Protein Synthesis, Folding, and Degradation Flashcards
Codons
The sets of 3 nucleotides that are translated to amino acids. mRNA transcripts are read 3 nucleotides at a time. Many amino acids have more than 1 codon
What does translation require? (3)
- mRNA
- tRNA- an anticodon and associated amino acid
- Ribosomes
mRNA
The message to be translated
tRNA structure
Around 80 nucleotides in length, folds into a very precise, 3D structure called a cloverleaf structure. This is due to RNA:RNA base pairing. tRNA also undergoes additional folding after base pairing. This folding is driven by hydrogen bonds and creates mature tRNA, which is an L-shaped molecule
Ribosomes
The site of translation (protein synthesis)
tRNA function
tRNA codon-anticodon pairing
Representation of tRNA
RNA polymerase 3
Transcribes tRNA, produced as large precursors which are trimmed
Chemical modification of tRNA
Results in altered nucleotides (1 in 10)
Some affect anticodon base pairing
Others affect amino acid attachment
Types of chemical modifications of tRNA
Wobble
Mismatch tolerance- some amino acids have only 1 tRNA that tolerates a mismatch at a third nucleotide position
Amino acid coding
Multiple codons can code for 1 amino acid. With respect to this, some amino acids have multiple tRNAs (each with specific codon).
tRNA base pairs
Inosine (I) can base pair with uracil (U), cytosine (C), or adenine (A) in prokaryotes
Wobble base pairing
Aminoacyl-tRNA synthetase
Amino acid “activation” of tRNA (3 steps)
- The amino acid is linked to AMP through ATP hydrolysis, which is unfavorable
- The AMP-linked carboxyl group of amino acid is transferred to OH on the 3’ end of tRNA
- All of these steps are catalyzed by synthetase
Amino acid specificity of synthetase
There is an active site pocket that is specific for each amino acid
Aminoacyl-tRNA synthetase specificity
The correct amino acid is of the highest affinity for the binding site
Aminoacyl-tRNA result
Results in correct amino acid added to 3’ end of tRNA (called aminoacyl-tRNA)
Aminoacyl-tRNA synthetase structure
Free ribosomes
Synthesize most soluble, cytosolic and nuclear proteins. These proteins will never leave the cell and will never enter a membrane
Membrane-bound (ER) ribosomes
Found on the cytosolic side of the ER membrane. They synthesize proteins destined to reside in the ER, membrane proteins, proteins destined for secretion, lysosomal proteins
Cytosol and ER
Ribosome subunits (2)
- Large subunit- catalyzes formation of peptide bonds
- Small subunit- framework on which tRNAs are matched to codons
Where are ribosomes made?
The subunits assemble in the nucleolus- ribosomal proteins join with rRNA. When they are not participating in translation, the subunits are separate- they join together on mRNA. Ribosomes are made from more than 50 different proteins
Ribosomes and protein synthesis
The small subunit of ribosomes provides a framework on which tRNAs can be accurately matched to codons. Large subunit catalyzes formation of peptide bonds. The subunits are usually separate but come together on an mRNA near 5’ end when protein synthesis occurs.
4 binding sites in the ribosome
- A site
- P site
- E site
- mRNA binding site
Ribosomes
N-terminus
C terminus
How the ribosome translates (4)
- Aminoacyl-tRNA binds A site, spent tRNA exits the E site
- A new peptide bond forms
- The large subunit shifts, leaves sites in hybrid or broken states
- The small subunit shifts 3 nucleotides over, re-joining the large
Then, the cycle begins anew
Elongation factors
They are GTPases. Includes:
1. EF-Tu or EF-1
2. EF-G or EF-2
Elongation factors mechanism
EF-Tu
- Enhances tRNA to ribosome (A site) – GTP bound
- Enhances/monitors anticodon-codon pairing –GTP bound
- Allows for amino acid incorporation –GTP hydrolysis. Only unbinds if match is correct, allowing tRNA conformational change for tighter base pairing
EF-G
- Binds ribosome when large subunit is shifted relative to small subunit – GTP bound
- Re-shifts ribosome to original orientation – GTP hydrolysis. Induces conformation change in ribosome
Most ribosome functions are mediated by
RNA. rRNAs position tRNA on mRNA and are responsible for the catalytic activity of the ribosome in forming peptide bonds. Proteins stabilize the core and facilitate changes in rRNA conformation