes separated.
135
Role of IF1 and 2?
IF-1 and 2 facilitates initiator-
tRNA binding to the correct site.
136
Altogether, IF3, IF2, IF1 and the other items form what?
This forms the 30S initiation
complex
137
What happens when the initiator factor proteins dissociate?
Initiation factor proteins
dissociate and the large
ribosome subunits binds.
* This forms the 70S initiation
complex.
138
What is elongation?
The synthesis of the polypeptide chain
139
What are the 4 key steps in elongation?
The key steps in elongation are:
1) entry of aa-tRNA into the A site of the ribosome,
2) peptide bond formation,
3) ribosome translocation, and
4) Exit of tRNA from E-site of the ribosome
140
What are the 4 requirements for elongation?
Requirements
1) aa-tRNAs (or charged tRNAs)
2) 70S initiation complex
3) Elongation factors (EFs)
4) GTP
141
What are the three ribosome sites used for tRNAs?
E, P and A
142
What is the A site and its role?
A-site (aminoacyl) - accepts the
incoming aa-tRNA carrying the next amino acid to be added to the chain.
143
What is the P site and its role?
P-site (peptidyl) - holds the tRNA carrying the growing polypeptide.
144
What is the E site and its role?
E-site (exit) - discharged tRNA's
leave the ribosome from this site.
145
What is the first step in elongation?
aa-tRNA is delivered to the A site:
- Incoming aa-tRNA bind to the A site.
- Elongation factors (EF) guides the incoming aa-tRNA to the
correct site.
- aa-tRNA anticodon base pairs with the mRNA codon.
146
What is the second step of elongation?
peptide bond fomation
147
What catalyzes peptide bond formation?
Peptidyl transferase catalyses peptide bond formation.W
148
What is peptide formation enzyme activity performed by?
Enzyme activity performed by rRNA in the large ribosome subunit (ribozyme).
149
Peptide bond formation is between what?
Peptide bond formation between amino acid attached to P-site tRNA and amino
acid attached to A-site tRNA..
150
pepide bond formation releases?
Peptide bond formation releases the amino acid from the tRNA at the P-site.
151
Where is the polypeptide attached?
Growing polypeptide now attached to A-site tRNA
152
third step of elongation?
Ribosome movement or translocation
153
What direction does the ribosome translocate
The ribosome translocates in the 5’ to 3’ direction on the mRNA.
154
What is translocation facilitated by?
Translocation facilitated by elongation factors.
155
What occurs to the P site and A site following translocation?
1. P-site tRNA is now located at E-site, this tRNA exits.
2. A-site tRNA now located at the P site.
3. A-site is now empty and ready to accept another aa-tRNA.
156
Three steps of elongation?
1. aa-tRNA binds to A-site
2. Peptide bond formation and release of amino acid from P-site tRNA
3. Translocation and exit of E-site tRNA. A-site is now open to accept another aa-tRNA
157
When does protein synthesis terminate?
Protein synthesis terminates when the ribosome translocates to a stop codon.
158
What aa-tRNA enters after the stop codon?
NO aa-tRNA enters an A site that has a stop codon.
159
What does the release factor do?
Release factor (RF) protein binds to the A site and triggers release of the polypeptide from the P-site tRNA.
160
Can multiple ribosomes translate the same mRNA?
Multiple ribosomes can simultaneously translate the same mRNA molecule.
161
Prokaryotic mRNA is...?
Prokaryotic mRNA: Polycystronic, i.e. multiple ORFs
162
Eukaryotic mRNA is..?
Eukaryotic mRNA: Monocystronic mRNA, i.e. single ORF
163
Eukaryotes ribosome complex?
Composition of the eukaryotic ribosome complex is different.
- 40S and 60S makes 80S
164
What consensus sequence is needed for eukaryotic ribosome binding?
No consensus sequence
for ribosome binding.
165
What facilitates binding of ribosome for eukaryotes?
5’ CAP and 3’ poly(A) tail
facilitate ribosome
binding.
- Initiation complex scans
along mRNA until 1st AUG
codon is encountered.
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