Protein Synthesis and Transport

Hi, in this post we'll be discussing protein synthesis and protein transport. I'll be discussing the basics of protein synthesis and how translation works. I'll also outline the post-translational modifications that occur to a protein after it has been synthesised as well as explain protein targeting.

Protein Synthesis

Protein synthesis refers to the creation of a protein from a mRNA template copied from DNA. Proteins are synthesised outside the nucleus on ribosomes. Protein synthesis starts with transcription (see previous post) which takes place in the nucleus, where RNA polymerase reads a DNA strand and synthesises a single strand of mRNA (messenger RNA) from a gene. This strand of mRNA migrates through nuclear pores to ribosomes located in the cytoplasm. 

A group of 3 consecutive nucleotides on a mRNA is termed a codon, eg. UAG. Some codons tell the ribosome where to start making the protein, these are called start codons (AUG). Some codons such as UAG, UAA, and UGA tell the ribosome where to stop adding amino acids to the polypeptide, these are termed stop codons. Codons also code for different amino acids, eg. GUU codes for Valine. When the mRNA molecule travels to the ribosomes, the ribosomes attach to the mRNA and read the codons. Transfer RNA molecules then bring the corresponding amino acid to the ribosome where it binds to other amino acids to form a polypeptide.

Transfer RNA has a three nucleotide sequence known as an anticodon which is complimentary to the codon for that particular amino acid. Aminoacyl-tRNA synthetase catalyses the attachment between each tRNA and its corresonding amino acid. There are 20 aminoacyl-tRNA synthetases, approximately 50 different tRNA molecules and 61 codons. If each codon can pair with only a unique anticodon, then 61 tRNAs would be needed. However, there are less than 61 tRNAs in the cell, so how are all the codons read?

The Wobble Hypothesis, proposed by Francis Crick in 1966, can explain this. Some codons code for the same amino acid, for example CGU and CGC both code for arginine but both pair with the same tRNA, how? A weak interaction between the third base in the codon of the mRNA (termed the wobble base) allows one anticodon to associate with several different codons. Some tRNA molecules have ionosine (I) as the wobble base which can pair with U, C or A. This means that fewer types of tRNA molecules are needed.


Translation

Translation involves three steps: initiation, elongation and termination.

Initiation: 

To begin translation, a large and small ribosomal subunit along with the initiating tRNA assemble onto the mRNA. The start signal for translation is the codon AUG which codes for methionine. The first AUG at the 5' end of the mRNA is not necessarily the start site for translation. Instead, the start codon is always preceded by a sequence specifying the AUG as the start site. Eukaryotes have specific sequences (A/GCCA/GCCAUGA/G) which base pair with a complimentary sequence near the 3' terminus of the ribosomal subunit. This positions the ribosome correctly on the mRNA molecule. Once this has occurred, elongation begins.

Elongation: 

An initiator tRNA is placed at the P site (the site where the growing protein will be) of the ribosome causing elongation to proceed. The next tRNA molecule comes in and binds to the A site (the place where the incoming tRNA will attach itself) which is next to the initiator tRNA at the P site. After this occurs, the ribosome shifts so that the tRNA is now in the P site and a new tRNA binds to the A site. Here, a peptide bond is formed between the two amino acids. The first tRNA is now released and the ribosome shifts again so that the tRNA carrying the two amino acids is now in the P site, and a new tRNA can bind to the A site. This repeats until the ribosome reaches a stop codon. 

Termination 

Translation ends when a stop codon and release factor are encountered, causing ribosomal subunits to dissociate.  

This whole process is explained well in the video below:

Post Translational Modification

Cells contain many different specialised compartments. In addition, a protein must be maintained in a translocation-competent state, that is it must not misfold or aggregate. The protein needs to be directed to its proper membrane or compartment. A particular type of protein, called chaperones, act to ensure that newly synthesised proteins remain unfolded and are delivered to their correct location. But how does a protein know where it is supposed to go?

The answer lies in the structure of the protein. Newly synthesised proteins contain specific signal sequences which are short regions of the protein that act as targeting signals to direct the protein to its correct location. The signal sequence is like the proteins "address".

Some proteins need to travel across the nuclear membrane. If the protein is small enough it can simply pass through the nuclear pores. However, if the protein is quite large it will require active transport and contain a nuclear localisation sequence (NLS). Nuclear import receptors bind to the NLS and carry the protein into the nucleus. 

 Proteins may also need to move across membranes in places such as the peroxisome, mitochondria or endoplasmic reticulum (ER). This requires energy in the form of ATP and an aqueous channel through the membrane. Transmembrane transport can be post translational, such as when travelling through the mitochondria and peroxisomes, or co translational, such as when travelling to the ER.

The ER performs two important functions for protein targeting:

1. N-linked glycosylation: this is the attachment of sugars to particular asparagine residues in proteins. The sugars act as recognition signals and are important for cellular recognition of proteins and protein folding.
2. Peptide folding: Chaperones in the cytoplasm and the lumen of the ER work together to give the polypeptide chain several opportunities to fold.

In addition, proteins may be modified after translation by:

• cleavage by proteolytic enzyme to convert the protein to a functional form
• acetylation
• phosphorylation
• ubiquitination

Proteins may be transferred from the rough ER to the Golgi complex to other areas of the cell. The ER acts as the gateway for protein transport into all the other membrane bound organelles of the secretory pathway of the cell. Proteins are transported through this pathway in small carriers called vesicles. But how do the vesicles know where to go? 

Vesicles from a donor organelle have proteins attached to the vesicles known as v-snares. The target organelle has t-snares. V-snares and t-snares have specific partners so the vesicle must have the correct v-snare to fuse with a specific organelle. The t-snares on each organelle act as "address labels" to ensure that the vesicles go to the right place.

Some proteins may be destined for the lysosome - a specialised compartment which acts as a degradative organelle. Lysosomes contain enzymes which break down proteins. A specific sugar acts as a sorting signal to target proteins to the lysosome.


That's it for this post, if you have any questions or feed back please leave a comment below.