Lecture 10 - Translation and Protein Synthesis

Protein | A macromolecular polymer of amino acids linked together by peptide bonds

Functions of proteins | Structure, biochemical reactions, and regulation of the body; e.g., enzymes, hormones, antibodies, transporters

Four groups attached to an amino acid's central carbon | 1) Amino group (NH2); 2) Carboxylic acid group (COOH); 3) Hydrogen atom (H); 4) Radical or R group

R group | The variable side chain (e.g., H, CH3, CH3-CH2) that determines an amino acid's properties and function in a protein

L-alanine vs D-alanine | Mirror-image forms (stereoisomers) of alanine around the central carbon

Five classes of the 20 amino acids | Nonpolar aliphatic, polar uncharged, aromatic, negatively charged, positively charged R groups

Nonpolar, aliphatic amino acids | Glycine, alanine, proline, valine, leucine, isoleucine, methionine

Polar, uncharged amino acids | Serine, threonine, cysteine, asparagine, glutamine

Aromatic amino acids | Phenylalanine, tyrosine, tryptophan

Negatively charged (acidic) amino acids | Aspartate, glutamate

Positively charged (basic) amino acids | Lysine, arginine, histidine

Messenger RNA (mRNA) in translation | Genetic coding template used by the translational machinery to determine the order of amino acids in an elongating polypeptide

Transfer RNA (tRNA) in translation | Covalently attaches to individual amino acids and recognizes the encoding sequence of the mRNA so amino acids are inserted correctly

Ribosomal RNA (rRNA) in translation | Assembles with numerous ribosomal proteins to form the ribosome, which engages the mRNA and forms a catalytic domain that tRNAs enter with their amino acids

Parts of the tRNA cloverleaf model | Attached amino acid at the 3' end (acceptor stem), D loop, T loop, variable loop, anticodon loop with the anticodon

Translation (general meaning) | Conversion from one language or form to another

Translation (biology) | The process by which proteins are synthesized using mRNA as a template

Ribosomes | Complexes of proteins and rRNA that catalyze joining of amino acids as directed by the mRNA; made of a small subunit and a large subunit

Bacterial ribosome subunits | Complete ribosome 70S = large subunit 50S + small subunit 30S

Aminoacyl-tRNA synthetases | Enzymes that attach amino acids to the appropriate tRNAs to form aminoacyl-tRNA

Role of aminoacyl-tRNA | Acts as the adaptor translating the mRNA nucleic acid sequence into the protein's amino acid sequence

Three stages of translation | Initiation, elongation, termination

Initiation (translation) | Components of the translational apparatus come together with the mRNA; small and large ribosomal subunits and a tRNA carrying the first amino acid bind the start codon

Elongation (translation) | Amino acids are brought to the mRNA as aminoacyl-tRNAs and added one at a time to a growing polypeptide chain

Termination (translation) | A stop codon is recognized by a protein release factor (GTP-dependent), and the apparatus comes apart to release the completed polypeptide

A site | Aminoacyl site: cavity where an incoming charged tRNA (carrying an amino acid) binds during elongation

P site | Peptidyl site: cavity where the tRNA linked to the growing polypeptide chain binds

E site | Exit site: where discharged tRNAs leave the ribosome

mRNA-binding site | Binds a sequence near the 5' end of the mRNA, positioning the mRNA to translate its first codon

Where are the ribosome binding sites located? | At or near the interface between the large and small subunits

mRNA regulatory elements (slide 5 diagram) | 5' UTR: cap binding complex (eIF-4E, 4G, 4A, 4B), IRE/IRP, IRES, uORFs (translational control, stability); 3' UTR: IRE/IRP, miRNA, CPE/EDEN/DICE, PABP on poly-A tail (translational control, subcellular localization, stability)

tRNA synthetase example (methionine) | Aminoacyl-tRNA synthetase binds methionine, ATP, and tRNA for methionine, releasing AMP + 2 Pi and producing charged tRNA-Met

Four elements of tRNA molecules | 1) Three major loops (T, D, variable and anticodon); 2) four base-paired regions; 3) an anticodon triplet; 4) a 3' terminal CCA sequence where the amino acid attaches by an ester bond

Modified nucleotides in tRNA | Inosine (I), methylinosine (mI), dihydrouridine (D), ribothymidine (T), pseudouridine (Ψ), and methylguanosine (Gm)

When are tRNA nucleotides modified? | During maturation of the tRNA, in tRNA-specific ways

tRNA 3D structure | An L-shaped "hockey stick": the amino acid attachment site is at one end (tip of the handle) and the anticodon at the other (blade)

Why is tRNA drawn 3' to 5'? | Because tRNA aligns that way relative to the mRNA

Wobble position | The first base of the anticodon, which pairs with the third base of the codon

How many aminoacyl-tRNA synthetases are there? | Twenty; each links amino acids to the correct tRNAs

How many amino acids does each aminoacyl-tRNA synthetase recognize? | Only one amino acid; some recognize only one tRNA, some recognize a few because of redundancy in the genetic code

Codons vs tRNAs | There are 61 possible (sense) codons but far fewer tRNAs

Codons for the same amino acid often differ where? | Only in the third position of the codon

Wobble | A slight shift in the position of guanine in a tRNA anticodon lets it pair with uracil instead of its normal partner (cytosine)

Wobble example | One tRNA-Leu (anticodon 3'-GAU-5') can read two leucine codons: CUA and CUG

Wobble rules (anticodon base → codon third-position bases) | A → U; C → G; U → A or G; G → C or U; I (inosine) → U, C, or A

Genetic code | The collection of codons; letters A, U, G, C in mRNA organized into codons

Triplet codons | Each codon has 3 nucleotides to give each amino acid specificity

Why 3 nucleotides per codon? | 1 nucleotide = 4 combinations; 2 = 16; 3 = 64 combinations, enough for 20 amino acids

How many codons vs amino acids? | 64 possible codons but only 20 amino acids

Start codon | AUG: methionine; also forms part of the initiation signal

Stop codons | UGA, UAA, UAG

Direction codons are read | 5' to 3'

Genetic code: 1-codon amino acids | Met: AUG; Trp: UGG

Genetic code: 2-codon amino acids | Phe: UUU, UUC; Tyr: UAU, UAC; His: CAU, CAC; Gln: CAA, CAG; Asn: AAU, AAC; Lys: AAA, AAG; Asp: GAU, GAC; Glu: GAA, GAG; Cys: UGU, UGC

Genetic code: Ile | AUU, AUC, AUA (3 codons)

Genetic code: 4-codon amino acids | Val: GUN; Pro: CCN; Thr: ACN; Ala: GCN; Gly: GGN (N = any base)

Genetic code: 6-codon amino acids | Leu: UUA, UUG, CUN; Ser: UCN, AGU, AGC; Arg: CGN, AGA, AGG

One-letter amino acid codes | Ala A, Arg R, Asp D, Asn N, Cys C, Glu E, Gln Q, Gly G, His H, Ile I, Leu L, Lys K, Met M, Phe F, Pro P, Ser S, Thr T, Trp W, Tyr Y, Val V

Genetic code table color key | Nonpolar residues tan, basic blue, acidic red, polar uncharged purple

Silent mutation | A nucleotide substitution that causes no change in the protein sequence (e.g., GTA → GTT, still Val)

Missense mutation | A substitution that changes an amino acid (e.g., CCC → ACC, Pro → Thr)

Nonsense mutation | A substitution that creates a stop codon (e.g., TAC → TAG, Tyr → Stop)

Frameshift mutation | Caused by insertions or deletions in coding sequences; shifts the reading frame or inserts a stop codon (e.g., deleting TA turns Ser-Val-Pro-Tyr into Ser-Val-Leu-Leu)

What bond do aminoacyl-tRNA synthetases form? | An ester bond between the amino acid's carboxyl group and the 3'-hydroxyl (OH) of the appropriate tRNA, in two chemical steps

Aminoacyl-tRNA synthetase step 1 | Amino acid and ATP enter the active site; ATP loses pyrophosphate and the AMP bonds covalently to the amino acid (aminoacyl-AMP intermediate); pyrophosphate is hydrolyzed into two phosphates

Aminoacyl-tRNA synthetase step 2 | The tRNA bonds covalently to the amino acid, displacing AMP; the aminoacyl-tRNA is released

Class I vs Class II synthetases | Class I attaches the amino acid to the tRNA's 2'-OH; Class II attaches it to the 3'-OH

Editing by synthetases | Pre-transfer editing (hydrolyzes wrong aminoacyl-AMP) and post-transfer editing (removes wrong amino acid from the tRNA)

Peptide bond formation between sites | The amino group of the A-site aminoacyl-tRNA attacks the carbonyl of the P-site peptidyl-tRNA

70S initiation complex formation | Occurs in three steps (prokaryotes)

Prokaryotic initiation step 1 | Three initiation factors (IF1, IF2, IF3) and GTP bind the small ribosomal subunit (30S)

Prokaryotic initiation step 2 | The initiator aminoacyl-tRNA and mRNA attach

What forms the prokaryotic mRNA-binding site? | At least partly a portion of the 16S rRNA of the small subunit

Shine-Dalgarno (SD) sequence | Ribosome binding site (RBS) on prokaryotic mRNA that base pairs with a pyrimidine-rich stretch at the 3' end of the 16S rRNA

Prokaryotic initiation step 3 | The large ribosomal subunit (50S) joins, forming the 70S initiation complex with fMet-tRNAfMet in the P site

IF2 | Binds the initiator tRNA-Met before the small ribosomal subunit

Where does the ribosome begin translation? | At a start codon AUG

Initiator tRNA anticodon | UAC (pairs with AUG)

Recycling of translational components | After termination, the ribosome subunits, mRNA, and factors are reused

Requirement for chain elongation | A peptidyl-tRNA, or in the first cycle an fMet-tRNAfMet, at the P site

Prokaryotic elongation step 1 | The second aminoacyl-tRNA binds the A site, escorted by EF-Tu with two bound GTPs; GTPs are hydrolyzed and EF-Tu is released

EF-Ts | Helps recycle EF-Tu

Prokaryotic elongation step 2 | A peptide bond forms between the carboxyl group of the terminal amino acid (or fMet) at the P site and the amino group of the new amino acid at the A site

Peptidyl transferase | Activity of the 23S rRNA in the large subunit that catalyzes peptide bond formation

Prokaryotic elongation step 3 | EF-G-GTP binds and GTP is hydrolyzed; the tRNA with the polypeptide translocates from the A site to the P site

What happens during translocation? | Discharged tRNA moves from P to E and leaves; the mRNA moves with the peptidyl-tRNA, bringing the next codon into the open A site; repeated for each amino acid

Release factors | Proteins that recognize the three stop codons and terminate protein synthesis

Termination mechanism | A stop codon (UAG, UAA, UGA) in the A site is bound by a release factor, which transfers the polypeptide to water, releasing it from the tRNA and dissociating the complex

Chloramphenicol | 50S inhibitor: binds 50S and reversibly inhibits peptide bond formation by peptidyl transferase

Macrolides, clindamycin, and streptogramins | 50S inhibitors: bind the peptide exit tunnel near the peptidyl transferase center, preventing chain elongation; can also inhibit 50S subunit formation

Linezolid | 50S inhibitor with a unique binding site; prevents formation of the 50S/30S initiation complex

Tetracyclines | 30S inhibitors: bind reversibly, blocking aminoacyl-tRNA binding to the acceptor site

Aminoglycosides | 30S inhibitors: bind tightly causing mRNA misreading; interfere with the initiation complex; break polysomes into nonfunctional monosomes; irreversible and bactericidal

Why are aminoglycosides ineffective against anaerobes? | They need oxygen-dependent active transport to cross the cell membrane

Antibiotic mnemonics | Tetracyclines prevent binding of tRNA; Linezolid doesn't let subunits bind; Aminoglycosides alter 30S shape and codon reading; Chloramphenicol Corrupts peptide bond formation; Macrolides, Streptograms & Clindamycin Share a Common Molecular Site

Ribosome composition | Two separate subunits composed of protein and rRNA

Eukaryotic vs prokaryotic ribosome size | Eukaryotic 80S (larger, more complex); prokaryotic 70S

When do ribosome subunits come together? | When they bind an mRNA near its 5' end

Direction of mRNA reading and protein synthesis | mRNA read 5' to 3'; protein made from N-terminal (amino) to C-terminal (carboxyl)

Where are ribosomes located? | In the cytosol, free or associated with the endoplasmic reticulum