The Central Dogma: DNA Encodes RNA; RNA Encodes Protein

The flow of genetic information in cells from DNA to mRNA to protein is described by the central dogma ([Figure 1]), which states that genes specify the sequences of mRNAs, which in turn specify the sequences of proteins.

The copying of DNA to mRNA is relatively straightforward, with one nucleotide being added to the mRNA strand for every complementary nucleotide read in the DNA strand. The translation to protein is more complex because groups of three mRNA nucleotides correspond to one amino acid of the protein sequence. However, as we shall see in the next module, the translation to protein is still systematic, such that nucleotides 1 to 3 correspond to amino acid 1, nucleotides 4 to 6 correspond to amino acid 2, and so on.

Transcription: from DNA to mRNA

Both prokaryotes and eukaryotes perform fundamentally the same process of transcription, with the important difference of the membrane-bound nucleus in eukaryotes. With the genes bound in the nucleus, transcription occurs in the nucleus of the cell and the mRNA transcript must be transported to the cytoplasm. The prokaryotes, which include bacteria and archaea, lack membrane-bound nuclei and other organelles, and transcription occurs in the cytoplasm of the cell. In both prokaryotes and eukaryotes, transcription occurs in three main stages: initiation, elongation, and termination.

Initiation

Transcription requires the DNA double helix to partially unwind in the region of mRNA synthesis. The region of unwinding is called a transcription bubble. The DNA sequence onto which the proteins and enzymes involved in transcription bind to initiate the process is called a promoter. In most cases, promoters exist upstream of the genes they regulate. The specific sequence of a promoter is very important because it determines whether the corresponding gene is transcribed all of the time, some of the time, or hardly at all ([Figure 2]).

Elongation

Transcription always proceeds from one of the two DNA strands, which is called the template strand. The mRNA product is complementary to the template strand and is almost identical to the other DNA strand, called the nontemplate strand, with the exception that RNA contains a uracil (U) in place of the thymine (T) found in DNA. During elongation, an enzyme called RNA polymerase proceeds along the DNA template adding nucleotides by base pairing with the DNA template in a manner similar to DNA replication, with the difference that an RNA strand is being synthesized that does not remain bound to the DNA template. As elongation proceeds, the DNA is continuously unwound ahead of the core enzyme and rewound behind it ([Figure 3]).

Termination

Once a gene is transcribed, the prokaryotic polymerase needs to be instructed to dissociate from the DNA template and liberate the newly made mRNA. Depending on the gene being transcribed, there are two kinds of termination signals, but both involve repeated nucleotide sequences in the DNA template that result in RNA polymerase stalling, leaving the DNA template, and freeing the mRNA transcript.

On termination, the process of transcription is complete. In a prokaryotic cell, by the time termination occurs, the transcript would already have been used to partially synthesize numerous copies of the encoded protein because these processes can occur concurrently using multiple ribosomes (polyribosomes) ([Figure 4]). In contrast, the presence of a nucleus in eukaryotic cells precludes simultaneous transcription and translation.

Eukaryotic RNA Processing

The newly transcribed eukaryotic mRNAs must undergo several processing steps before they can be transferred from the nucleus to the cytoplasm and translated into a protein. The additional steps involved in eukaryotic mRNA maturation create a molecule that is much more stable than a prokaryotic mRNA. For example, eukaryotic mRNAs last for several hours, whereas the typical prokaryotic mRNA lasts no more than five seconds.

The mRNA transcript is first coated in RNA-stabilizing proteins to prevent it from degrading while it is processed and exported out of the nucleus. This occurs while the pre-mRNA still is being synthesized by adding a special nucleotide “cap” to the 5′ end of the growing transcript. In addition to preventing degradation, factors involved in protein synthesis recognize the cap to help initiate translation by ribosomes.

Once elongation is complete, an enzyme then adds a string of approximately 200 adenine residues to the 3′ end, called the poly-A tail. This modification further protects the pre-mRNA from degradation and signals to cellular factors that the transcript needs to be exported to the cytoplasm.

Eukaryotic genes are composed of protein-coding sequences called exons (ex-on signifies that they are expressed) and intervening sequences called introns (int-ron denotes their intervening role). Introns are removed from the pre-mRNA during processing. Intron sequences in mRNA do not encode functional proteins. It is essential that all of a pre-mRNA’s introns be completely and precisely removed before protein synthesis so that the exons join together to code for the correct amino acids. If the process errs by even a single nucleotide, the sequence of the rejoined exons would be shifted, and the resulting protein would be nonfunctional. The process of removing introns and reconnecting exons is called splicing ([Figure 5]). Introns are removed and degraded while the pre-mRNA is still in the nucleus.

The Protein Synthesis Machinery

In addition to the mRNA template, many other molecules contribute to the process of translation. The composition of each component may vary across species; for instance, ribosomes may consist of different numbers of ribosomal RNAs (rRNA) and polypeptides depending on the organism. However, the general structures and functions of the protein synthesis machinery are comparable from bacteria to human cells. Translation requires the input of an mRNA template, ribosomes, tRNAs, and various enzymatic factors ([Figure 1]).

In E. coli, there are 200,000 ribosomes present in every cell at any given time. A ribosome is a complex macromolecule composed of structural and catalytic rRNAs, and many distinct polypeptides. In eukaryotes, the nucleolus is completely specialized for the synthesis and assembly of rRNAs.

Ribosomes are located in the cytoplasm in prokaryotes and in the cytoplasm and endoplasmic reticulum of eukaryotes. Ribosomes are made up of a large and a small subunit that come together for translation. The small subunit is responsible for binding the mRNA template, whereas the large subunit sequentially binds tRNAs, a type of RNA molecule that brings amino acids to the growing chain of the polypeptide. Each mRNA molecule is simultaneously translated by many ribosomes, all synthesizing protein in the same direction.

Depending on the species, 40 to 60 types of tRNA exist in the cytoplasm. Serving as adaptors, specific tRNAs bind to sequences on the mRNA template and add the corresponding amino acid to the polypeptide chain. Therefore, tRNAs are the molecules that actually “translate” the language of RNA into the language of proteins. For each tRNA to function, it must have its specific amino acid bonded to it. In the process of tRNA “charging,” each tRNA molecule is bonded to its correct amino acid.

The Genetic Code

To summarize what we know to this point, the cellular process of transcription generates messenger RNA (mRNA), a mobile molecular copy of one or more genes with an alphabet of A, C, G, and uracil (U). Translation of the mRNA template converts nucleotide-based genetic information into a protein product. Protein sequences consist of 20 commonly occurring amino acids; therefore, it can be said that the protein alphabet consists of 20 letters. Each amino acid is defined by a three-nucleotide sequence called the triplet codon. The relationship between a nucleotide codon and its corresponding amino acid is called the genetic code.

Given the different numbers of “letters” in the mRNA and protein “alphabets,” combinations of nucleotides corresponded to single amino acids. Using a three-nucleotide code means that there are a total of 64 (4 × 4 × 4) possible combinations; therefore, a given amino acid is encoded by more than one nucleotide triplet ([Figure 2]).

Three of the 64 codons terminate protein synthesis and release the polypeptide from the translation machinery. These triplets are called stop codons. Another codon, AUG, also has a special function. In addition to specifying the amino acid methionine, it also serves as the start codon to initiate translation. The reading frame for translation is set by the AUG start codon near the 5′ end of the mRNA. The genetic code is universal. With a few exceptions, virtually all species use the same genetic code for protein synthesis, which is powerful evidence that all life on Earth shares a common origin.

The Mechanism of Protein Synthesis

Just as with mRNA synthesis, protein synthesis can be divided into three phases: initiation, elongation, and termination. The process of translation is similar in prokaryotes and eukaryotes. Here we will explore how translation occurs in E. coli, a representative prokaryote, and specify any differences between prokaryotic and eukaryotic translation.

Protein synthesis begins with the formation of an initiation complex. In E. coli, this complex involves the small ribosome subunit, the mRNA template, three initiation factors, and a special initiator tRNA. The initiator tRNA interacts with the AUG start codon, and links to a special form of the amino acid methionine that is typically removed from the polypeptide after translation is complete.

In prokaryotes and eukaryotes, the basics of polypeptide elongation are the same, so we will review elongation from the perspective of E. coli. The large ribosomal subunit of E. coli consists of three compartments: the A site binds incoming charged tRNAs (tRNAs with their attached specific amino acids). The P site binds charged tRNAs carrying amino acids that have formed bonds with the growing polypeptide chain but have not yet dissociated from their corresponding tRNA. The E site releases dissociated tRNAs so they can be recharged with free amino acids. The ribosome shifts one codon at a time, catalyzing each process that occurs in the three sites. With each step, a charged tRNA enters the complex, the polypeptide becomes one amino acid longer, and an uncharged tRNA departs. The energy for each bond between amino acids is derived from GTP, a molecule similar to ATP ([Figure 3]). Amazingly, the E. coli translation apparatus takes only 0.05 seconds to add each amino acid, meaning that a 200-amino acid polypeptide could be translated in just 10 seconds.

Termination of translation occurs when a stop codon (UAA, UAG, or UGA) is encountered. When the ribosome encounters the stop codon, the growing polypeptide is released and the ribosome subunits dissociate and leave the mRNA. After many ribosomes have completed translation, the mRNA is degraded so the nucleotides can be reused in another transcription reaction.

Prokaryotic versus Eukaryotic Gene Expression

To understand how gene expression is regulated, we must first understand how a gene becomes a functional protein in a cell. The process occurs in both prokaryotic and eukaryotic cells, just in slightly different fashions.

Because prokaryotic organisms lack a cell nucleus, the processes of transcription and translation occur almost simultaneously. When the protein is no longer needed, transcription stops. As a result, the primary method to control what type and how much protein is expressed in a prokaryotic cell is through the regulation of DNA transcription into RNA. All the subsequent steps happen automatically. When more protein is required, more transcription occurs. Therefore, in prokaryotic cells, the control of gene expression is almost entirely at the transcriptional level.

The first example of such control was discovered using E. coli in the 1950s and 1960s by French researchers and is called the lac operon. The lac operon is a stretch of DNA with three adjacent genes that code for proteins that participate in the absorption and metabolism of lactose, a food source for E. coli. When lactose is not present in the bacterium’s environment, the lac genes are transcribed in small amounts. When lactose is present, the genes are transcribed and the bacterium is able to use the lactose as a food source. The operon also contains a promoter sequence to which the RNA polymerase binds to begin transcription; between the promoter and the three genes is a region called the operator. When there is no lactose present, a protein known as a repressor binds to the operator and prevents RNA polymerase from binding to the promoter, except in rare cases. Thus very little of the protein products of the three genes is made. When lactose is present, an end product of lactose metabolism binds to the repressor protein and prevents it from binding to the operator. This allows RNA polymerase to bind to the promoter and freely transcribe the three genes, allowing the organism to metabolize the lactose.

Eukaryotic cells, in contrast, have intracellular organelles and are much more complex. Recall that in eukaryotic cells, the DNA is contained inside the cell’s nucleus and it is transcribed into mRNA there. The newly synthesized mRNA is then transported out of the nucleus into the cytoplasm, where ribosomes translate the mRNA into protein. The processes of transcription and translation are physically separated by the nuclear membrane; transcription occurs only within the nucleus, and translation only occurs outside the nucleus in the cytoplasm. The regulation of gene expression can occur at all stages of the process ([Figure 1]). Regulation may occur when the DNA is uncoiled and loosened from nucleosomes to bind transcription factors (epigenetic level), when the RNA is transcribed (transcriptional level), when RNA is processed and exported to the cytoplasm after it is transcribed (post-transcriptional level), when the RNA is translated into protein (translational level), or after the protein has been made (post-translational level).

The differences in the regulation of gene expression between prokaryotes and eukaryotes are summarized in [link].

Evolution in Action

Alternative RNA SplicingIn the 1970s, genes were first observed that exhibited alternative RNA splicing. Alternative RNA splicing is a mechanism that allows different protein products to be produced from one gene when different combinations of introns (and sometimes exons) are removed from the transcript ([Figure 2]). This alternative splicing can be haphazard, but more often it is controlled and acts as a mechanism of gene regulation, with the frequency of different splicing alternatives controlled by the cell as a way to control the production of different protein products in different cells, or at different stages of development. Alternative splicing is now understood to be a common mechanism of gene regulation in eukaryotes; according to one estimate, 70% of genes in humans are expressed as multiple proteins through alternative splicing.

How could alternative splicing evolve? Introns have a beginning and ending recognition sequence, and it is easy to imagine the failure of the splicing mechanism to identify the end of an intron and find the end of the next intron, thus removing two introns and the intervening exon. In fact, there are mechanisms in place to prevent such exon skipping, but mutations are likely to lead to their failure. Such “mistakes” would more than likely produce a nonfunctional protein. Indeed, the cause of many genetic diseases is alternative splicing rather than mutations in a sequence. However, alternative splicing would create a protein variant without the loss of the original protein, opening up possibilities for adaptation of the new variant to new functions. Gene duplication has played an important role in the evolution of new functions in a similar way—by providing genes that may evolve without eliminating the original functional protein.