FINAL! Flashcards
(139 cards)
1
Q
Where does transcription occur?
A
Nucleus
2
Q
Where does translation occur?
A
on ribosomes in the cell cytoplasm
3
Q
Translation
A
-“decoding” a messenger RNA (mRNA) and using its information to build polypeptide
-Process by which ribosomes read genetic message in mRNA and produce a protein product according to the message.
4
Q
What does translation involve?
A
Non-coding RNA:
-rRNA: makes up ribosomes, the protein factories
-snoRNA: facilitates necessary mods to rRNA (and other RNA)
tRNA: adaptors that bind amino acid at one end and interact with mRNA at the other end.
5
Q
Expression
A
Production of a final product
i.e. for a protein-coding gene expression= protein produced
6
Q
Open reading frame or coding region
A
Region of the mRNA from start codon to stop codon that codes for a protein
ALWAYS made up of exons*
7
Q
What are the untranslated regions?
A
5’ UTR and 3’ UTR
Also exons, bud do not code for protein; regulatory in nature
8
Q
Ribosome
A
-The large subunit ineracts with the aminoacylated end of the tRNA
-Small subunit interacts with mRNA and anticodon loop of the tRNA
9
Q
Two subunits of E. coli and its coefficient?
A
70S ; Small 30S and decodes mRNA; 50s Large links amino acids together through peptide bonds
10
Q
E. coli 50S SU contains
A
55 rRNA, 23S rRNA, 34 proteins (L1-L34), and a catalytic subunit
11
Q
E. coli 30S SU contains
A
16S rRNA, 21 proteins, ensures proper tRNA/mRNA base pairing
12
Q
Eukaryotic cytoplasmic ribosomes are
A
Larger and contain more RNAs and proteins
13
Q
Ribozyme
A
Ribosome capable of acting as an enzyme
RNA only ribosome still catalyzes peptide bond formation
Henry Noller
14
Q
What are the characteristics of rRNA?
A
-Highly structured
-Contain many types of modified ribonucleotides that allow complex structure
15
Q
Translation Steps and order
A
Initiation (beginning), Elongation (middle), and Termination (end)
16
Q
Initiation
A
Ribosome gets together with the mRNA and the first tRNA so translation can begin
-Small subunit on mRNA binding site is joined by large subunit and aminoacyl-tRNA binds
17
Q
Elongation
A
Amino acids brought to the ribosome by tRNAs and linked together to form a chain
-Ribosome moves along mRNA, extending protein by transfer from peptidyl-tRNA to aminoacyl-tRNA
18
Q
Termination
A
The finished polypeptide is released to go and do its job in the cell
-Polypeptide chain is released from tRNA, and ribosome dissociates from mRNA
19
Q
What are the key ingredients of initiation?
A
-A ribosome (which comes in two pieces, large and small)
-An mRNA with instructions for the protein we’ll build
-An “initiator” tRNA carrying the first amino acid in the protein, which is almost ALWAYS methionine (Met)
20
Q
What happens to the key ingredients during initiation?
A
The pieces must come together to form initiation complex
21
Q
Initiation complex
A
Ribosome, mRNA, and “initiatior” tRNA
Molecular setup needed to start making a new protein
22
Q
What two important events must occur before translation initiation can take place?
A
1) Generate supply of aminoacyl-tRNAs
-AAs must be covalently bound to tRNAs
-Process of bonding tRNA to AAs is called tRNA charging
2) Dissociation of Ribosomes into their subunits
-Cell assembles the initiation complex on the small ribosomal subunit
-Two SUs must separate to make assembly possible
23
Q
tRNA structure
A
-All share common secondary structure represented by cloverleaf
-Four base-paired stems define three stem-loops: D loop, Anticodon loop, T loop
-Acceptor stem is site to which AAs are added in the charging step
24
Q
Specificity of tRNA
A
All tRNA have similar structure, but sequence and modification within the tRNA allow for specificity in:
-AA charging
-Anticodon loop binding mRNA
-Acceptance into ribosome
25
What does the shape of tRNA do?
Maximizes stability
26
What does the anticodon base-pair with in tRNA?
The corresponding codon in mRNA
27
What unique base-pairing does tRNA utilize?
Wobble Base-pairing
-3rd position have the least influence -- wobble position
-Same tRNA can base pair with multiple codons via nonWatson-Crick base pairs
28
Wobble Hypothesis
-Codon-Anticodon recognition involves wobbling
-Multiple codons that encode the same AA most often differ at the third base position
-Pairing between first base of anticodon and third base of codon can vary from standard Watson-Crick base pairing according to specific wobble rules
29
Most of the time, existing nts in tRNA are what instead of replaced? What does this do for the structure of tRNA?
-Modified
-Regulate stability and recognition by proteins and rRNA
30
tRNA Charging
AA attached by ester bond between its carboxyl group and 2' or 3' hydroxyl group of terminal adenosine of tRNA
31
Aminoacyle-tRNA synthetases
-Join AAs to their cognate tRNAs
-Differences in tRNAs recognized by synthetases to charge with proper AA
- i.e. D loop nt seq or mods may be unique enough to allow only one synthetase to recognize it
- i.e nt adjacent to acceptor stem allow only that tRNA to fit into the synthetase
32
Initiator tRNA
-All initiation begins with AUG codon, but not ALL AUGs are initiation codons
- 2 Different tRNA recognize intiatior AUGs and internal AUGs
33
Initiator tRNA
-All initiation begins with AUG codon, but not ALL AUGs are initiation codons
- 2 Different tRNA recognize intiatior AUGs and internal AUGs-
34
N-formyl-methionyl-tRNA (tRNAfMet)
The aminoacyl-tRNA that initiates bacterial polypeptide translation
-Amino group of the methionine is formylated
-Met-tRNA = internal AUG
35
Initiation in simplified terms
-elongating ribosome (bacteria) = 70S
-At termination, the 2 SUs separate
-SUs reassociate to 70S during initiation
36
Ribosome: "Where do I start?"
-Prokaryotes: based on sequence
-Eukaryotes: Based on structure
37
Determining Ribosome Binding Site on mRNA in Prokaryotes
-Start codon AUG on an mRNA is chosen as initiation codon due to presence of SHINE DALGARNO SEQUENCE upstream of translation site (prokaryotes only)
38
Shine Dalgarno Sequence
- Ribosome binding site
-Base pairs with 16S rRNA ( in small subunit)
-Upstream of the translation site (in prokaryotes only)
39
Why use Shine-Dalgarno sequences?
-Many AUG sequences in mRNA
-Bacterial RNAs are often polycistronic (cistron is coding region), so one bacterial mRNA can contain the coding sequence for several genes
- SD sequence marks start of each coding sequence, letting the ribosome find the right start codon for each gene.
40
What does initiation in bacteria need?
30S subunits and Accessory factors
-Initiation of translation requires separate 30S and 50S ribosome SUs
-Initiation also requires INITIATION FACTORS which bind to 30S SUs
41
Initiation factors
IF-1, IF-2, and IF-3
42
Bacterial IF-3
-Binds to free 30S SU to inhibit binding of 30S to 50S
-30S SU carrying initiation factors binds to an initiation site on mRNA to form an initiation complex
-IF-3 MUST be released to allow 50S SUs to join the 30S-mRNA complex
43
What is use of fMet-tRNA controlled by?
IF-2 and the ribosome
44
IF-2
Control use of fMet-tRNA
-Binds initiatior fMet-tRNAf and allows it to associate with the 30S SU
45
Prokaryotic Initiation Steps
1) Dissociation of 70S into 50S and 30S
2) IF1, IF2, and IF3 bind cooperatively to 30S SU
3) IFs direct binding of mRNA (at SD) and initiator tRNA, forming 30S initiation complex
4) IF1 and IF3 leave
5) IF2 hydrolyzes GTP and then leaves
6) 50S associates with 30S-- forms active 70S complex
46
Eukaryotic Translation
- No SD sequences to indicate the start codon
-The ribosome recognizes the 5' cap
-Transcripts are largely monocistronic
-5' UTR is short
47
Scanning Model of Initiation
-Eukaryotic 40S ribosomal SUs locate start codon by BINDING 5' CAP and scanning downstream to find the 1st AUG in a favorable context
-Kozak's Observations (Rules) not always correct
- 5-10% of the time, most ribosomal SUs bypass 1st AUG scanning for a more favorable one - Leaky Scanning
48
Kozak's Observations
-Internal AUGS not used
-Initiation does not occur. ata fixed distance from 5' end (5'UTR varying length)
-Most of the time, first AUG downstream from cap is initiator
-Cap promotes translation
49
Gist of Eukaryotic Initiation
1) tRNA carrying methionine attaches to the small ribosomal subunit
2) Together, they bind to the 5' end of the mRNA by recognizing the 5' GTP cap
3) They "walk" along the mRNA in the 3' direction, stopping when they reach the start codon (often but not always the first AUG)
50
Leaky Scanning
-When Kozak's rules don't apply
-5-10% of the time, most ribosomal SUs bypass 1st AUG scanning for a more favorable one
51
What allows the right AUG to be chosen in leaky scanning?
AUG needs to be in CONTEXT
52
AUG in CONTEXT
-A of AUG as -1 position
- Purine in -3 position
-G in +4 position
53
Effects of mRNA secondary structure
-Secondary structure near the 5'-end of an mRNA can have either positive or negative effects on start codon selection
-Hairpin just past AUG can force a pause by ribosomal SU and stimulate translation
-Very stable stem loop between cap and initiation site can block scanning and inhibit translation
54
What can "hiding" a start codon in a STABLE hairpin do?
Prevent recognition of THAT AUG, leading to a more downstream AUG being used
55
What does selecting different start sites make?
Different isoforms
56
eIFs
Eukaryotic initiation factors ; similar to prokaryotes
57
Eukaryotic Initiation factors
-Initiator tRNA and specific initiation codon
-- Met on initiator tRNA is NOT formylated
-tRNAi-Met
58
What is required for ALL stages of initiation, including binding the initiator tRNA, attachment of the 40S SU to mRNA, joining of 60S SU, and movement of the ribosome?
Initiation factors
59
43S preinitiation complex
eIF2, eIF3, Met-tRNAi, eIF1, eIF1A
60
eIF2
Binds Met-tRNA to ribosomes (similar to prokaryotic IF2)
61
eIF1 and eIF1A
Aid in scanning to initiation codon
62
eIF3
Binds to 40S ribosomal subunit, inhibits reassociation with 60S SU (similar to prokaryotic IF3)
63
eIF4
Cap-binding protein allowing 40S SU to bind 5'-end of mRNA
64
eIF5
Encourages association between 60S ribosome SU and 48S complex
65
eIF6
Binds to 60S SU, blocks reassociation with 40S SU
66
Three parts of the eIF4F Cap-Binding Complex
1) eIF4E, has actual cap-binding activity
2) eIF4A: RNA helicase, ATP-dependent unwinding of hairpins found in the 5'-leaders of eukaryotic mRNA
3) eIF4B: has RNA-binding domain, can stimulate binding of eIF4A to mRNA
67
eIF4G
-Scaffold protein capable of binding to other proteins including: eIF4E, eIF3, and PAB1
- Recruits 40S SUs to mRNA and stimulates translation
68
Viral RNAs do what to eukaryotic initiation factors ?
Hijack them to facilitate translation of viral RNA
69
Internal Ribosome Entry Site (IRES)
A eukaryotic mRNA sequence that allows a ribosome to initiate polypeptide translation WITHOUT migrating from the 5' end
70
What does a dicistronic assay indicate with PV?
That internal ribosome entry site (IRES) activity is present in the PDCD8 5' UTR
71
What does Polio virus do to translation?
Shuts down host mRNA translation, but translated its own RNA using IRES
72
Why control initiation on a translational level given the amount of control at transcriptional and post-transcriptional level?
SPEED
New gene products can be produced quickly
Simply turn on translation of pre-existing mRNA
-Valuable in eukaryotes, transcripts relatively long, and take correspondingly long time to make
Most control of translation happens at the initiation step
73
Translation Elongation
-similar in bacteria and eukaryotes
- Three Sites of the Ribosome: E site, P site, and A site
74
In what direction is a polypeptide synthesized?
3' to 5'
75
In what direction does the ribosome read the RNA?
5' to 3'
76
What is the nature of the genetic code that dictates which amino acids will be incorporated in response to the mRNA?
The anticodon sequence
77
E Site
-Elongation site on ribosome
- Exit site
-Where tRNAs leave the ribosome
78
P site
Elongation site on ribosome
-Pepidyl-tRNA site
-Holds the tRNA carrying the growing polypeptide chain
79
A site
-Elongation site on ribosome
-Aminoacyl-tRNA site
-Where incoming tRNA binds
80
Elongation Cycle (three steps)
1) Aminoacyl-tRNA to the ribosomal A site (Ef-Tu)
2) Peptide bond between peptide in P site and newly arrived aminoacyl-tRNA in the A site (peptidyl transferase); Lengthens peptide by one amino acid and shifts it to the A site
3) Translocates the growing polypeptidyl-tRNA with its mRNA codon to the P Site ( EF-G)
81
EF-Ts are involved in what?
The first elongation step
-T = transfer
-Transfers aminoacyl-tRNAs to the ribosome
-Actually TWO different proteins: Tu, u = unstable and Ts, s= stable
82
EF-G
Participates in the third step of elongation;
G, GTPase activity
83
EF-Tu
*elongation factor
-Monomeric G protein whose active form (bound to GTP) binds to aminoacyl-tRNA
84
EF-Tu-GTP-aminoacyl-tRNA Complex
Binds to the ribosome's A site
85
What triggers Ef-Tu to hydrolyze GTP and what happens after that?
tRNA hydrogen binding to mRNA triggers Ef-Tu and after hydrolyzation, shifts tRNA in the ribosome so it can accept the amino acids chain
86
Ef-Tu bound to GDP is what?
Released from ribosome
87
EF-Ts does what?
Regenerates GTP to allow recycling of EF-Tu to EF-Tu-GTP
88
Elongation Step 1:
Codon Recognition
-Incoming aatRNA to A Site
-H bonds from between the mRNA codon and tRNA anticodon
-Energy required
89
When does proofreading for translation occur?
In the first step of elongation
90
Ef-TU-GTP hydrolysis and release are what?
slow; peptide bond formation is slow.
91
What does the slowness of EF-TU-GTP hydrolysis allow?
Proofreading; time for incorrect tRNAs to leave the A site before incorporation of the wrong AA into the polypeptide
92
Weakness of incorrect codon-anticodon base pairing ensures what?
That Dissociation of tRNA from mRNA/ribosome complex occurs more rapidly than peptide bond formation
93
How is speed of translation related to accuracy?
Inversely; faster translation = more errors
94
Elongation Step 2
The polypeptide chain
- 50S SU has peptidyl transferase activity, provided by rRNA ribozyme
- Nascent PP chain is transferred from peptidyl-tRNA in P site to aminoacyl-tRNA in A site
-Peptide bond synthesis generates deacylated tRNA in P site and peptidyl tRNA in A site
95
Difference between ribosome and ribozyme?
The ribosome is a ribozyme but EACH individual SU/rRNA on its own is NOT a ribozyme
96
Elongation Step 3
Translocation Moves the Ribosome
-Ribosomal translocation moves the mRNA through the ribosome by three nucleotides
-Translocation moves deacylated tRNA into the E site and the peptidyl-tRNA into the Psite and empties the A site
-Process requires elongation factor EF-G which hydrolyzes GTP after translocation is complete
97
Elongation factors bind alternatively to Ribosome
-Once A site is empty, EF-Tu can bring another charged tRNA
-Ribosomes cannot bind EF-G and EF-Tu simultaneously
98
Elongation Summary
-Translocation requires EF-G, whose structure resembles the aminoacyl-tRNA-EF-Tu-GTP complex
-Binding of EF-Tu and EF-G to the ribosome is mutually exclusive --> EF-G blocks EF-Tu
- Translocation requires GTP hydrolysis, which triggers a change in EF-G, which, in turn, triggers a change in ribosome structure
99
Antibiotics and toxins often act on what that affects translation?
The ribosome
100
Puromycin
Antibiotic that inhibits translation
101
How does puromycin work?
Ribosome treats Puromycin similar to aminoacyl-tRNA and allows polypeptide to transfer from the P site to the Puromycin in the A site; once Puromycin moves to the P site it initiates translation termination
102
Translational Termination
*elongation cycle repeats to grow polypeptide
-Eventually, ribosome encounters STOP codon
103
Stop Codon
-Signals time for last step
-No tRNA for stop codon
-Release factor binds to A site instead
104
What are termination codons recognized by?
Protein Release Factors
-NOT BY aminoacyl-tRNAs
105
Protein Release Factors
-Proteins that have a conformation mimicking tRNA: bind to A site and recognize stop codon; cannot accept a polypeptide chain, so chain is released
-Prokaryotic termination factors: RF1 and RF2
106
Peptide Release
-Prokaryotic and Eukaryotic RF involved in this step
107
RF1
Recognizes UAA and UAG
108
RF2
Recognizes UAA and UGA
109
RF3
Is a GTP-binding protein facilitating binding of RF1 and RF2 to the ribosome
110
RRF
Ribosomal Release Factor
Separates Ribosomal subunits
111
eRF1
Recognizes all three termination factors
112
eRF3
Ribosome-dependent GTPase helping eRF1 release the finished polypeptide
113
Release of Ribosomes from mRNA
-Does not happen spontaneously after termination
-Recycling factors help release mRNA, tRNA, and ribosomal SUs from complex
-Prokaryotic ribosomes use RRF and EF-G
Eukaryotes use different proteins that resemble RRF
114
Once ribosomal subunits are released they associate with what?
Initiation factors (i.e eIF1 and 3, and IF3) and are free to start another round of translation
-The same 2 SUs do not come back together (random)
-More efficient translation termination, the more efficient translation
115
Aberrant Termination (euk)
-Pre-mature stop (nonsense-mediated decay)
-No Stop (non-step mediated decay)
-Stalled ribosome (no-go decay)
116
What is quality control of mRNA translation performed by?
Cytoplasmic Surveillance Systems
117
Exon Junction Complexes (EJCs)
Involved in non-mediated decay
- recognition of a termination codon as PREMATURE involves EJCs (downstream and in mammals) and 3' UTR structure or length
118
Pioneer Round of Translation
-First translation event for a newly synthesized and exported mRNA
119
NMD Triggers what?
Decay of the mRNA, degradation of the nascent polypeptide, and removes the ribosome
120
NMD is stimulated by what?
Lef-over EJC
***Premature stop codons in the last exon will not be recognized by NMD****
121
Non-Stop Decay (NSD)
Targets mRNAs lacking an in-frame termination codon
122
Eukaryotic Ribosomes stalled at the end of the Poly(a) tail contain what?
0-3 nt of Poly(A) tail
-Stalled ribosome state is recognized by carboxyl-terminal domain of protein called Ski7p
-Ski7p associates tightly with cytoplasmic exosome
-Non-stop mRNA recruit Ski7p-exosome complex to the vacant A site
-Ski complex is recruited to the A-site
123
What degrades the RNA in NSD?
The exosome, positioned just at the end of non-stop mRNA; aberrant PP is presumably destroyed
124
No-Go Aberrant Termination
-When ribosomes are stalled on an mRNA (e.g. secondary structure)
-mRNA decay begins with an endonucleolytic cleavage near stalled ribosome
-Exonucleases degrade cleaved mRNA
-Provides another potential means of post-transcriptional control by selective degradation of mRNAs
125
Dominant and Recessive describe what?
Phenotypes; the mutation contributes to the phenotype
126
Recessive Phenotypes
-Occur with loss of function mutations
-Loss of function usually means that the protein is no longer made
127
Dominant Phenotypes
-Occur when protein is made, but it's presence disrupts the process
-Think about receptor that usually works as a dimer. If one SU cannot bind the ligand, it doesn't matter if the other one can, both are now inacvtive
128
Mutations of the same gene can cause?
Moderate or Severe phenotypes
129
Maternal-Zygotic Transition
mRNAs present in early development derived from oocyte (maternal)
-mRNAs stored in a translationally repressed state until needed
-Upon necessity (signal), translation of specific maternal mRNA can resume
-Requires A LOT of regulation
130
In the blastula, zygotes transcription ....
Turns on and maternal mRNAs are degraded
-This shifts gene expression responsibilities to zygote
131
Repressing translation through eIF4 binding is what?
A point of translation regulation
132
Regulating translation during the cell cycle:
-4E-BP production turned on at start of mitosis by growth factor stimulation
-Helps reduce protein synthesis while cells are dividing
133
eIF2 alpha-SU is a target of what and how?
Translational control
-Phosphorylation inhibits its ability to GTP --> GDP
-Heme-starved reticulocytes activate HCR: heme -controlled repressor (HCR): phosphorylates eIF2
-Interferon and dsRNA activate DAI: dsRNA activated inhibitor: viral protection mechanism that leads to phosphorylation of eIF2
134
Repression of translation from individual mRNA by mRNA-binding protein
-Ferritin mRNA (codes for iron storage protein) translation is subject to inductio by iron
-Only affects transcripts with an IRE -- selective not global translational regulation
135
let-7 miRNA
Shifts polysomal profile target mRNAs in human cells toward smaller polysomes (blocks translation initiation in human cells)
136
What type of translation initiation is not affected by let-7 miRNA?
Cap-dependent due to presence of IRES, or a tethered initiation factor
-miRNA blocks binding of eIF4E to cap of target mRNAs in human cells
137
What can be an indication of global translational activity?
A polysome profile
138
NSP14: Sars-CoV-2 Viral Protein
-Transfection of cells with NSP14 affects global translation
-M2 mutant of NSP14 restores translation
-Other results show that NSP14 does not affect amount of mRNA present
139
Post-Translational Regulation
-Folding is a co-translational event occurring as nascent PP is being made: proteins must fold properly, membrane proteins must be inserted into membrane; ribosome attached to ER and inserts in the membrane there; ribosomes often translate near location where protein will be used/processed
-Most newly-made PPs do not fold properly alone: require folding help from molecular chaperones
