Protein Synthesis

Front 1

Process of protein synthesis

Back 1

a. mRNA enters cytoplasm, associates with ribosome
b. mRNA aligns itself on ribosome so first codon is read 1st
c. tRNA w/ complementary amino acid to 1st codon binds to 1st codon (tRNA with AA for 2nd binds to 2nd codon, etc.)
d. mRNA codons continue to be read toward carboxyl terminus
e. protein is released from mRNA and ribosomal particle disassembles

Front 2

Releasing factors

Back 2

a. Help terminate protein synthesis

Front 3

Signal peptide

Back 3

a. Amino acid sequence that signals where the protein will be targeted
b. 10-20 amino acids at amino terminal

Front 4

Structure and function of tRNA

Back 4

a. Folded into a cloverleaf structure
b. Accomplished by H-bonding between complementary bases

Front 5

tRNA regions

Back 5

a. 3’ end of molecule→ terminates in CCA→ amino acid binding site
b. TψC loop→ tRNA binds to ribosome
c. D loop→ contains a high percentage of dihydrouridine residues (recognized by tRNA synthetase)
d. Anticodon loop→ loop contains the anticodon which is complementary to the codon of the messenger RNA

a. 3’ end of molecule→ terminates in CCA→ amino acid binding site
b. TψC loop→ tRNA binds to ribosome
c. D loop→ contains a high percentage of dihydrouridine residues (recognized by tRNA synthetase)
d. Anticodon loop→ loop contains the anticodon which is complementary to the codon of the messenger RNA

Front 6

Possible functions of rare or minor bases in tRNA

Back 6

a. Disallow base pairing at certain points in order to form cloverleaf structure
b. Disallow unwanted base pairing with the mRNA
c. Protect the tRNA molecules from the action of ribonucleases

Front 7

Isoacceptor tRNA

Back 7

i. Greater than 20 types of tRNA because of redundancy of amino acid code
ii. Recognize several different codons, but all recognize a single amino acid

Front 8

Base pairing

Back 8

i. Anti-parallel
ii. tRNA→ 3’-5’
iii. mRNA→ 5’-3’

Front 9

Wobble hypothesis

Back 9

i. Third nucleotide of the codon is degenerate
ii. U in anticodon can recognize A or G
iii. G in anticodon recognizes C or U
iv. I in anticodon recognizes U, C, or A

Front 10

Inosine

Back 10

i. Formed by adenosine deaminase after tRNA is formed on certain tRNAs

Front 11

Tertiary structure of tRNA molecules

Back 11

i. L-shaped

Front 12

Activation

Back 12

a. Involves coupling of an amino acid to its corresponding tRNA
b. Catalyzed by amino acyl tRNA synthetases
c. Ester bond between amino acid and tRNA molecules is a high-energy bond→ AA bound to 2’ or 3’ -OH group of ribose of terminal residue of tRNA
d. Activation requires equivalent of 2 ATPs

Front 13

Aminoacyl-tRNA synthetase

Back 13

i. Specific to tRNA and amino acid
ii. 20 tRNA synthetases
iii. Recognize multiple points on tRNA structure

Front 14

Error correction

Back 14

1. Occurs while tRNA is bound to the synthetase
2. Editing mechanism where correct tRNA is recognized and corrected by the synthetase itself
3. One protein per thousand will contain incorrect AA due to misacylation of tRNA

Front 15

Role of amino acid in recognition of anticodon for amino acid

Back 15

a. NONE
b. If an amino acid attached to tRNA is chemically altered, the altered form will be incorporated into the protein

Front 16

Ribosomes in initiation

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i. Both prokaryotic and eukaryotic ribosomes consist of 2 subunits
ii. Each subunit is composed of proteins and rRNA

Front 17

Initiation process

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i. Initiation factors dissociates the 70S (80S in eukaryotes) ribosomes into 50S and 30S (40S and 60S)
ii. tRNAf met (formyl methionine) is the initiation codon for prokaryotes→ binds to IF2-GTP (required for binding to 30S subunit)
iii. 50S reassembles with 30S and tRNAf met introduces the first amino acid→ binds to initiation codon at P site, IF2 is lost from complex with GTP hydrolysis

Front 18

IF 1, 2, 3

Back 18

1. IF1 and 3 bind to free 30S subunit
2. mRNA then also binds to 30S ribosome subunit
3. IF2 (GTP) binds to tRNAf met which binds AUG at the P site
4. IF2 is lost as GTP is hydrolyzed and 70S ribosome is formed and IF1 and 3 are lost

Front 19

e1F-4E

Back 19

mRNA cap binding factor which is essential for lining up the start codon on the ribosome

Front 20

A&P (E sites)

Back 20

i. P=peptide site
ii. A=acceptor site
iii. E=exit site for tRNA ejection

Front 21

Start codon recognition

Back 21

i. AUG in both pro and eukaryotes
ii. Protein synthesis begins by positioning AUG codon in P site with tRNAf met or tRNA met bound to it
iii. mRNA is translated starting at AUG at the 5’ end of mRNA

Front 22

Shine-Delgarno sequence

Back 22

1. AGGAG
2. Found between 5’ end of mRNA and initiation codon
3. Permits binding and proper orientation of mRNA with ribosome

Front 23

Eukaryotic start codon

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i. AUG nearest 5’ cap
ii. 1 per mRNA molecule
iii. Only one protein can be made per eukaryotic mRNA molecule→ monocystronic

Front 24

Kozac consensus sequence

Back 24

i. Determines relative efficiency of eukaryotic message translation
ii. Messages in close proximity to Kozac sequence will translate efficiently

Front 25

Elongation binding

Back 25

i. Aminoacyl tRNA possessing the anticodon to the codon in A site forms complex with EF-Tu and GTP
ii. Complex binds to ribosome such that aminoacyl tRNA is positioned at A site
iii. GTP of EFTu-GTP complex is hydrolyzed and EFTu is released

Front 26

Peptide bond formation

Back 26

i. Formed by nucleophilic attack of alpha-amino group of AA in A site on ester bond between AA and tRNA in P site
ii. Catalyzed by ribosomal RNA
iii. Energy fro synthesis from activated amino acid attached to tRNA

Front 27

Translocation

Back 27

i. Movement of ribosome one codon on mRNA with release of empty tRNA present in P site and transfer of petide-bearing tRNA from a to P site
ii. Requires elongation factor EFG and GTP hydrolysis
iii. A site becomes vacant and the next amino acid charged tRNA enters and binds
iv. On average, 10 EF-Tu-GTP amino acyl tRNAs are tried in the A site before the correct one matches

Front 28

Termination

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i. Termination codon reaches A site (UAA, UAG, UGA)
ii. Releasing factors RF1, RF2, and RF3 causes release of polypeptide chain from ribosome
iii. Releasing factors cause peptidyl transferase activity to switch to a hydrolase activity to release the completed polypeptide

Front 29

Polycistronic mRNA

Back 29

i. In prokaryotes, if AUG is not too far from terminal codon, 70S will not dissociate but will form a new initiation complex which will lead to translation of second protein
ii. One mRNA can code for several proteins and each one might be translated

Front 30

Polyribosomes

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a. As ribosome translates the mRNA, the 5’ end becomes exposed
b. Second initiation complex can form
c. Distance between ribosomes is about 100 nucleotides

Front 31

Activation energetics

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i. Synthesis of AA tRNA uses 2 ATP equivalents for each AA introduced to the protein
ii. Energy of tRNA-AA bond drives peptide bond formation

Front 32

Energetics of elongation and translocation

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i. 2 GTP/AA
ii. 1 GTP per residue at A site
iii. 1 GTP per translocation
iv. Energy used to change conformation of translation machine and not for forming new covalent bonds

Front 33

Prokaryotes v. eukaryotes-- initiation

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i. Initiation amino acid in eukaryotes is methionine
ii. Regulation occurs by phosphorylation of eIF-2→ translation inhibition

Front 34

Pro v. eu-- factors

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i. Eukaryotic cells require more initiation and elongation factors than prokaryotic cells

Front 35

Pro v. eu-- 5'-cap

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i. Binding of mRNA to 40s subunit requires 5’-cap
ii. Proper placement of 5’-cap requires cap binding protein CBP (eIF-4F)

Front 36

Pro v. eu-- cistronic

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i. Eukaryotic mRNA is monocystronic
ii. Prokaryote mRNA is polycystronic

Front 37

Pro v. euk--- ribosomes

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i. Eukaryotic cells have both free and bound ribosomes
ii. Prokaryotes have only free ribosomes

Front 38

Pro v. euk-- signal peptide

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i. Signal for a ribosome to attach to membrane of ER is in synthesized protein
ii. Found in first 15-30 AA residues

Front 39

Protein trafficking

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i. Many proteins of eukaryotic cells must be transported across the ER or integrated into the ER
ii. Proteins destined for secretion follow this pathway

Front 40

SRP

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i. Signal recognition particle binds to signal peptide as it exits the ribosomal complex
ii. SRP blocks translation until ribosome has docked w/ ER membrane
iii. SRP contains 6 proteins and an RNA molecule as central component

Front 41

SRP receptor

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i. Found only in ER
ii. Binds SRP when it is part of ribosomal complex
iii. GTP binds to SRP and SRP receptor→ protein targeting
iv. Forms a gated channel which opens upon ribosome docking

Front 42

Ribphorin

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i. Ribosomal receptor binds the ribosome

Front 43

Translocation

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i. Signal peptide enters ER lumen
ii. Protein elongation continues
iii. Signal peptide is removed by signal peptidase
iv. ATP required to transport protein through the membrane

Front 44

Secreted protein

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i. Proteins destined for secretion enter ER lumen and are transported to Golgi for modification and glycosylation

Front 45

Membrane proteins

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i. Contain stop-transfer sequences→ signal a stop to translocation of growing peptide chain into ER lumen

Front 46

Protein synthesis in mitochondria

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a. Mitochondria contain mtDNA
b. Resembles prokaryotic DNA
c. mtDNA codes for nine mitochondrial polypeptides

Front 47

Transport of protein into mitochondria

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i. Most coded for by nuclear DNA
ii. N-terminal of proteins targeted for mitochondria are post-translationally imported into mitochondria

Front 48

Cycloheximide

Back 48

1. Inhibits peptide bond formation in eukaryotic cells

Front 49

Diptheria toxin

Back 49

1. Catalyzes the reaction of NAD+ with a eukaryotic elongation factor, resulting in inhibition of translocation

Front 50

Chloramphenicol

Back 50

1. Inhibits peptide bond formation
2. Can inhibit mitochondrial protein synthesis at high levels
pro/euk

Front 51

Puromycin

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1. Cause premature termination by acting as an aminoacyl-tRNA analogue
2. The AA chain in the P site is transferred to puromycin rather than to the A site
pro/euk

Front 52

Tetracycline

Back 52

1. Inhibits the binding of aminoacyl tRNAs to the A site on the ribosome→ inhibits elongation
eukaryotic

Front 53

Streptomycin

Back 53

1. Binds to 30s subunit and causes dissociation of the translation process
prokaryotic

Front 54

Erythromycin

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1. Binds to the 50s subunit and inhibits translocation
prokaryotic