8 - Translation in Molecular and Cell Biology LF206

Translation

Recodes the 4-letter nucleotide code of the mRNA template into the 20-letter amino acid code of the polypeptide product.

is this 4 letter code DNA or RNA?

GATC

DNA

is this 4 letter code DNA or RNA?

GAUC

RNA

20 letter code of all amino acids.

Ala, A Arg, R Asn, N Asp, D 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

Gene

A sequence of DNA that is transcribed into mRNA.

Transcription

The process of synthesizing mRNA from a DNA template.

mRNA

Messenger RNA that carries the genetic information from DNA to the ribosome.

Polypeptide formation

The process by which amino acids are linked together to form a polypeptide chain.

Ribosome

A molecular machine that catalyzes the formation of polypeptides from mRNA.

Aminoacyl tRNA

tRNA that is attached to its corresponding amino acid.

Peptide bonds

Covalent bonds that link amino acids together in a polypeptide.

Ribosomal RNA (rRNA)

RNA component of the ribosome, crucial for its structure and function.

Bacterial ribosome

Composed of 50S and 30S subunits, with specific rRNA and protein components.

Eukaryotic ribosome

Composed of 60S and 40S subunits, larger and more complex than bacterial ribosomes.

acronym for the 3 sites on a ribosome?

APE

a - acceptor

p - peptidyl

e - exit

Acceptor site (A site)

The site on the ribosome where incoming aminoacyl tRNA binds.

Peptidyl site (P site)

The site on the ribosome that holds the tRNA carrying the growing polypeptide chain.

Exit site (E site)

The site on the ribosome where uncharged tRNA exits after delivering its amino acid.

Compensatory mechanism

A mutation that restores base pairing after a disruption caused by a previous mutation.

Watson-Crick base pairing

The specific pairing of nucleotides in DNA and RNA, maintaining structural integrity.

Structural conservation

The preservation of specific structures in rRNA across different species.

Secondary structures

The local folded structures that form within a polypeptide due to interactions between atoms of the backbone.

Tertiary structures

The overall three-dimensional structure of a polypeptide, formed by the folding of secondary structures.

High energy cost to cell

The significant energy expenditure required for protein synthesis, involving over 150 proteins and ~ 40 tRNAs.

tRNA

Transfer RNA, a molecule that helps decode a messenger RNA sequence into a protein.

D loop

A loop in tRNA containing 8-12 unpaired bases and 2-3 dihydrouracil residues.

T-loop

A loop in tRNA that contains modified nucleotides including thymine.

CCA terminus

also known as?

The 3’ end of the tRNA molecule that is flexible and important for editing.

AKA acceptor stem

anticodon

A sequence of three bases in tRNA that pairs with the corresponding codon in mRNA.

aminoacyl-tRNA synthetase

An enzyme that attaches the appropriate amino acid to its corresponding tRNA.

aminoacyl-tRNA synthetase mechanism [4]

1. a specific amino acid and ATP binds to a specific amino acyl-tRNA synthetase

2. amino acid is activated by covalent binding of AMP - pyrophosphate is released

3. a specific tRNA binds to the enzyme, AMP is released as the amino acid covalently attaches to tRNA by an ester bond

4. charged tRNA is released

what is meant by charged tRNA?

amino acid is covalently attached to tRNA

Non-Canonical Base Pairs

Base pairs in tRNA that include non-conventional A-U and G-C pairs held together by multiple hydrogen bonds.

amino acylation

The process of attaching an amino acid to its corresponding tRNA by specific synthetases.

initiation factor

A protein that recognizes the initiator tRNA (fmet tRNA) during the initiation of translation.

transformylase

An enzyme that recognizes the initiator tRNA and modifies it.

Class I aminoacyl-tRNA synthetases

Enzymes that approach/contact tRNA at the minor groove of the tRNA acceptor stem and the anticodon.

Class II aminoacyl-tRNA synthetases

Enzymes that approach/contact tRNA at the major groove of the tRNA acceptor stem and the anticodon.

double sieve is a _________ mechanism that allows for the ________ of incorrect aminoacyl-___intermediates.

this can occur at which two stages?

proofreading

hydrolysis

AMP

1. hydrolysis of ester bond at incorrect aminoacyl-AMP

2. editing sites on most aminoacyl-tRNAs hydrolyse aminoacyl-tRNAs smaller than cognate aa

editing site

A site in aminoacyl-tRNA synthetases that hydrolyzes aminoacyl-tRNAs which are smaller than the cognate amino acid.

ester bond

A type of covalent bond formed between an amino acid and tRNA during aminoacylation.

hydrolysis

The chemical process of breaking down a compound by reaction with water, used in proofreading.

charged tRNA

tRNA that has an amino acid covalently attached, ready for incorporation into a protein.

pyrophosphate

A byproduct released during the activation of the amino acid by binding to AMP.

specific amino acid binding site

A site on the aminoacyl-tRNA synthetase that ensures the correct amino acid is attached to the tRNA.

specific anticodon binding site

A site on the aminoacyl-tRNA synthetase that ensures the correct tRNA is selected based on its anticodon.

invariant modifications

Modifications to bases in tRNA that are consistent across all known tRNAs.

hydrolytic sites

Sites in aminoacyl-tRNA synthetases that facilitate the breakdown of incorrect aminoacyl-tRNAs.

Overall fidelity of charging in vivo

99.99%

Ribosome binding sites for tRNA

Ribosomes have three binding sites for tRNA: A (Acceptor), P (Peptidyl), and E (Exit).

Peptide bond is built by which activity?

Requires a peptidyl synthase activity.

A site

Acceptor site of codon-directed binding of incoming aa tRNA.

P site

Peptidyl site; holds codon directed peptidyl tRNA.

E site

Exit site; not associated with mRNA.

Peptidyl transferase activity - is it RNA or enzyme activity? and why?

RNA

the closest protein of the growing polypeptide chain is 18.4Å from the active site

Provided by an invariant adenine residue A2451, which is provided by RNA, not protein.

Bacterial 70S ribosome

The ribosome is a giant ribozyme.

Protein folding starts where?

Starts in the ribosome polypeptide exit tunnel.

Erythromycin and other _______

target ______

it binds to the small 23S _____ of the ___S ribosomal subunit and inhibit elongation of the ______

they block the exit tunnel of ________ ribosomes

macrolides

elongation rRNA

50S

peptide

bacterial

Streptomycin inhibits _______ and causes _______

initiation

misreading.

Doxycycline and other _________

target ________

they bind to the ___ subunit and inhibit the binding of ______-tRNA to the _ site of the ribosome.

tetracyclines

elongation

30S

aminoacyl

A

Chloramphenicol and other ________

target _________

they inhibit ______ transferase activity of the ___ ribosomal subunit in ____karyotes

amphenicols

translocation

peptidyl

50S

prokaryotes

Diphtheria toxin

binds to a receptor with what domain? what follows?

Binds its receptor via its receptor-binding domain (R) followed by endocytosis.

Furin cleavage

what does it process? what domains are still connected & by what?

The diphtheria toxin (DTx) is processed by furin cleavage, but the C and TR domains are still connected by a disulfide bridge.

Translocation domain (T)

forms what? where?

of diphtheria toxin (DTx) forms a pore in the membrane of an acidified endosome.

Ricin

binds to a receptor with what? what follows?

Binds its receptor via its B chain (RTB) followed by endocytosis.

RTA

what is it?

what does it do?

Catalytic A chain of ricin that hydrolyses the N-glycosidic bond of adenine 4324 (A4324) within the 28S rRNA.

Protein synthesis inhibition by RTA

Active RTA irreversibly hydrolyses the N-glycosidic bond of A4324, stopping protein synthesis.

t-RNA structure

t-RNA forms an L shaped tertiary structure with a key CCA terminus for charging with an amino acid at one end and the anticodon at the other.

t-RNA synthetases

Most have both an activating domain and an editing domain to ensure correct charging of amino acids.

Secondary structures in polypeptides

Some can be formed by a polypeptide exiting the 50S ribosome subunit.

_______ synthesis can be used as a target for anti-microbial discovery

protein

mechanism of antibiotic streptomycin and other __________.

they target initiation.

they bind the small ___ rRNA of the 30S ribosomal ______ irreversibly, distorting it and interfering with the binding of _____-methionyl-tRNA to the ___ subunit.

aminoglycosides

16S

subunit

formyl

30S