Objectives
By the end of this lesson you should be able to:
- Describe steps in DNA transcription and translation
- Identify the general mechanism by which proteins fold
- Identify methods of post-translational modification
- Differentiate between different types of mutation
The DNA contains the code for making all proteins. In order for proteins to be synthesized, the code on the DNA for making the protein of interest must be transcribed. Imagine trying to make a cake (the protein) that you have never made before. Consider that the book with the recipe is in a rare book that cannot leave the library. However, copies of various sections of the book can be made, which could then leave the library. Once you have the copy of the recipe you want, you can take it home and make the cake in your kitchen. In molecular biology you could consider the nucleus of the cell to be the library and the genes on the DNA to be the books in the library. The DNA cannot leave the nucleus but parts of it (genes) may be copied and sent to the cytosol where the message can be translated and protein made. The copying of the DNA is called transcription and the reading and interpretation of the transcript is called translation.
Parts of the DNA Involved in Transcription
The DNA that will be transcribed consists of the following main parts,
- Activator or Repressor Region: Part of the DNA to which proteins bind, activating or hindering activity of RNA polymerase, and thereby activating or promoting transcription
- Promoter Region (also called the TATA box): Part of the DNA located between 25 and 35 base pairs upstream from the coding region
- Coding Region: Part of the DNA where genes get transcribed
Steps in Transcription
DNA transcription consists of the following major steps, i.e., initiation, elongation, termination, and splicing.
Initiation: RNA polymerase binds to the promotor site along with transcription factors, causing the DNA to unwind
Elongation: RNA polymerase reads the template strand from the 3′ to 5′ end and writes down new nucleotides from 5′ to 3′ (Remember: “Read up and write down rule”). The new template is called pre-mRNA. Remember that RNA does not have thymine but uracil instead. Therefore, during transcription, wherever there is an A on the template, U will be “typed” instead of T. As soon as transcription begins, the 5′ end is capped by the addition of methyl guanosine. This process, along with polyadenylation helps in (1) preventing degradation of the mRNA before it can be transcribed, (2) facilitating export of the mature mRNA from the nucleus, and (3) helping ribosomes attach to the 5’ end of the mRNA once it reaches the cytoplasm.
The process of initiation and elongation occurs simultaneously in prokaryotes due to the absence of a nucleus. No transcription factors are needed in prokaryotes. RNA binds directly to the promotor.
Termination: Once RNA polymerase reaches a termination sequence (AAUAAA), the pre-mRNA is cleaved. Poly-A polymerase then binds to the 3’ end and adds a series of adenine residues to form a poly-A tail. Once that process is complete, the pre-RNA is ready for splicing.
What we have described so far is true of eukaryotes. Prokaryote termination may either be rho-dependent or rho-independent.
Rho-Dependent
In rho-dependent termination, the protein called rho, travels in the direction of RNA polymerase as it transcribes the DNA. When it eventually catches up with, and bumps into the RNA polymerase, it signals it to stop transcription.
Rho-Independent
In rho-independent translation, rho is not involved. Instead, translation is signaled to stop when the RNA forms a hair-pin loop due to the presence of inverted repeats.
The new pre-mRNA is also referred to as heterogenous nuclear RNA (hnRNA). This is because it consists of RNA that will be eventually expressed to make proteins (exons) and others that are considered as “junk” and will be thrown out (introns). Processing involves getting rid of the introns via splicing. A complex biological machine consisting of proteins and small nuclear RNA (snRNA) called a spliceosome, cuts away the introns and join the exons together.
Three parts of an intron are important to remember, i.e., its start (GU), end AG), and a branch point (A). During the excision process, the spliceosome cuts off the intron at GU. The GU sequence then binds to the branch point (A) forming a lariat (structure looking like a rope with a loop). The lariat is then cleaved at the AG site and the two exons are ligated together. The mRNA is now ready for translation.
DNA Translation
Three sequences of nucleotides called a codon on the mRNA code for amino acids. There are 64 codons. Sixty-one of them code for amino acids including AUG which is a start codon corresponding to methionine. Three codons are referred to as STOP codons. Stop codons terminate translation of mRNA.
The stop codons are UGA, UAA, and UAG. Remember,
- UGA – “You go away”
- UAA – “You are away”
- UAG – “You are gone”
The transfer RNA (tRNA) is used in translation to transfer amino acids corresponding to codons on the mRNA. As you can see from the figure below, the tRNA forms the shape somewhat of a “t”. Amino acids attach to the tRNA at the 3′ end where there is the nucleotide sequence, AAC. Two arms called the D and T-loop on the tRNA play an important role in the function of the tRNA and its interaction with the ribosome. The sequence of the tRNA that reads the codon on the mRNA is called the anticodon.
In order to bind the tRNA, amino acid first reacts with ATP to produce amino acyl-AMP with the help of amino acyltransferase. The amino acid is then transferred to tRNA with the help of aminoacyl tRNA synthetase. AMP is released in the process.
Along with mRNA and tRNA, ribosomes complete the machinery needed for translation. Ribosomes are a combination of rRNA and proteins. There are two subunits of the ribosome, a large and a small. In eukaryotes, the large unit is called the 60s subunit, and the small is called the 40s subunit.
The small subunit positions itself onto the mRNA with the help of initiation factors, and the tRNA is brought into position (also with the help of initiation factors).
The large subunit is engaged with the help of initiation factors to complete the initiation complex. The tRNA is now ready to begin translating, starting at the peptidyl site (or p-site) where the start codon AUG codes for methionine.
After the initial installation of the first amino acid (methionine), another tRNA reads the codon on the aminoacyl site (or A site). The movement of tRNA to this site is facilitated by elongation factors.
The first amino acid that was translated is transferred to the second using peptidyl transferase.
Now tRNA in the p-site is empty (has no amino acid) while the second has two amino acids. This is the beginning of a chain of amino acids that will make the new protein.
The “empty” tRNA moves over to the exit (or e-site) while the tRNA that was previously in the a-site moves over to the p-site.
The empty tRNA in the e-site then exits the complex, leaving behind the tRNA in the p-site.
The process continues with a string of amino acids being joined together each time a new tRNA enters the A site. Eventually, a STOP codon is reached. No tRNA binds to these codons.
Instead, a release factor comes in and binds the STOP codon resulting in the release of the protein a dissociation of the complex.
Protein Folding and Post-Translational Modifications
Translation determines the sequence of amino acids in proteins. The sequence of amino acids (primary protein structure) in turn determines how it folds. Post-translational modifications may be necessary before the protein becomes functional. These modifications may include the following:
- Phosphorylation: Additional of phosphate groups by protein kinases
- Methylation: Addition of methyl groups by methyltransferases. This increases hydrophobicity of proteins and is important in epigenetic regulation
- Glycosylation: Adding sugar components to the protein. This affects conformation and folding. Glycosylated proteins may act as receptors for certain biological molecule
- Proteolysis: Breaking of proteins by proteases to enable activation, e.g. the conversion of zymogens (pro-enzymes) to active enzymes
- N-Acetylation: The transfer of an acetyl group onto the nitrogen of amino acids. Acetylation of histones reduce their ability to fold and hence opens up the DNA for transcription
- Lipidation: Addition of lipid components into peptides. This enable proteins to be translocated to hydrophobic regions of the cell, e.g. membranes
Mutations Affecting Protein Structure and Function
Changes in the genetic information of a cell (mutation) may occur due to errors during DNA replication or recombination, and with exposure to mutagens such as radiation, chemicals and viruses. Even small-scale changes can lead to detrimental effects. Examples of small-scale mutations are shown in the figure below. The original codon, UAC produces the amino acid tyrosine. However, if a nucleotide is added or deleted, this causes a frameshift mutation. That means every codon that follows will be affected resulting in the protein producing completely different amino acids. A change in only one nucleotide may result in either a missense or a silent mutation. A missense mutation produces a new amino acid, in this case, asparagine. This change may or may not be critical to the protein conformation and function. A silent mutation is harmless since it ends up coding for the same amino acid. A nonsense mutation occurs when a stop codon is produced, resulting in premature termination of translation. This almost certainly will lead to a nonfunctional protein.
Reference: Reece, J. B., & Campbell, N. A. (2011). Campbell biology. Boston: Benjamin Cummings / Pearson.
