CHAPTER 21: Unit 9. Gene Expression and Protein Synthesis

Gene expression is the process by which information from a gene is used in the synthesis of a functional gene product. These products are often proteins, but in non-protein coding genes such as transfer RNA (tRNA) or small nuclear RNA (snRNA) genes, the product is a functional RNA.

Genes encode proteins and proteins dictate cell function. Moreover, each step in the flow of information from DNA to RNA to protein provides the cell with a potential control point for self-regulating its functions by adjusting the amount and type of proteins it manufactures.

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Through a cell biology lens, the study of gene expression is tightly linked to our understanding of proteins. Since the early work of Christian Anfinsen in the 1950s, we know that the sequence of amino acids in a protein determines its final three-dimensional structure. Following from that, scientists have repeatedly observed that protein structure dictates where it will act and what it will do. Nowhere has this been more obvious than with the function of enzymes. The shape and structure of proteins is a crucial aspect of gene expression biology and links our understanding of gene expression to the biology of the cell. While primarily concerned with protein molecules that act on DNA and RNA sequences, such as transcription factors and histones, the study of gene expression also focuses on where in the cell expression is modulated. In fact, the modulation of gene expression can occur in the nucleus, the cytoplasm, or even at the cell membrane due to the impact of proteins on RNA in those cellular subregions.

How do scientists study protein shape and function?

A technique called mass spectrometry permits scientists to sequence the amino acids in a protein. After a sequence is known, comparing its amino acid sequence with databases allows scientists to discover if there are related proteins whose function is already known. Often similar amino acid sequences will have similar functions within a cell. The amino acid sequence also allows scientists to predict the charge of the molecule, its size, and its probable three-dimensional structure. The charge and size can later be confirmed experimentally (via SDS-PAGE and double-dimension gels). To deduce the intricacies of three-dimensional structure, scientists will try to crystallize the protein to confirm its molecular structure through X-ray crystallography and/or nuclear magnetic resonance spectroscopy (pNMR).

How do scientists study the impact of proteins on genes or other proteins?

A good way to study the function of the protein is to see what happens in the cell when the protein is not present. For this, scientists use model systems, such as cell culture or whole organisms, wherein they can test the function of specific proteins or genes by modifying or mutating them. The expression level of a gene can be calculated by measuring the transcribed mRNA (northern blot), the expressed protein (Western Blot), or by directly staining the protein or mRNA when it is still in the cell. New techniques have changed the way we study gene expression — DNA microarrays, serial analysis of gene expression (SAGE), and high-throughput sequencing allow larger screens of multiple molecules simultaneously and have opened up the possibility of new and broader kinds of questions. To analyze large datasets and see how networks of molecules interact, a new discipline called systems biology provides the framework for these larger and more integrated understandings of regulatory networks.

Interestingly, proteins are not the only gene regulators. Regulatory molecules come in the form of RNA and act on other nucleic acids by changing or disrupting them. One example is the family of riboswitches, ribonucleic acid molecules that form three-dimensional structures that halt or interfere with transcription, given the proper external signal. Another example of RNA acting on other RNA is the mechanism of RNA interference (RNAi), whereby double-stranded RNA molecules degrade mRNA before translation, thus effectively interfering with protein expression. The dissection of this mechanism and its subsequent experimental imitation has been a boon to those interested in manipulating gene function.

Ultimately, results from these kinds of studies have fundamental relevance, from the basic understanding of normal cell function, such as cell differentiation, growth, and division, to informing radically new approaches for treating disease. In fact, some human diseases can arise simply from a defect in a protein’s three-dimensional structure. Through the study of gene expression and proteins, it is easy to see how minute changes at the molecular level have a reverberating impact.

Protein Synthesis
Protein synthesis is the process in which cells make proteins. It occurs in two stages: transcription and translation. Transcription is the transfer of genetic instructions in DNA to mRNA in the nucleus. It includes three steps: initiation, elongation, and termination.

Protein synthesis is the process all cells use to make proteins, which are responsible for all cell structure and function. There are two main steps to protein synthesis. In transcription, DNA is copied to mRNA, which is used as a template for the instructions to make protein.

Proteins are synthesized stepwise by the polymerization of amino acids in a unidirectional manner, beginning at the N-terminus and ending at the C-terminus. The amino acids are linked by the formation of peptide bonds, and the resulting polypeptide chain contains one of 20 different amino acids at each position. For protein synthesis, a messenger RNA (mRNA) molecule copied from DNA provides the instruction for the synthesis of a specific protein. The information encoded in the sequence of bases in the mRNA is translated by transfer RNA (tRNA) molecules that bind to the mRNA at one end, and carry specific amino acids at the other end. The synthesis of the growing polypeptide chain is carried out on ribosomes, that contain RNA and associated proteins. Additional specific protein factors aid in the initiation, elongation and termination of protein synthesis. Genetic information is encoded as a series of three bases, or triplets, in the mRNA. The 64 triplets and the amino acids they specify are called the genetic code. In most organisms three (and sometimes two) of the triplets signal chain termination.

Protein Synthesis

Protein synthesis is the process all cells use to make proteins, which are responsible for all cell structure and function. In transcription, DNA is copied to mRNA, which is used as a template for the instructions to make protein. In the second step, translation, the mRNA is read by a ribosome.We can regard protein synthesis as a chemical reaction, and we shall take this approach at first. Then we shall take a three-dimensional look at the physical interactions of the major components.In protein synthesis as a chemical reaction:

1. Each amino acid is attached to a tRNA molecule specific to that amino acid by a high-energy bond derived from ATP. The process is catalyzed by a specific enzyme called a synthetase (the tRNA is said to be “charged” when the amino acid is attached):

There is a separate synthetase for each amino acid.

2. The energy of the charged tRNA is converted into a peptide bond linking the amino acid to another one on the ribosome:

3. New amino acids are linked by means of a peptide bond to the growing chain:

4. This process continues until aa n (the final amino acid) is added. The whole thing works only in the presence of mRNA, ribosomes, several additional protein factors, enzymes, and inorganic ions.

Steps in Protein Synthesis

STEP 1: The first step in protein synthesis is the transcription of mRNA from a DNA gene in the nucleus. At some other prior time, the various other types of RNA have been synthesized using the appropriate DNA. The RNAs migrate from the nucleus into the cytoplasm.Prior to the beginning of the protein synthesis, all of the component parts are assembled in the ribosome which is the brown/tan structure in the left graphic.STEP 2: Initiation:In the cytoplasm, protein synthesis is actually initiated by the AUG codon on mRNA. The AUG codon signals both the interaction of the ribosome with m-RNA and also the tRNA with the anticodons (UAC). The tRNA which initiates the protein synthesis has N-formyl-methionine attached. The formyl group is really formic acid converted to an amide using the -NH2 group on methionine (left most graphic)The next step is for a second tRNA to approach the mRNA (codon – CCG). This is the code for proline. The anticodon of the proline tRNA which reads this is GGC. The final process is to start growing peptide chain by having amine of proline to bond to the carboxyl acid group of methinone (met) in order to elongate the peptide.STEP 3: Elongation:Elongation of the peptide begins as various tRNA’s read the next codon. In the example on the left the next tRNA to read the mRNA is tyrosine. When the correct match with the anticodons of a tRNA has been found, the tyrosine forms a peptide bond with the growing peptide chain .The proline is now hydrolyzed from the tRNA. The proline tRNA now moves away from the ribosome and back into the cytoplasm to reattach another proline amino acid.Step 4: Elongation and Termination:When the stop signal on mRNA is reached, the protein synthesis is terminated. The last amino acid is hydrolyzed from its t-RNA.The peptide chain leaves the ribosome. The N-formyl-methionine that was used to initiate the protein synthesis is also hydrolyzed from the completed peptide at this time. The ribosome is now ready to repeat the synthesis several more times.

Reference: http://chemistry.elmhurst.edu/vchembook/584proteinsyn.html