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Central Dogma exam 3 > Translation (I) > Flashcards

Translation (I) Flashcards

1
Q

What are the three stages of translation?

A

Initiation, elongation, and termination

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2
Q

What direction do ribosomes read mRNA in?

A

5’ to 3’

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3
Q

What direction does protein synthesis occur relative to the protein?

A

N-term to C-term

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4
Q

Where does translation begin?

A

With the AUG start codon
~10 nt downstream from a shine dalgarno sequence

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5
Q

What are polysomes?

A

-Ribosomes that are arranged on mRNAs like beads on a string to form assemblies (called polysomes)
-Active translation occurs on polysomes

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6
Q

How are ribosomes organized on mRNA?

A

Adjacent ribosomes are densely packed, with peptide exit channel facing the cytosol and staggered

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7
Q

After a ribosome clears the initiation site on an mRNA, what happens?

A

Another ribosome initiates at that site

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8
Q

What are the three sites of a ribosome?

A

aminoacyl (A), peptidyl (P), and exit (E)

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9
Q

How does the nascent peptide grow in a ribosome?

A

The nascent peptide grows by being transferred from the peptidyl-tRNA to the incoming aminoacyl-tRNA to form a peptidyl-tRNA with one more amino acids

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10
Q

Basic idea of polymerization in a ribosome

A

-In the P site is the growing chain of amino acids attached to a tRNA
-There is a new amino acid in the A site that has only one amino acid
-The N-terminal part of the amino acid in the A site attacks the c-terminal of the newest amino acid in the P site, which causes a peptide bond to form, the tRNA in the A site to be without any amino acids, and the peptide chain to be added to the top of the amino acid in the A-site
-After the formation of a peptide bond, the uncharged/deacylated tRNA in the P site moves to the E site to be released
-The outgoing tRNA is replaced by the newly formed peptidyl-tRNA from site A, effectively moving from the A site to the P site, permitting another round of peptide bond formation

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11
Q

What are the 5 stages of protein synthesis

A

-Activation of amino acids
-Initiation
-Elongation
-Termination
-Protein folding

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12
Q

What start codon is required for initiation in prokaryotes?

A

-AUG
-Typically codes for methionine (Met)
-The initial methionine is formylated (fMET)

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13
Q

What tRNAs (concerning Met) are used in translated?

A

-tRNA that recognizes the initial AUG: tRNA^fMet
-tRNA for internal Met: tRNA^Met

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14
Q

Formation of tRNA fMet and Met

A

-tRNA^fMet is aminoacylated by the same MetRS that charges tRNA^Met
-Both are charged with Met
-Met-tRNA^fMet is N-formylated after charging

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15
Q

What happens to fMet part way through translation?

A

-A deformylase hydrolytically deformylates the fMet residue
-This means that the N-terminus is removed from fMet
-In many proteins, the entire initiator Met is removed
-Mature E. coli proteins all lack fMet

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16
Q

How is the start site selected in prokaryotes (as mRNAs contain many AUGs?

A

-Non-start AUGs are often masked by mRNA secondary structure
-Interactions between mRNA and 16s rRNA (dominant mechanism) (includes Shine-Dalgarno sequence)

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17
Q

How does the Shine-Dalgarno sequence work?

A

-Ribosome binding site (a.k.a. Shine-Dalgarno sequence) on mRNA is centered ~10 nt upstream of start codon; consensus: AGGAGG
-Sine-Dalgarno sequence is complementary to the 16S rRNA
-Base pairing occurs between Shine-Dalgarno sequence and anti-Shine Dalgarno sequence in 16S rRNA permits ribosome to select proper AUG

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18
Q

Are sequence or distance important to Shine-Dalgarno sequence in prokaryotes?

A

Both sequence and distance from AUG are important

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19
Q

Why are IFs important for initiation to ribosomes in prokaryotes?

A

-Need Initation Factors (IFs) to bind mRNA (IF-1, IF-2, IF-3)
-IFs are temporarily associated with ribosome
-30S and 50S dissociate after one cycle of synthesis

20
Q

Steps of initiation in prokaryotes

A

-IF-1 and IF-3 bind 30S, block the A site and prevent reassociation with 50S respecively
—mRNA binds 30s, AUG is guided to the P-site by the Shine-Dalgarno sequence interacting with 16S
-A complex is formed between:
—mRNA
—IF-2 bound to GTP and fMet-tRNA^fMet
—IF-1, IF-3, and 30S
-IF-1 and IF-3 are released, the 50S subunit joins, IF-hydrolyzes its GTP and its release
-Result is formation of an mRNA-Ribosome-fMet-tRNA^fMet complex

21
Q

What is the result of the steps of initiation in prokaryotes

A

-Result is formation of an mRNA-Ribosome-fMet-tRNA^fMet complex
-fMet-tRNA^fMet occupies ribsome P site; unique for aminoacyl tRNA
-A site poised to accept next incoming tRNA

22
Q

Pactamycin

A

-Binds in the E site of the ribosome
-rRNA interacts with the Shine-Dalgarno sequence just past the E site
-Pactamycin binding causes movement of the E site triplet by 12.5 A, likely abolishing initiation

23
Q

Kasugamycin

A

-Binds the codon of mRNA: rRNA between the E site and P site
-Causes a bulge which leads to less efficient interaction of fMet-tRNA with AUG codon

24
Q

Edeine

A

Interacts with mRNA and fMet-tRNA to prohibit initiation

25
Q

What are the three overarching steps of chain elongation?

A

-Decoding: ribosome selects and binds an aminoacyl-tRNA
-Transpeptidation: peptide on P-stire tRNA is transferred to aminoacyl group in A-site via formation of a peptide bond
-Translocation: A-site and P-site tRNAs are transferred to P and E sites, and mRNA with its base-paired tRNA is ratcheted through the ribosome by one codon

26
Q

What are the three elongation factors used during chain elongation?

A

-EF-Tu
-EF-Ts
-EF-G

27
Q

What happens with Aminoacyl-tRNA binds A site without EF-TU

A

Aminoacyl-tRNAs can bind ribosomal A site without EF-Tu, but too slowly to support growth

28
Q

Abundance of EF-Tu

A

-EF-Tu is the most abundant protein in E. coli
-Likely all aminoacyl-tRNAS are bound by EF-Tu

29
Q

Steps of decoding

A

-Elongation Factor Ef-Tu, bound to GTP, binds an aminoacyl-tRNA, then binds the ribosome
-With hydrolysis of GTP, aminoacyl-tRNA is bound in a codon-anticodon complex to the ribosomal A site, and EF-Tu is released
-EF-Ts, acting as a GTP exchange factor (GEF), exchanges the GDP for GTP on EF-Tu to recycle it

30
Q

Chloramphenicol (Cam)

A

-Inhibits bacterial peptidyl transfer on large subunit
-Toxic at high doses because of effects on mitochondria
-Binds in exit tunnel close to A site, thought to interfere with ribosome interaction with aminoacyl-tRNA in A site

31
Q

Streptomycin

A

-Different effects at different doses
-Binds 30s subunit rRNA and protein
-Low dose: causes misreading the genetic code (can’t distinguish pyrimidines)
-High dose: inhibits chain initiation

32
Q

What does the 23S rRNA in the large subunit of the ribosome do?

A

-Catalyzes the reaciton by binding and aligning the tRNAs in the A and P sites in the proper orientations
-May also have addition roles in nucleophilic attack

33
Q

Important characteristics of transpeptidation

A

-Ribosome is a ribozyme (RNA acts as the catalyst)
-Does not require ATP because the ester linkage between nascent peptide and P-site tRNA is a high energy bond
-Involves nucleophilic attack of amino group on carbonyl of an ester
-H-bond between 2’-OH of P site tRNA and attacking group is critical

34
Q

What is an important bond in transpeptidation?

A

-H-bond between 2’-OH of P site tRNA and attacking amino group

35
Q

What is the current model for transpeptidation?

A

23S rRNA enhances rate of peptide bond formation by orienting its substrates in a “proton shuttle mechanism”

36
Q

Steps of translocation

A

-Ribosome moves one codon at a time toward the 3’ end of the mRNA
-EF-G-GTP shifts the anticodon of the dipeptidyl-tRNA from the A site to the P site
-This shifts the deacylated tRNA from the P site to the E site
-EF-G hydrolyses GTP and disassociates
-The A site is left open for the next round of aminoacyl-tRNA decoding
-Ribosome translocation requires EF-G and energy provided GTP hydrolysis

37
Q

What does EF-G mimic

A

-Structure of EF-G mimics the structure of the EF-Tu-tRNA complex
-EF-G binds the A site and displaces the peptidyl-tRNA

38
Q

When does translation end?

A

At stop codons

39
Q

What recognizes stop codons?

A

-Stop codons are the only codons that have no corresponding tRNAs
-Instead they are recognized by release factors (RFs)
—RF-1 recognizes UAA and UAG
—RF-2 recognizes UAA and UGA

40
Q

What happens when PXT tripeptide in RF-1 is swapped with SPF tripeptide in RF-2?

A

-This interchanges their stop codon specificities
-Suggests that these tripeptides mimic anticodons

41
Q

General overview of termination in prokaryotes

A

-On binding to the stop codon, RF-1/RF-2 induce the transfer of the peptidyl group from the tRNA to water (rather than to an aminoacyl-tRNA)
-This releases the completed strand

42
Q

Steps of termination in prokaryotes

A

-RF-1 or RF-2 recognize the stop codon
-Peptidyl group is transferred to water
-Ribosome Release Factor (RRF) and EF-G-GTP bind to the A site
-EF-G’s hydrolysis of GTP and association with IF-3 causes tRNAs to be released and 30S and 50S subunits to separate

43
Q

What is the evidence for proofreading

A

-The ribosome has an error rate of 10^-4, yet codon-anticodon interactions cannot fully explain accuracy beyond 10^-2
—Therefore evidence of proofreading, must be a two-step process
-Aminoacyl-tRNAs are selected by ribosome only according to codon-anticodon interactions (no selection based on aminoacyl group)
-DNA polymerases and AARS have a second proofreading site, ribosome does not

44
Q

What are the two mechanisms to ensure accurate codon-anticodon interactions?

A

-Codon-anticodon interactions are stabilized by interactions with conserved ribosomal bases
-Accommodation: subjects codon-anticodon to strain that only a correct pair can withstand

45
Q

Codon-anticodon interactions are stabilized by interactions with conserved ribosomal bases specifics

A

-These first rRNA nucleotides undergo a conformational change upon formation of correct codon-anticodon
-Incorrect codon-anticodon does not provide sufficient free energy to keep the tRNA bound to the ribosome; tRNA dissociates
-Correct interactions stimulate GTP hydrolysis of EF-Tu-GTP
-Ex: first codon-anticodon base pair is stabilized by 16S rRNAs A 1493

46
Q

Accommodation mechanism to ensure accurate codon-anticodon interactions

A

-tRNA initially binds only to 30S, not to 50S
-After EF-Tu hydrolyzes its bound GTP, protein disassociates
-tRNA must swing (~70A) into the A/A state
-This steps subjects codon-anticodon interaction to a strain that only a correct pairing can withstand

47
Q

How do tetracyclines interact with protein synthesis?

A

-Inhibit protein synthesis in bacteria by blocking the A site
-Bind to 16s rRNA
-Allow for initial screening of codon:anticodon interaction but does not allow for accommodation in the A/A site
-Also causes unproductive hydrolysis of GTP during these attempts, which drains energy

Central Dogma exam 3 (5 decks)

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