Protein biosynthesis
Protein biosynthesis, or protein synthesis, is a core biological process, occurring inside cells, balancing the loss of cellular proteins through the production of fresh proteins. Proteins perform a number of critical functions as enzymes, structural proteins or hormones. Protein synthesis is a very similar process for both prokaryotes and eukaryotes but there are some distinct differences.
Protein synthesis can be divided broadly into two phases: transcription and translation. During transcription, a section of DNA encoding a protein, known as a gene, is converted into a molecule called messenger RNA. This conversion is carried out by enzymes, known as RNA polymerases, in the nucleus of the cell. In eukaryotes, this mRNA is initially produced in a premature form which undergoes post-transcriptional modifications to produce mature mRNA. The mature mRNA is exported from the cell nucleus via nuclear pores to the cytoplasm of the cell for translation to occur. During translation, the mRNA is read by ribosomes which use the nucleotide sequence of the mRNA to determine the sequence of amino acids. The ribosomes catalyze the formation of covalent peptide bonds between the encoded amino acids to form a polypeptide chain.
Following translation the polypeptide chain must fold to form a functional protein; for example, to function as an enzyme the polypeptide chain must fold correctly to produce a functional active site. To adopt a functional three-dimensional shape, the polypeptide chain must first form a series of smaller underlying structures called secondary structures. The polypeptide chain in these secondary structures then folds to produce the overall 3D tertiary structure. Once correctly folded, the protein can undergo further maturation through different post-translational modifications, which can alter the protein's ability to function, its location within the cell and its ability to interact with other proteins.
Protein biosynthesis has a key role in disease as changes and errors in this process, through underlying DNA mutations or protein misfolding, are often the underlying causes of a disease. DNA mutations change the subsequent mRNA sequence, which then alters the mRNA encoded amino acid sequence. Mutations can cause the polypeptide chain to be shorter by generating a stop sequence which causes early termination of translation. Alternatively, a mutation in the mRNA sequence changes the specific amino acid encoded at that position in the polypeptide chain. This amino acid change can impact the protein's ability to function or to fold correctly. Misfolded proteins have a tendency to form dense protein clumps, which are often implicated in diseases, particularly neurological disorders including Alzheimer's and Parkinson's disease.
Transcription
Transcription occurs in the nucleus using DNA as a template to produce mRNA. In eukaryotes, this mRNA molecule is known as pre-mRNA as it undergoes post-transcriptional modifications in the nucleus to produce a mature mRNA molecule. However, in prokaryotes post-transcriptional modifications are not required so the mature mRNA molecule is immediately produced by transcription.Initially, an enzyme known as a helicase acts on the molecule of DNA. DNA has an antiparallel, double helix structure composed of two, complementary polynucleotide strands, held together by hydrogen bonds between the base pairs. The helicase disrupts the hydrogen bonds causing a region of DNAcorresponding to a geneto unwind, separating the two DNA strands and exposing a series of bases. Despite DNA being a double-stranded molecule, only one of the strands acts as a template for pre-mRNA synthesis; this strand is known as the template strand. The other DNA strand is known as the coding strand.
Both DNA and RNA have intrinsic directionality, meaning there are two distinct ends of the molecule. This property of directionality is due to the asymmetrical underlying nucleotide subunits, with a phosphate group on one side of the pentose sugar and a base on the other. The five carbons in the pentose sugar are numbered from 1' to 5'. Therefore, the phosphodiester bonds connecting the nucleotides are formed by joining the hydroxyl group on the 3' carbon of one nucleotide to the phosphate group on the 5' carbon of another nucleotide. Hence, the coding strand of DNA runs in a 5' to 3' direction and the complementary, template DNA strand runs in the opposite direction from 3' to 5'.
The enzyme RNA polymerase binds to the exposed template strand and reads from the gene in the 3' to 5' direction. Simultaneously, the RNA polymerase synthesizes a single strand of pre-mRNA in the 5'-to-3' direction by catalysing the formation of phosphodiester bonds between activated nucleotides that are capable of complementary base pairing with the template strand. Behind the moving RNA polymerase the two strands of DNA rejoin, so only 12 base pairs of DNA are exposed at one time. RNA polymerase builds the pre-mRNA molecule at a rate of 20 nucleotides per second enabling the production of thousands of pre-mRNA molecules from the same gene in an hour. Despite the fast rate of synthesis, the RNA polymerase enzyme contains its own proofreading mechanism. The proofreading mechanisms allows the RNA polymerase to remove incorrect nucleotides from the growing pre-mRNA molecule through an excision reaction. When RNA polymerases reaches a specific DNA sequence which terminates transcription, RNA polymerase detaches and pre-mRNA synthesis is complete.
The pre-mRNA molecule synthesized is complementary to the template DNA strand and shares the same nucleotide sequence as the coding DNA strand. However, there is one crucial difference in the nucleotide composition of DNA and mRNA molecules. DNA is composed of the bases: guanine, cytosine, adenine and thymine. RNA is also composed of four bases: guanine, cytosine, adenine and uracil. In RNA molecules, the DNA base thymine is replaced by uracil which is able to base pair with adenine. Therefore, in the pre-mRNA molecule, all complementary bases which would be thymine in the coding DNA strand are replaced by uracil.
Post-transcriptional modifications
Once transcription is complete, the pre-mRNA molecule undergoes post-transcriptional modifications to produce a mature mRNA molecule.There are 3 key steps within post-transcriptional modifications:.
- Addition of a 5' cap to the 5' end of the pre-mRNA molecule
- Addition of a 3' poly tail is added to the 3' end pre-mRNA molecule
- Removal of introns via RNA splicing
This modified pre-mRNA molecule then undergoes the process of RNA splicing. Genes are composed of a series of introns and exons, introns are nucleotide sequences which do not encode a protein while, exons are nucleotide sequences that directly encode a protein. Introns and exons are present in both the underlying DNA sequence and the pre-mRNA molecule, therefore, to produce a mature mRNA molecule encoding a protein, splicing must occur. During splicing, the intervening introns are removed from the pre-mRNA molecule by a multi-protein complex known as a spliceosome. This mature mRNA molecule is then exported into the cytoplasm through nuclear pores in the envelope of the nucleus.
Translation
During translation, ribosomes synthesize polypeptide chains from mRNA template molecules. In eukaryotes, translation occurs in the cytoplasm of the cell, where the ribosomes are located either free floating or attached to the rough endoplasmic reticulum. In prokaryotes, which lack a nucleus, the processes of both transcription and translation occur in the cytoplasm.Ribosomes are complex molecular machines, made of a mixture of protein and ribosomal RNA, arranged into two subunits, which surround the mRNA molecule. The ribosome reads the mRNA molecule in a 5'-3' direction and uses it as a template to determine the order of amino acids in the polypeptide chain. To translate the mRNA molecule, the ribosome uses small molecules, known as transfer RNAs, to deliver the correct amino acids to the ribosome. Each tRNA is composed of 70–80 nucleotides and adopts a characteristic cloverleaf structure due to the formation of hydrogen bonds between the nucleotides within the molecule. There are around 60 different types of tRNAs, each tRNA binds to a specific sequence of three nucleotides within the mRNA molecule and delivers a specific amino acid.
The ribosome initially attaches to the mRNA at the start codon and begins to translate the molecule. The mRNA nucleotide sequence is read in codons. Each tRNA has an exposed sequence of three nucleotides, known as the anticodon, which are complementary in sequence to a specific codon that may be present in mRNA. For example, the first codon encountered is the start codon composed of the nucleotides AUG. The correct tRNA with the anticodon binds to the mRNA using the ribosome. This tRNA delivers the correct amino acid corresponding to the mRNA codon, in the case of the start codon, this is the amino acid methionine. The next codon is then bound by the correct tRNA with complementary anticodon, delivering the next amino acid to ribosome. The ribosome then uses its peptidyl transferase enzymatic activity to catalyze the formation of the covalent peptide bond between the two adjacent amino acids.
The ribosome then moves along the mRNA molecule to the third codon. The ribosome then releases the first tRNA molecule, as only two tRNA molecules can be brought together by a single ribosome at one time. The next complementary tRNA with the correct anticodon complementary to the third codon is selected, delivering the next amino acid to the ribosome which is covalently joined to the growing polypeptide chain. This process continues with the ribosome moving along the mRNA molecule adding up to 15 amino acids per second to the polypeptide chain. Behind the first ribosome, up to 50 additional ribosomes can bind to the mRNA molecule forming a polysome, this enables simultaneous synthesis of multiple identical polypeptide chains. Termination of the growing polypeptide chain occurs when the ribosome encounters a stop codon in the mRNA molecule. When this occurs, no tRNA can recognise it and a release factor induces the release of the complete polypeptide chain from the ribosome.