ALL THE THINGS Flashcards
1
Q
What are the 3 RNA species involved in translation?
A
tRNA
rRNA
mRNA
2
Q
What function/role do tRNAs play in translation?
A
function as adaptors between mRNA codons and specific amino acids
3
Q
What % of total cellular RNA is tRNA?
A
15%
4
Q
What function/role do rRNAs play in translation?
A
form part of the structure of ribosomes and participate in protein synthesis
5
Q
What % of total cellular RNA is rRNA?
A
80%
6
Q
What function/role do mRNAs play in translation?
A
code for proteins
7
Q
What % of total cellular RNA do mRNAs play in translation?
A
5%
8
Q
How many total codons are there in the Genetic Code?
A
64
9
Q
What is the start codon?
A
AUG
10
Q
What are the 3 stop codons?
A
UAA, UAG, UGA
11
Q
What is the Wobble Position?
A
means there is “steric freedom” at this site; other bases may pair
12
Q
Codons that differ in the first two bases will have their own what?
A
tRNA
13
Q
How many reading frames are possible for a given mRNA?
A
3
14
Q
What causes alteration of the reading frame?
A
non-N3 insertions/deletions cause alteration of the reading frame
15
Q
When the tRNA arrives at the mRNA, pairing between what occurs?
A
pairing between codon and anticodon; antiparallel alignment
Codon is 5’-3’
Anticodon is 3’-5’
16
Q
When the anticodon has an inosine at position 1, it can pair with how many different codons?
A
3
Position 1 is always at the 5’ end
17
Q
What are the 3 different mutations affecting translation?
A
Missense
Nonsense
Insertions
18
Q
What types of insertions/deletions are possible?
A
Frameshift mutations
3n insertions/deletions
19
Q
What are silent mutations?
A
DNA base-pair substitutions that do not affect the amino acid coding
20
Q
What are a special class of insertions/deletions?
A
splicing errors caused by splice site mutations
21
Q
Aminoacyl-tRNA synthetases are enzymes that carry out how many coupled reactions?
A
2
22
Q
Describe the 2 coupled reactions carried out by aminoacyl-tRNA synthetases
A
(1) An amino acid is activated by reaction with ATP to give an aminoacyl-AMP intermediate
(2) Amino acids are coupled to tRNA’s by an ester linkage between the carboxyl group of the amino acid and the 2’ or 3’ OH of the 3’ A-residue of the tRNA
23
Q
Why do aminoacyl-tRNA synthetases have to be very precise?
A
they bridge between 3-letter codon code and specific amino acid
24
Q
Each aminoacyl-tRNA synthetase is specific for one what and one or more corresponding?
A
specific for one amino acid and one or more corresponding tRNAs
25
tRNA specificity is based on what?
specific bases in the anticodon and other bases elsewhere in the tRNA
26
What is a "Charged tRNA?"
tRNA carrying an amino acid
27
What are the 2 steps in aminoacylation of tRNA by aminoacyl-tRNA synthetases?
(1) amino acid activation by ATP with the release of phosphate
(2) binding of the activated amino acid to terminal A residue of the tRNA
28
tRNA synthetases can remove what kinds of errors?
coupling errors; if the incorrect amino acid binds the synthesis site, it gets moved to the editing site to be removed and replaced
29
The Initiator tRNA (tRNAf) is first charged with what?
methionine
30
A formyl group is transferred to the methionyl - tRNAfMet from what?
N10 - formyltetrahydrofolate
31
What role does formylation of the amino group play?
the attached methionine can no longer be inserted in the interior of the polypeptide chain
All other methionines added will be devoid of the formyl group
32
Before translation begins in Prokaryotes, the 3'-end of mRNA must pair with what?
16S rRNA
33
What is the pairing to the 16S rRNA mediated by in Prokaryotes?
the Shine-Dalgarno sequence
34
Where is the Shine-Dalgarno sequence in Prokaryotes?
6 bases upstream of the AUG
35
After pairing with 16S RNA, what else must the mRNA pair with before translation begins in Prokaryotes?
the mRNA AUG must pair with the anticodon of the initiator methionyl-tRNAfMet
36
What are the initiation factors for Prokaryotic translation?
IF1, IF2, IF3
37
Multiple internal Shine-Dalgarno sequences are located where?
near the start sites for each of the encoded proteins in a polycistronic mRNA
38
What are the 2 ribosomal subunits in Prokaryotes?
Small 30S
Large 50S
Together make the 70S ribosome
39
In the first step of bacterial translation initiation, what does IF3 prevent?
the 30S and 50S subunits from combining prematurely
40
In the first step of bacterial translation initiation, what does IF1 prevent?
prevents the use of the A site in initiation
41
In the second step of bacterial translation, what does IF2 do?
binds to an initiator tRNA and controls the entry of tRNA onto the P-site. IF2 bound to GTP binds to the 30S P-site.
IF2 allows addition of the initiator methionyl-tRNAfMet to the P-site
42
In the third step of bacterial translation, what happens?
50S subunit binds. It hydrolyzes GTP bound to IF2 and leads to the release of all the IFs
43
During protein synthesis, the nascent protein is always covalently bound to what?
a tRNA
44
What does bacterial protein synthesis begin with?
a peptidyl-tRNA in the P-site. An aminoacyl-tRNA guided by EF-Tu binds the A-site
45
In bacterial elongation, with both P & A sites occupied, what is formed and what catalyzes its formation?
a new peptide bond catalyzed by 23S rRNA
46
23S rRNA is a known what with what kind of activity?
ribozyme with peptidyl-transferase activity
47
In bacterial elongation, what translocates the ribosomal subunits to move the deacylated tRNA to the E-site?
elongation factor G
48
Once in the E-site, the deacylated tRNA is free to do what in bacterial elongation?
dissociate
49
Peptide bond formation in bacterial elongation is followed by shifting a tRNA position on the 50S subunit. What kind of binding state is formed?
hybrid binding state
50
In the Translocation step of bacterial elongation, energy from the hydrolysis of what bound to what drives the step?
GTP bound to EF-G
51
Termination of translation in bacteria is by what?
by release factors that are proteins with tRNA shapes
52
Binding of release factors force the peptidyl transferase to add what to the peptidyl tRNA?
to add water instead of an amino acid
53
Two high energy bonds of what are cleaved when charging tRNA with an amino acid?
ATP
54
To the bind the charged tRNA to the ribosomal A-site, one high end energy bond of what is cleaved?
GTP
55
What is cleaved for the translocation step in bacteria?
GTP
56
For each amino acid incorporated in translation, how many total bonds are consumed?
4
57
What are the ribosomal functional sites?
A site
P site
E site
58
What is the A-site?
acceptor site; associated with mRNA codon
59
What is the P-site?
peptidyl transferase site, associated with mRNA codon
60
What is the E-site?
exit site, not associated with mRNA codon, less well defined in Eukary ribosomes
61
Both large and small ribosomal subunits contribute to form which sites?
A and P-sites
62
Where is the complementary sequence for the Shine-Dalgarno sequence?
withi the 16S rRNA
63
In the 50S subunit, what rRNA is the peptidyl transferase (ribozyme)?
23S rRNA
64
What rRNA is the peptidyl transferase in Eukary and which subunit is it found in?
28S rRNA
60S subunit
65
In bacteria, what ion is associated with activation of amino acids, initiation, and elongation?
Mg2+
66
What are the 5 agents mentioned in class that inhibit protein synthesis?
```
Streptomycin
Puromycin
Tetracyclin
Chloramphenicol
Eryhtromycin
```
67
How does Streptomycin inhibit protein synthesis?
binds to 30S subunit and causes misreading of the genetic code and inhibits initiation of translation at higher concentrations
68
How does Puromycin inhibit protein synthesis?
structurally similar to the 3'-end of aminoacyl tRNA
binds to the A site and participates in peptide bonding: peptidyl puromycin --- prematurely ending synthesis
69
How does Tetracyclin inhibit protein synthesis?
blocks A-site
70
How does Chloramphenicol inhibit protein synthesis?
binds to the large ribosomal subunit and blocks bacterial peptidyl transferase
71
How does Erythromycin inhibit protein synthesis?
binds to the large ribosomal subunit, blocking translocation
72
Which agents specifically inhibit the 50S ribosomal subunit?
Erythromycin
| Chloramphenicol
73
Which agents specifically inhibit the 30S ribosomal subunit?
Tetracyclin
| Streptomycin
74
Bacterial resistance to antibiotics is mediated by what?
plasmids encoding enzyme(s) --- which alter the host
75
Staphylococcal resistance to erythromycin is the result of what?
conversion of a single adenosine in 23S rRNA to N6-dimethyladenosine, so that erythromycin can no longer bind
76
The conversion of the adenosine to N6-dimethyladenosine is mediated by what plasmid borne enzyme?
RNA methylase
77
What are the eukaryotic orthologs of EF-Tu and EF-G initiation factors?
eEF1
| eEF2
78
When forming the eukary initation complex, what binding protein is bound to the 3'-end of the mRNA?
PABP - poly(A) binding protein
79
With PABP bound, what binds to the 5'-cap in eukaryotic initiation?
eIF4E
80
With eIF4E bound to the cap and PABP at the 3'-end, what binds next to tie the ends of the mRNA together?
eIF4G
81
The eIF proteins are also involved in the binding of the initiation complex to what ribosomal unit?
40S subunit
82
Another eIF protein scans the mRNA with the 40S subunit for what codon?
AUG codon
83
In order to find the AUG, what does the eIF protein need to have to resolve secondary structure in the 5'UTR of the mRNA?
it needs to have helicase activity; it also NEEDS ATP
84
In eukaryotic initation, is the methionine bound to the initiator tRNAMet formylated?
No
85
What are the two potent toxins that affect Eukaryotic protein synthesis?
Diptheria toxin
| Ricin
86
What does Diptheria toxin do/cause?
inactivates eukary elongation factor 2 (eEF2, similar to bacterial EF-G) by modification of a histidine residue (by ADP-ribosylation)
87
What kind of illness is Diptheria?
respiratory track illness
88
What Ricin do/cause?
depurinates a specific adenosine residue in the 28S rRNA, thereby inactivating its peptidyl transferase activity
89
What are heat shock proteins?
synthesized upon cellular heat exposure. Hsp's have affinity for exposed hydrophobic patches of incompletely folded proteins and use ATP for binding and release of these patches
90
Both Hsp 60 & 70 use lots of what to ensure correct protein folding?
ATP
91
Where can you find chaperones like Hsp 60 & Hsp 70?
in both the cytoplasm and inside organelles
92
What are some of the modifying groups involved in posttranslational modifications?
Phosphate on Ser, Thr, or Tyr
Methyl on Lys
Acetyl on Lys
Palmityl on Cys
N-acetylglucosamine on Ser or Thr
Ubiquitin on Lys
93
What affect does the phosphate on Ser, Thr, or Tyr have? (Function of the modification)
drives the assembly of a protein into larger complexes
94
What effect does methylation of Lys have (Postranslationally)
Helps to create histone code in chromatin through forming either mono-, di-. or tri-methyl lys
95
What effect does an acetyl group have on lys? (Postranslationally)
Helps to create histone code in chromatin
96
What effect does a palmityl group on Cys have? (Postranslationally)
this fatty acid addition drives protein association with membranes
97
What effect does N-acetylglucosamine have on Ser or Thr? (Postranslationally)
controls enzyme activity and gene expression in glucose homeostasis
98
Ubiquitin on Lys will have what affect postranslationally?
Monoubiquitin addition regulates the transport of membrane proteins in vesicles
A polyubiquitin chain targets a protein for degradation
99
In the formation of the functional final protein, what is removed?
the formyl group in prokaryotes, the amino terminal methionine, and often additional amino terminal terminal amino acids
100
In 50% of eukary proteins, the amino group of the N-terminal residue is what? (How is it modified)
N-acetylated
101
Are carboxy terminals susceptible to terminal modifications?
they may be trimmed and modified
102
How are proteins targeted to intracellular organelles or for secretion?
they lose their signal sequences often through cleavage at a specific site
103
What enzyme acts to cleave the signal sequence on a growing polypeptide chain?
signal peptidase - specific N-terminal aa sequence is cleaved off
104
What are the 3 common phosphorylated amino acids discussed in class?
Phosphoserine
Phosphothreonine
Phosphotyrosine
(All these groups have an OH in their side chains ;] )
105
What do protein kinases and phosphatases do?
Protein Kinases add a phosphate group via an ATP molecule
Phosphatases remove phosphate groups
106
Protein phosphorylation and dephosphorylation acts like what kind of switch?
They act like on/off switches.
The example given in class was with Casein. When the Ser residue on Casein is phosphorylated it is turned on and does its function. When acted on by a phosphatase, it is switced off
107
Carboxylation is an enzymatic mechanism dependent on what vitamin?
Vitamin K
108
Where is carboxylation an important modification?
at the Glutamate residue in the Gla region of certain blood-clotting factors
109
In blood clotting, what does modification via vitamin-K dependent carboxylation confer?
confers membrane binding properties required for proper functioning of the blood clotting cascade
110
Vitamin-K antagonists would do what?
slow down clotting
111
Hydroxylation of a proline residue to make hydroxylated proline is an important constituent of what?
fibrillar collagen
112
What are the two types of oligosaccharide linkages found in glycoproteins?
N-linked oligosaccharides: carbohydrate side chains are enzymatically attached to the amide nitrogen of an Asn residue
O-linked oligosaccharides: carbohydrate side chains can be attaced to the hydroxyl of Ser or Thr residues
113
What kinds of proteins contain oligosaccharide side chains?
proteins that function extracellularly like plasma proteins and lubricating proteoglycans
114
Acetylation of lysine regulates the interaction of histones with?
DNA
115
Methylation of lysine regulates the interaction of histones with other proteins, mainly what?
transcription factors
116
Which modification removes the positive charge on Lysine?
acetylation
117
The Hsp 70 chaperone system is working how? Hsp 60 system what?
Hsp 70 works co-translationally
Hsp 60 uses a folding chamber
118
Addition of fatty acids to amino terminal amino or cystein groups does what?
anchors proteins to membranes
119
Many enzymes are synthesized as inactive precursors that are activated how? BONUS: What are the inactive forms of the proteins called?
by proteolysis
BONUS: Zymogens
120
The binding and tagging of a target protein is carried out by what?
ubiquitin ligase
121
Ubiquitin is how many amino acids long?
76
122
Target proteins for ubiquitin ligase first undergo what to mark them for polyubiquitylation?
phosphorylated
123
A polyubiquitin chain on a lysine residue directs protein to what for degradation?
the proteasome
124
What is a proteasome?
a cylindrical ATP-dependent protease
125
A target protein with a polyubiquitin chain is degraded where in the proteasome?
it is brought through the active site.
the ubiquitin residues are removed before entering the active site
126
Protein folding diseases are caused by misfolding due to mutations and subsequent aggregation in protease resistant protein aggregates like amyloid. Name some of these diseases.
These are dominant diseases:
Sickle Cell Anemia
Alpha-1-antitrypsin deficiency
Huntington's and Alzheimer's
127
Changes in what structure can cause proteins to resist proteolysis?
Changes in secondary protein structure
128
Prion diseases are caused by what and what?
misfolding and aggregation of prion proteins
129
Prion diseases are described as having a highly ____ nature.
infectious
130
What are some common prion diseases?
CJD: Creutzfeldt-Jacob disease in humans
BSE: Bovine spongiform encephalopathy in cattle
Scrapie in sheep
131
Protein misfolding diseases all result in conversion of protein secondary structure into what?
beta sheets which stack one above another
