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
How does translation begin?
With the start codon, which is usually AUG. The first amino acid is always Met
Translation in prokaryotes vs. eukaryotes
Initiator tRNA carries
Distinct from normal Met tRNA
Usually removed later
How does translation end?
Through the stop codon- includes UAA, UAG, and UGA. These codons do not code for a protein and are not recognized by tRNA. Release factors bind the stop codons when they are present in the A site. This causes the end of translation and the release of the newly-made protein and ribosome
Translation initiation in eukaryotes (8 steps)
- eIF2 binds initiator tRNA-Met
- Initiator tRNA-Met binds the P site in the small subunit
- 2 IFs are bound to mRNA since nuclear export
- IFs aid the small subunit in binding to the 5’ end of RNA
- tRNA searches for RNA along AUG
- Once at start site, eIF2 and other IFs dissociate
- Large ribosome subunit now joins
- Translation has now begun with the addition of a second amino acid
Translation initiation in bacteria
Bacteria mRNA lacks a 5’ cap. They have a Shine-Dalgarno sequence instead. This means that the ribosome can recognize internal start codons on mRNA
5’ cap
The site in RNA, in addition to eIFs, that signals the ribosome where to start searching for the start site in eukaryotes
Shine-Dalgarno sequences
Ribosome binding few nucleotides upstream of start codon
Bacterial mRNA structure
Translation initiation in prokaroytes
- Initiator tRNA-Met meets the P site in the small subunit
- IFs guide the small subunit in binding the Shine-Dalgarno sequence
- IF2-GTP is hydrolyzed, IFs dissociate
- tRNA searches along RNA for AUG
- Large ribosome subunit now binds
- Translation has now begun with the addition of the 2nd amino acid
Translation termination
- Stop codon in A site
- Release factor (molecular mimic of tRNA) binds the stop codon
- The large subunit shifts
- Peptidyl transferase catalyzes water to OH
- C-terminus of the protein is made
- The protein is released
- The ribosome falls apart
Polyribosomes
Amino acid structure
2 acidic amino acids
Asp, Glu
3 basic amino acids
Lys, Arg, His
3 small side chain amino acids
Gly, Pro, Ser
4 bulky side chain amino acids
His, Tyr, Phe, Trp
8 hydrophobic amino acids
Ala, Val, Leu, Ile, Met, Phe, Tyr, Trp
How many hydrophilic amino acids are there?
12
Polypeptides
Primary structure
A linear chain of amino acids
Secondary structure
The spatial arrangement of amino acids driven by hydrogen bonding. The amino acids can be arranged into alpha helix or beta sheet structures
Hydrogen bonding in alpha helices
The NH of an amino acid forms an H-bond with the CO of the amino acid 4 residues earlier
Hydrogen bonding in beta sheets
The sheets can be anti-parallel or parallel
Tertiary structure
The 3D structure of the protein
Tertiary structure is dictated by
Non-covalent and covalent bonds
Disulfide bonds
Only form between 2 cysteines. Methionine contains sulphur as well, but can’t form disulfide bonds due to the strength of the S-CH3 bond.
Quaternary structure
Hemoglobin
2 types of post-translational modifications
- Formyl group of N-term fMet removed (prokaryotes)- Methionine deformylase
- Entire fMet/Met hydrolyzed/removed (prokaryotes/eukaryotes)
Methionyl amino peptidase (MAP) – fMet-specific peptidase
Methionine amino peptidase 2 (METAP2)
Mature polypeptide
The information for the secondary and tertiary structure formation is found in
The amino acid sequence. Technically, some proteins can fold properly entirely on their own, and some do, especially small, rapidly folding proteins
Co-translational folding
Molten globule
Chaperone proteins
Proteins that transiently associate with newly synthesized proteins and prevent misfolding while bound. They also prevent aggregation. Some are complexes that actively direct protein folding and are useful for the proper folding of misfolded proteins
How do chaperones know if a protein needs help folding?
Chaperone protein functions
Trigger factor (prokaryotes)
Attached to large subunit (50S) of ribosome
Nascent polypeptide-associated complex (NAC) - eukaryotes
Peptidyl-prolyl cis/trans isomerase
Promotes correct orientation proline residues
Proline residue site (in trigger factor)
Nonspecific sites
Unfolded, hydrophobic
Important – binding to these sites prevents folding
Allows proline residue site to do its job
Trigger factor and NAC
Hsp70 family (prokaryotes & eukaryotes)
ATP bound Hsp70 binds hydrophobic regions as the protein exits the ribosome. ADP-bound HSP70 binds more tightly and guides folding
DnaK
A monomeric protein with 2 domains. Its N-terminus has ATPase activity and binds to co-chaperone GrpE. The C-terminus binds polypeptides and co-chaperone DnaJ (Hsp40). DnaJ modulates ATPase activity
DnaK mechanism
- DnaK binds hydrophobic portions and DnaJ induces ATPase activity. Protein folding is guided
- GrpE exchanges ADP for ATP. DnaK is recharged and releases the fully folded protein.
Hsp60 family
A barrel-like family of proteins present in prokaryotes and eukaryotes. Requires ATP hydrolysis for energy. Helps to unfold and re-fold misfolded proteins, following translation and unsuccessful folding attempts/aggregation. In prokaryotes, it includes GroE (GroEL/ES) and eukaryotes have different members in different organelles
Hsp60 mechanism (5)
- Incorrectly or incompletely folded proteins are captured by hydrophobic interactions
- ATP and cap binding expand the rim (stretching/unfolding the client protein)
- Protein folding catalyzed
- ATP hydrolysis weakens the complex, and new ATP binds, releasing the folded protein
- More cycles will occur if the protein is still incorrectly or not completely folded
Hsp60
Which proteins are considered abnormal?
Proteins with mutations, proteins that are misfolded, or proteins without partners (individual subunits of oligomeric proteins)
What can cause protein misfolding?
Stress responses, including those to temperature or pH
Which proteins recognize misfolded proteins?
ATP-dependent proteases recognize and degrade misfolded proteins. Small peptides can be additionally degraded by ATP-independent proteases
Lon proteases
A free, cytosolic protease that recognizes misfolded proteins. It is homotetrameric- each subunit has a serine active site. It degrades proteins to acid-soluble peptides that are less than 1500 Da. Lon proteases are inactivated by ATP hydrolysis (when they are bound by ADP). Once a new substrate is bound, ADP is swapped for ATP
ClpXP or ClpAP
ClpXP or ClpAP – ssrA-SspB
Ubiquitylation
The addition of a ubiquitin (Ub) chain to a protein. Ub chains are linked by Lys, and target the protein for destruction. Has other functions in cell, but targeting for destruction is best studied. The proteins tagged for destruction are degraded by the proteasome
Ubiquitylation mechanism
- E1 binds (activates) Ub
- E1-Ub binds ubiquitin ligase (E2/E3)
- Ub binds Ub ligase, E1 dissociates
- Ub-Ub ligase recognizes degradation signal
- Ub-Ub ligase binds
- Ub is added to Lysine
- Process continues with more E1-Ub until poly-ubiquitinated
And what does ubiquitylation do?
It targets proteins to the proteasome, a large proteolytic complex that digests proteins in eukaryotic cells
Proteasome structure
It is a central, hollow cylinder. Has 2 caps
19S proteasome cap
Unfoldase
The proteasome cap is an unfoldase. ATP hydrolysis creates strain in ring, unfolding and pulling in protein
