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1. MOLECULAR CELL BIO > deborah (L6-7) > Flashcards

deborah (L6-7) Flashcards

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1
Q

compensating base changes

A

If one of the bases changes (A to G), another base changes to balance it out (G to A somewhere else)

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2
Q

tRNA’s 3 binding pockets

A

A (acceptor site of codon directed binding of incoming amino acid tRNA)
P (petidyl site, holds codon directed peptidyl tRNA)
E (exit site, not associated with mRNA)

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3
Q

the P site of the tRNA

A

–> where transfer occurs
Center is where the peptidyl transferase site sits (PT in purple) - surrounded by rna with the proteins on the edges
The proteins are too far away to catalyse so it is a ribozomal activity

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4
Q

isoaccepting tRNAs

A

Trnas are being charged with one amino acid only, but they can recognise different codons

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5
Q

Aminoacyl trna synthetases

A

enzymes that catalyse the transfer of the amino acid onto a trna to charge them. they show specificity for the tRNAs they charge, and the correct interaction is with cognate tRNAs

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6
Q

TψC loop

A

7 unpaired bases

involved in binding to the ribosome A site

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7
Q

3’ end of the tRNA

A

has an invariant ACC end
4th base can change though
this end recognises and binds to the amino acid

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8
Q

STEMS of the tRNA

A

stems between loops (H bonds)
gives structure
closely controls the size and composition of it

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9
Q

tertiary structure of tRNA (bend and flexibility)

A

Distance between the anticodon and the amino acid is 70 A
Bends in the structure are formed by the T and D loops
Areas where you don’t have any H bonds gives flexibility for their functions (binding of amino acid and binding of anticodon to codon)

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10
Q

shared reactions of all tRNAs

A
  • interaction with elongation factor except initiator tRNA
  • binding to the ribosome A site
  • CCA terminal addition
  • invariant modifications to bases
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11
Q

unique reactions of individual tRNAs

A

amino acylation by synthetases

  • codon anticodon interaction
  • recognition of initiator (fmet tRNA of bacteria) by initiation factor
  • recognition of initiator by transformylase
  • unique base modifications
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12
Q

tRNA charging by aminoacyl-tRNA synthetases

A
  • specific amino acid and ATP bind to the aminoacyl tRNA synthetase
  • amino acid is activated by the covalent binding of AMP and pyrophosphate is released
  • the correct tRNA binds to the synthetase. the amino acid is covalently attached to the tRNA. AMP is released
  • the charged tRNA is released
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13
Q

aminoacyl tRNA synthetases classes

A

they are a diverse group of enzymes
40 to 100kDa in size
may be monomeric, diimeric or tetrameric

2 general groups:

  • class I enzymes (contains 10 enzymes, contacts tRNA at minor groove of the acceptor stem and anticodon, N terminal has subunits aBaB)
  • class II enzymes (contains 10 enzymes, contacts tRNA at major groove of the acceptor stem and anticodon, central has a B surrounded a subunit)

the 2 classes recognise different faces of the tRNA molecule, and the CCA arm adopts different conformations with the 2 classes.

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14
Q

identity elements

A

features of individual tRNAs which are recognised by their cognate synthetase.
they lie in th eanticodon bases most of the time, others in other areas of the tRNA loop.

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15
Q

proofreading

A

occurs at 2 stages (so called double sieve)

  • by hydrolysis of the ester bond of an incorrect aminoacyl-AMP intermediate triggered by the binding of the cognate tRNA
  • by hydrolysis of the ester bond of a miss matched aminoacyl-tRNA
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16
Q

editting sites of aminoacyl-tRNA synthetase

A

called a hydrolytic site
they possess this in addition of the acylation site
usually, the acylation site rejects an amino acid if it is larger than the cognate aa, due to insufficient room
the editing site hydrolyses aminoacyl-tRNAs which are smaller than the cognate aa

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17
Q

CCA arm function

A

it is flexible to move the amino acid between the activation site and the editing site
if it fits well into the editing site, the aa is removed by hydrolysis

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18
Q

colicin E3

A

protein that inhibits growth of bacterial cells that lack the Col plasmid. it acts by cleaving rRNA 50 nucleotides from its 3’ end.
the cleaved fragment has a sequence complementary to the ribosome binding site of the mRNA
so ribosomes cannot initiate protein synthesis

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19
Q

how do antibiotics target the transferase activity in ribosomes

A

they bind directly to the RNA which means that it’s easy for the bacteria to make a single base change and make it resistant

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20
Q

dyphteria toxin

A

produced by pathogenic strains of cyanobacterium diphteriae
highly toxic
acts catalytically on elongation factor 2 (EF-2), the eukaryotic homologue of EF-G
all EF-2s contain a posttranslationally modified histidine residue called diphtamide
the toxin transfers ADP ribose from NAD+ to the imidazol ring
COMPLETELY INHIBITS TRANSLOCATION

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21
Q

puromycon

A

resembles the aminoacyl tRNA.
it enters the vacant A site without the involvment of EF-Tu.
it’’s a substrate for peptidyl puromycin (which is not anchored to the A site, so it dissociated from the ribosome, resulting in premature chain termination)

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22
Q

features of prokaryotic mrna

A

PROKARYOTIC MRNA
Have the ribosome binding site right at the start of the 5’end - called the shine-dalgarno sequence (early 70s)
The start signal is AUG (or GUG) preceded by several bases that pair with 16S rRNA.
the purine rich region (shine dalgarno) is complementary to initiator sites of mrna
Ribosome can’t link on mRNA without this sequence. Done by mutagenesis

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23
Q

e coli contains 2 types of tRNA methionine (the trna that binds to the start codon ATG, which codes for methionine)

A

tRNAf^met : Met residues attached to this are formylated
Initiate polypeptide chains only
Recognises AUG and GUG (GUG internally codes for valine)

tRNAm^met : Met residues are only attached, not formylated.
Recognises the codon AUG only
Used as a source of internal met residues.

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24
Q

Chromatography of the high salt supernatant revealed??

A

Chromatography of the high salt supernatant revealed that three proteins (factors) are necessary for initiation. Termed IF-1, IF-2 and IF-3.

Test that proved this - take 30s subunit, wash with high salt, denatures most proteins from 30s subunit, cannot translate protein.

25
Q

function of initiation factors in the translation initiation in prokaryotes using the 30S initiation complex

A

IF 3 prevents the activation of the whole ribosome
IF 1 sits on the side of the 30S, modifies the A site, so first fMent-tRAN cant interact to the A site, only the P site (this is where the initiation differs to the rest of the elongation that codon binds directly to the P site not A site)
IF2 is the biggest protein. Reacts directly with tRNA. GTP is loaded onto protein, which is now charged for the activation step, it interacts with mRNA, GTP is hydrolysed, allows 50S subunit to join together

26
Q

function of initiation factors in the translation initiation in prokaryotes using the 70S initiation complex

A

Once you have the dissociation of the IF1 and IF3, you have the incoming mrna, the whole thing is put together, all the initiation factors are released, we now have the ribosome with the mRNA through the center of it, primed with the first methionine tRNA in place, ready to start to make protein changes
Here streptomycin inhibits the protein synthesis

27
Q

the 3 steps of elongation reactions of protein synthesis

A
  1. Codon-directed binding of the incoming aminoacyl-tRNA.
  2. Peptide bond formation
  3. Translocation (movement) of the ribosome along the mRNA in a 5’ 🡪 3’ direction by the length of one codon.
28
Q

LEARN MECHANISM OF TRANSLOCATION IN ELONGATION

A

LECTURE 7 SLIDE 10

29
Q

role of elongation factor and its structure

A

Binding of the incoming aminoacyl-tRNA requires a soluble supernatant factor, elongation factor T (EF-T) and GTP

EF-T is composed of two polypeptides:

  • EF-Tu 45kDa, heat unstable
  • EF-Ts 30kDa, heast stable
30
Q

EF-Tu role

A

EF-Tu is very abundant (~20 mols per ribosome). Most aa tRNAs in the cell are already complexed with EF-Tu → Protection of the bond from water until it reaches the ribosomal pocket for it to do the peptidyl transferase

EF-Tu does not react with fmet-tRNAmet explaining why this is not bound during elongation (because this elongation factor doesn’t recognise it and we can’t use for anything other than the initiation)

When bound to EF-Tu the labile ester bond between the tRNA and amino acyl residue is protected from hydrolysis.

31
Q

sites of the ribosome

A 
A/T: site for the aa-tRNA bound to EF-Tu
P: peptidyl site
E: Exit site
dc: decoding centre
A: aminoacyl site
GAC: GTPase-associated centre
32
Q

decoding region function

A

Movement as the EFTU protected tRNA comes in, you have the decoding region - where it is checking that it is the codon that is on the mRNA correctly matched with the anticodon of tRNA

  • if not it is chucked out for another one to come in
  • if it is right, then get the release and the shift in the shape, so rather than it being a distortion, the incoming tRNA is upright and closely associated with the peptidyl site tRNA. The 2 are close enough for the transfer of the peptide chain across)
33
Q

Proofreading role of EF-Tu in translation

A

It takes a few milliseconds for GTP hydrolysis to occur, and a few more milliseconds for EF-Tu-GDP release. Only after EF-TU-GDP release can peptide bond formation occur. These intervals provide the opportunity for a weakly bound, non-cognate aa-tRNA (incorrect codon-anticodon match) to dissociate from the ribosome.

34
Q

mechanism of translocation

A

Movement of the ribosome along the mRNA in a 5’ 🡪 3’ direction by the length of one codon
This is effected by the ELONGATION FACTOR G/GTP (EF-G/GTP) in bacteria or similarly EF-2 in eukaryotes

35
Q

structure of EF-G

A

The structure of EF-G is very similar to the EF-Tu

The structure of the N-terminal region of EF-G (red) mimics the tRNA

Structure of EFTu with tRNA in it, is similar to the EFG structure (which is a tertiary protein) - this is molecular mimicery (where the G is all protein, but the chain and positioning of charges are important and very similar to the positions and shapes and charges between the tRNA and EFTu, this means that they can fit into the same pockets)

36
Q

translocation step of EF-G/GTP binding

A

EF-G/GTP binds to the pre-translocation ribosome at a site including L7/L12, L11 and the sarcin/ricin loop of 23S rRNA.

The tRNA-like domain interacts with the 30S subunit close to the partial A site.

GTP hydrolysis induces a conformational change in EF-G, forcing its arm deeper into the 30S subunit, which forces the peptidyl tRNA from the A site into the P site, carrying the mRNA and deacylated tRNA with it. RESULT:-ribosome moves along the mRNA by length of one codon

37
Q

probability of forming a protein with no errors

A

Note: the probability of forming a protein with no errors depends on n, the number of amino acids, and ɛ, the frequency of insertion of a wrong amino acid: p = (1-ɛ)n.

38
Q

ELONGATION MECHANISM

A

LECTURE 7 SLIDE 18

39
Q

TERMINATION MECHANISM

A

LECTURE 7 SLIDE 20

40
Q

initiation of protein synthesis in eukaryotes

A

AUG is almost always used as the initiation codon.

A special initiator tRNA, tRNAimet is used as the initiator.
(Does not become formylated). tRNAmmet is used to insert internal methionines.

The “first” AUG is usually use for initiation (~90%).
Context dependent.
There is no ribosome binding site as in prokaryotes

41
Q

role of eIF-4E

A

You have a cap on the 5’ end on the mRNA
Recognised by initiation factor IF4E
form the CAP-eIF4E complex

42
Q

role of eIF-4A

A

eIF-4A is an ATP-dependent RNA helicase that removes secondary structure near the 5’ end.
Needed for scanning movement of the 40S subunit along the mRNA

43
Q

role of eIF-4G

A

eIF-4G is a “scaffold” subunit
links together the initiation complex .
Cleavage by protease results in inhibition of initiation.

IF4G it is necessary to hold the messenger RNA in the right shape
it interacts with the poly-A binding protein
that allows the the mrna to form a circle and allows the the polysome formation

44
Q

INITIATION OF PROTEIN SYNTHESIS IN EUKARYOTES

A

LECTURE 7 SLIDE 25-27

45
Q

IRES

A

Internal Ribosome Entry Sites

The vast majority of eukaryotic mRNAs are translated through the ribosome scanning mechanism
An alternative mechanism is internal ribosome entry
The mRNAs lack a 5’ cap, and translation is initiated at internal ribosome entry sites (IRES)
IRESs have a complicated tertiary structure and bind 40S subunits in close proximity to an AUG codon

46
Q

2 types of internal ribosome entry

A
  • the polysome has a cap binding and the mrna strand as a closed loop
    used 90% of the time
  • the 4G he is still having the scaffolding with the polyA binding protein but the cap and the rest of the initiation complex is missing
    it is also a stem-loop structure
    used 10% of the time
47
Q

Regulation of protein synthesis during the cell cycle

A

G2/M transition

  • decreased by 75% total protein synthesis.
  • caused by cell cycle-dependent dephosphorylation of eIF-4E (Cap binding)
  • decreased affinity of ribosomes for the cap

IRES-containing RNAs are unaffected
- relative IRES translation rates increase in M phase.
- in apoptosis, eIF-4G is cleaved: caspase 3
all translation decreases

48
Q

translation in apoptosis

A

in apoptosis, translation decreases, but there is still some translation happening for the proteins that are necessary for the apoptosis to occur

49
Q

picornavirus

A

shuts off ~ 90% of host protein synthesis
Gives virus maximum competition with the host.
Viruses use IRES, so cell cycle independent

These viruses have a protease that cleaves the 4E. so it cannot bind or give initiation by CAP binding anymore, so the only translation that can occur in the cell are infected by the picornavirus and used in the IRES part.
all the picornaviruses own genes are IRES structured mRNAs so it means that the virus has max competition for all the translation systems in the cell (so that they can then replicate themselves and they aren’t affected by the state of the host cycle, and are cell cycle independent)

50
Q

Structure of a eukaryotic release factor

A

Mimics the structure of the amino acid
acceptor stem of tRNA (CCA terminus)

The sequence Gly Gly Gln at the tip of the acceptor stem binds a water molecule, which is carried into the peptidyl transferase center of the ribosome. This water hydrolyses the ester bond of peptidyl tRNA releasing the polypeptide

51
Q

Release of polypeptide chains

A
  • The water molecule bound to the release factor hydrolyses the ester bond in the peptidyl tRNA, releasing the completed polypeptide.
  • NB. During normal chain elongation , water is excluded from the peptidyl transferase centre of the ribosome
  • Water bound by release factor - this attacks the tRNA that is holding on to the end of the polypeptide chain
  • the hydrolysis results in the release of the trna and both the polypeptide
  • Initiation factor protects the bonds in the AA until it reaches the ribosome
  • Usually water is excluded from ribosomes env to prevent it hydrolysing those bonds
52
Q

Translational control mechanisms (look at slide 35 for diagrams)

A

Regulation of the activities of initiation and/or elongation factors by phosphorylation (pro- and eukaryotes).

Blocking / opening of ribosome binding sites by reversible changes in secondary structure (prokaryotes).

Autogenous regulation. Protein product of a gene translation binds to ribosome binding site in mRNA, preventing initiation (prokaryotes).

Reversible binding of a repressor protein to a response element in 5’ UTR (eukaryotes).

Differential stability of mRNA

53
Q

Control of initiation by eIF2a phosphorylation

A

EIF2A is highly controlled by phosphorylation in eukaryotes
Cells sense env signals (like UV radiation or heat shock). Through cell surface receptors, you get initiation of phosphorylation cascades through diff kinases, that then act in eIF2 itself, to affect its activity
you also get the exchange factor that controls the position of the eif2 to between the GDP and GTP bounds. this can be also affected by phosphorylation

54
Q

Control of initiation by eIF4E phosphorylation

A 
the 4E (the bit that binds the cap is also regulated by phosphorylation)
Can be autoregulated (can be assimilation to initiate translation)
you also get for 4E binding proteins which is phosphorylation dependent
external stimuli on the cells will hit the receptors tyrosine kinases which will stimulate one of two cascades depending on which side is activated, to get increase or decrease in translation

Helps us design drugs for major diseases

55
Q

define autogenous control of ribosomal protein synthesis in E. coli

A

When rRNA is in short supply (e.g. when cells are nutrient limited) levels of free r-proteins increase.

One r-protein from each operon then binds to the polycistronic mRNA near to the ribosome binding site of one of the first genes of the operon. This prevents translation of this and the other downstream ORFs of the operon

This is called AUTOGENOUS (self limiting) control

56
Q

Regulation of translation of mammalian ferritin and transferrin mRNAs by iron-response element binding proteins

A

Ferritin is a cytosolic protein that binds iron ions and prevents accumulation of toxic levels of Fe2+/Fe3+.

However when Fe is limiting, ferritin poses a problem – it competes for Fe with iron-requiring enzymes.

Mammalian cells therefore modulate the synthesis of ferritin – expressed under excess Fe, repressed under Fe scarcity

The transferrin receptor (a cell surface protein responsible for Fe uptake into cells) shows reciprocal regulation of synthesis to that of ferritin

57
Q

Regulation of translation by iron response elements

both iron starvation and excess iron situations

A

Iron starvation:
Iron response element is bound to both the ferritin (the protein that mops up the excess iron in the cell) and to the transferrin (receptor that sits on the cell surface to allow iron ions to come into the cell)
Ferritin is shut down - anything that comes into the cell is needed by the cell to use to make preemptive enzyme
Transferrin is increased - more receptors on the cell

Excess iron:
Iron reacts with aconitase, changes its structure, it’s then released by both mRNAs, ferritin is now activated, excess iron is mopped up and held up by ferritin
In the case of the transferrin receptor, if there is more iron around, you don’t want to make more receptors to pull it in, signals are sent out for the mRNA to be degraded, and shuts down its production

58
Q

Eukaryote mRNA decay

A

Slow degradation process

Nearly all mRNAs subjected to poly (A) tail shortening

When tail < 30 A’s residues in length, Poly (A) binding protein is lost and 3’ end no longer associates with cap (decapping signals for a fast degradation of the mRNA)

This leads to decapping followed by degradation.

1. MOLECULAR CELL BIO (6 decks)

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