Proteins Flashcards
What is the main sequence of events during translation?
- Amino acid activation / tRNA charging, amino acids are linked to tRNA.
- Initiation where a small ribosomal subunit bought together with an mRNA and the first amino acid is bound to the AUG start codon. This is followed by the large subunit binding.
- Elongation the next tRNA binds to the next codon and the initial amino acid transfers from the tRNA to the second amino acid starting the chain. The ribosome translocates along the mRNA
What’s the difference between the smooth and rough endoplasmic reticulum?
The rough ER is where protein synthesis takes place as it has ribosomes. The smooth ER produces lipids, phospholipids (membranes) and steroids.
What is different about cells that produce lots of proteins?
Large quantities of ribosomes and prominent nuclei.
What are lysosomes?
Organelles that are membrane bound vesicles containing digestive enzymes that degrade proteins. Enzymes are active at low pH and are adapted to be active in white blood cells for example.
What is the Greek derivative of protein
“proteios” which means of the first rank
What is the central dogma of biology?
The central dogma of molecular biology is a theory stating that genetic information flows only in one direction, from DNA, to RNA, to protein, or RNA directly to protein.
What is the start codon and its corresponding amino acid?
AUG -> methionine
What direction does transcription and protein synthesis occur?
5’ -> 3’
N terminus -> C terminus
What is the generic structure of an amino acid?
Zwitterion
An alpha carbon with an amine group (NH3+), a carboxyl group (CO2-), a functional R group / residue and usually a H.
How is protein folding variation facilitated?
Varying properties of R groups dictate the folding of proteins. For example a hydrophobic R group will be pushed into the centre of a protein away from H2O.
What are the types of protein structure?
Fibrous
Transmembrane
Globular
Natively unfolded
What are the types of fibrous proteins?
Cytoskeletal such as actin, myosin and troponin.
Extracellular matrix proteins such as collagen and elastin.
(self assembly)
What do globular proteins do?
Largest category of proteins but some common examples:
Enzymes
Immune system proteins
Cell adhesion proteins
What are natively unfolded proteins for?
Generally signalling peptides.
What is the Shine-Dalgarno box?
A consensus sequence that can initiate translation. 16S RNA in the small ribosomal subunit will bind to the box attaching the mRNA to the small subunit to the mRNA. The Shine-Dalgrano boxes allows for prokaryotes to express polycistronic genes as it allows ribosomes to bind throughout the transcript and initiate transcription. these boxes are also important for allowing translation and transcription to occur simultaneously as the ribosomal subunits are only seeking out the Shine-Dalgrano boxes.
What are the initiation factors in bacteria and their roles?
IF1 blocks tRNA from entering the ribosome subunit too early.
IF2 binds specifically to the initiator fMet tRNA to bring it to the start codon
IF3 ensures that there is a match between codon and anticodon for the fMet.
Together these ensure correct translation and stops it occurring prematurely.
These all dissociate when the large subunit binds and are critical for ensuring translation starts correctly.
What is different about what AUG codes in bacteria compared to eukaryotes?
AUG is the start codon that codes for methionine but in bacteria, it codes for methionine with a formyl group (fMet) on a specialised tRNA. This specialisation allows it to bind in the middle p-site of the ribosome instead of the a-site. Once this binds it recruits the large subunit completing the ribosome. (reminder: in a ribosome amino acids are added like so E <- P <- A )
What are the main differences between eukaryotic and bacterial translation?
Eukaryotes do not have Shine-Dalgarno boxes, instead, a small ribosome subunit binds to the guanosine 5’ cap and moves along the mRNA until it reads an AUG and initiates translation.
In eukaryotes, a normal methionine on an initiator tRNA is recruited before the assembly of the large ribosomal subunit.
What two eukaryotic initiation factors are required to ensure the mRNA transcript is complete and how?
eIF4E replaces the 5’ cap on the transcript
eIF4G binds to the poly(A) binding proteins and eIF4E.
The binding of these two transcription factors acts as a proof read to signal that the transcript is in intact and in good condition.
Once the first AUG is read the transcription factors dissociate allowing the large subunit to bind.
There are many other eukaryotic transcription factors with important roles.
In eukaryotes why are translation and transcription separated
Transcription occurs in the nucleus and translation in the cytoplasm.
The 5’ cap and poly A tail modifications are required to initiate translation and these only happen once the mRNA is exported from the nucleus.
How, in eukaryotes, are multiple copies of a protein made?
Polyribosomes will bind. This is enabled by the mRNA forming a spiral due to the 3’ and 5’ mdoifications joining via the eFIG and eFIE allowing the initiation factors and ribosomal subunits can leave and rejoin the transcript in close proximity.
What is the function of micro RNA and how do they work?
Micro RNA transcripts are normally negative regulators of gene expression. They block expression either by
-binding to the mRNA and blocking initiation, elongation or termination.
- or degrading the mRNA transcript so no translation can occur.
How is the peptide bond formation between amino acids facilitated during elongation?
The joining of an amino acid to the peptide chain is mediated by the RNA enzyme, peptidyl transferase, which is part of the large subunit.
The enzyme mediates the nucleophilic attack from the nitrogen terminal of the new amino acid onto the ester bond between the polypeptide chain and its tRNA breaking the bond and creating a new peptide bond between the chain and the amino acid on the aminoacyl-tRNA.
How does aminoacyl tRNA become polypeptidyl-tRNA?
When the amino acid attached to the aminoacyl tRNA (in the A site) is added to the chain and the chain is transferred to this tRNA from the one in the P site. This is followed by the large subunit translocating then the small putting the unbound tRNA in the exit site, the peptidyl tRNA in the P site and leaving the A site free for a new aminoacyl tRNA
What direction does polypeptide chain elongation propagate in?
N - terminal (amine group) to C - terminal (ester group).
Amino acids are always added to the c terminus of a chain.
Describe the four steps of elongation.
- tRNA ejected from E site, peptide chain bound to tRNA in p site, tRNA enters A site and amino acid joins chain.
- Chain is transferred to tRNA in A site.
- Large subunit translocates moving unbound tRNA into e-site and peptidyl tRNA into p-site.
- The small ribosomal subunit translocates to pair up with the large subunit. A site is empty ready for the next aminoacyl tRNA and the e-site ready to eject the used tRNA.
What are elongation factors?
GTPases that improve translation efficiency by increasing the translocation speed of ribosomal subunits and they also have a role in proofreading. Without these translation would be hella slow stylllll.
What is EF-Tu and its function?
An elongation factor that helps load the A site.
It is bound to an aminoacyl-tRNA and helps bring it to the A site. If the codon and anticodon match the ribosome will cause the EF-Tu to hydrolyse the GTP bound to it into GDP causing a conformational change to occur in EF-Tu. This leads to it dissociating allowing the amino acid to join the peptide chain. The hydrolysis of GTP acts as a proof reading mechanism as it only occurs with correct anticodon-codon matching, so the aminoacyl will not bind to the A site unless if there’s not a match
What is the function of EF-G?
This elongation factor will help move tRNAs during translocation. Each ribosomal binding site has an affinity for a tRNA in the appropriate site (E: unbound, P: peptidyl, A: aminoacyl) but at one point the A site amino acid becomes peptidyl instead of aminoacyl and is in a hybrid state and therefore prefers the P site and the unbound P site tRNA prefers the E site. EF-G bound to GTP will enter the A site and bind to hybrid state to stabilise it. The GTP is then hydrolysed causing a conformational change in the protein that forces the hybrid A/P site tRNA into the P site only which in turn forces the unbound tRNA into the exit site. This followed by the tRNA being ejected and EF-G dissociating.
How is translation terminated?
Stop codons end translation (termination sites are for transcription). The general three stop codons are UAA, UAG and UGA. Stop codons are not recognised by tRNA but by a release factor. Release factors are protein that have molecular properties that mimic an aminoacyl-tRNA which allow them to enter the ribosome. They release the polypeptide by adding a water molecule to the end of the chain instead of an amino acid. This causes the protein to be released from the P site followed by the disassembly of the ribosome by the action of other release factors.
How are distant regions of polypeptide chains linked?
Often two cystine amino acids will covalently bond via disulfide linkages.
Where does an amino acids negative and positive charge come from, how do these groups interact at different pHs?
The amine group provides a negative charge and the carboxyl group a positive charge.
At acid pH the amino group the amino group is protonated and becomes slightly positive.
At basic pH the carboxyl group is ionised giving a slight negative charge.
What happens at neutral pH (physiological pH) to amino acid and how is this defined?
The carboxyl group is ionised and the amino group is protonated giving the molecule an overall neutral charge and so can act as both a negative and positive molecule. This is called a zwitterion. (can be altered by the properties of the R group).
What is the 3D structure of an amino acid generally?
They have an asymmetric chiral structure. Most are L:laevis which means they are left handed asymmetric. A chiral structure cannot be superimposed on its counterparts (think hands). The CORN acronym can be used to check this: going clockwise around the alpha carbon it goes CO group, R group then the H group.
