15.1 The Genetic Code
By the end of this section, you will be able to do the following:
- Explain the “central dogma” of DNA-protein synthesis
- Describe the genetic code and how the nucleotide sequence prescribes the amino acid and the protein sequence
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 on ribosomes converts nucleotide-based genetic information into a protein product. That is the central dogma of DNA-protein synthesis. Protein sequences consist of 20 commonly occurring amino acids therefore, it can be said that the protein alphabet consists of 20 “letters” (Figure 15.2). Different amino acids have different chemistries (such as acidic versus basic, or polar and nonpolar) and different structural constraints. Variation in amino acid sequence is responsible for the enormous variation in protein structure and function.
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 15.3), which states that genes specify the sequence of mRNAs, which in turn specify the sequence of amino acids making up all proteins. The decoding of one molecule to another is performed by specific proteins and RNAs. Because the information stored in DNA is so central to cellular function, it makes intuitive sense that the cell would make mRNA copies of this information for protein synthesis, while keeping the DNA itself intact and protected. The copying of DNA to RNA is relatively straightforward, with one nucleotide being added to the mRNA strand for every nucleotide read in the DNA strand. The translation to protein is a bit more complex because three mRNA nucleotides correspond to one amino acid in the polypeptide sequence. However, the translation to protein is still systematic and colinear , such that nucleotides 1 to 3 correspond to amino acid 1, nucleotides 4 to 6 correspond to amino acid 2, and so on.
The Genetic Code Is Degenerate and Universal
Each amino acid is defined by a three-nucleotide sequence called the triplet codon. Given the different numbers of “letters” in the mRNA and protein “alphabets,” scientists theorized that single amino acids must be represented by combinations of nucleotides. Nucleotide doublets would not be sufficient to specify every amino acid because there are only 16 possible two-nucleotide combinations (4 2 ). In contrast, there are 64 possible nucleotide triplets (4 3 ), which is far more than the number of amino acids. Scientists theorized that amino acids were encoded by nucleotide triplets and that the genetic code was “degenerate.” In other words, a given amino acid could be encoded by more than one nucleotide triplet. This was later confirmed experimentally: Francis Crick and Sydney Brenner used the chemical mutagen proflavin to insert one, two, or three nucleotides into the gene of a virus. When one or two nucleotides were inserted, the normal proteins were not produced. When three nucleotides were inserted, the protein was synthesized and functional. This demonstrated that the amino acids must be specified by groups of three nucleotides. These nucleotide triplets are called codons . The insertion of one or two nucleotides completely changed the triplet reading frame , thereby altering the message for every subsequent amino acid (Figure 15.5). Though insertion of three nucleotides caused an extra amino acid to be inserted during translation, the integrity of the rest of the protein was maintained.
Scientists painstakingly solved the genetic code by translating synthetic mRNAs in vitro and sequencing the proteins they specified (Figure 15.4).
In addition to codons that instruct the addition of a specific amino acid to a polypeptide chain, three of the 64 codons terminate protein synthesis and release the polypeptide from the translation machinery. These triplets are called nonsense codons , or 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. Following the start codon, the mRNA is read in groups of three until a stop codon is encountered.
The arrangement of the coding table reveals the structure of the code. There are sixteen "blocks" of codons, each specified by the first and second nucleotides of the codons within the block, e.g., the "AC*" block that corresponds to the amino acid threonine (Thr). Some blocks are divided into a pyrimidine half, in which the codon ends with U or C, and a purine half, in which the codon ends with A or G. Some amino acids get a whole block of four codons, like alanine (Ala), threonine (Thr) and proline (Pro). Some get the pyrimidine half of their block, like histidine (His) and asparagine (Asn). Others get the purine half of their block, like glutamate (Glu) and lysine (Lys). Note that some amino acids get a block and a half-block for a total of six codons.
The specification of a single amino acid by multiple similar codons is called "degeneracy." Degeneracy is believed to be a cellular mechanism to reduce the negative impact of random mutations. Codons that specify the same amino acid typically only differ by one nucleotide. In addition, amino acids with chemically similar side chains are encoded by similar codons. For example, aspartate (Asp) and glutamate (Glu), which occupy the GA* block, are both negatively charged. This nuance of the genetic code ensures that a single-nucleotide substitution mutation might specify the same amino acid but have no effect or specify a similar amino acid, preventing the protein from being rendered completely nonfunctional.
The genetic code is nearly universal. With a few minor exceptions, virtually all species use the same genetic code for protein synthesis. Conservation of codons means that a purified mRNA encoding the globin protein in horses could be transferred to a tulip cell, and the tulip would synthesize horse globin. That there is only one genetic code is powerful evidence that all of life on Earth shares a common origin, especially considering that there are about 10 84 possible combinations of 20 amino acids and 64 triplet codons.
Link to Learning
Transcribe a gene and translate it to protein using complementary pairing and the genetic code at this site.
RNA viruses promote activation of the NLRP3 inflammasome through a RIP1-RIP3-DRP1 signaling pathway
The NLRP3 inflammasome functions as a crucial component of the innate immune system in recognizing viral infection, but the mechanism by which viruses activate this inflammasome remains unclear. Here we found that inhibition of the serine-threonine kinases RIP1 (RIPK1) or RIP3 (RIPK3) suppressed RNA virus-induced activation of the NLRP3 inflammasome. Infection with an RNA virus initiated assembly of the RIP1-RIP3 complex, which promoted activation of the GTPase DRP1 and its translocation to mitochondria to drive mitochondrial damage and activation of the NLRP3 inflammasome. Notably, the RIP1-RIP3 complex drove the NLRP3 inflammasome independently of MLKL, an essential downstream effector of RIP1-RIP3-dependent necrosis. Together our results reveal a specific role for the RIP1-RIP3-DRP1 pathway in RNA virus-induced activation of the NLRP3 inflammasome and establish a direct link between inflammation and cell-death signaling pathways.
A. Replication of poliovirus is not affected by inhibitors of fatty acid synthase (orlistat) lipid droplet-associated lipases (DEUP) and lysosome acidification (bafilomycin). HeLa cells were infected with 50 PFU/cell of poliovirus and incubated for 4 h in the presence of 10μM orlistat, 400μM DEUP or 2μM bafilomycin. Expression of the viral non-structural protein 2C is shown, actin is shown as a loading control. B. Lypophagy is not required for activation of PC synthesis upon infection. HeLa cells were infected with poliovirus at an MOI of 10 PFU/cell, and were incubated with 2μM of bafilomycin. At 5 h p.i., the incubation medium was replaced with fresh pre-warmed balanced Earle solution containing propargylcholine. The cells were fixed at 6 h p.i. and processed for click-chemistry-based detection of incorporated propargylcholine and staining of nuclear DNA with Hoechst 33332 for normalization. Propargylcholine incorporation was normalized to that in mock-infected cells. C. Non-significant variability of poliovirus replication in independent choline deprivation experiments. HeLa cells pre-incubated in choline-free medium for
72h were infected with poliovirus and were incubated after infection either in choline-free or choline-supplemented medium. Expression of the viral non-structural protein 2C is shown. The right panel shows viral replication in the experiment used for EM images presented on Fig 7.
A. No significant recruitment of MGL to lipid droplets in either infected or mock-infected HeLa cells. HeLa cells were infected (mock-infected) with poliovirus at an MOI of 10 PFU/cell and at 4 h p.i., they were fixed and processed for immunofluorescent analysis of MGL. B. Recruitment of ATGL to lipid droplets early during poliovirus replication cycle. HeLa cells were infected (mock-infected) with poliovirus at an MOI of 10 PFU/cell and at 3 h p.i., they were fixed and processed for immunofluorescent analysis of a viral antigen 2B and ATGL. Arrows indicate recruitment of ATGL to lipid droplets.
S3 Fig. Translocation of GBF1 and PI4KIIIβ does not depend on membrane synthesis.
HeLa cells pre-incubated in choline-free medium for
72h were infected with poliovirus at an MOI of 10 PFU/cell and were incubated after infection either in choline-free or choline-supplemented medium for 4 h. GBF1 and PI4KIIIβ are concentrated in the Golgi area of mock-infected cells and translocate to perinuclear ring-like structures upon infection in cells incubated in either cholen-free or choline-supplemented media. Note the normal morphology of mock-infected cells incubated for
78h in choline-free medium.
S4 Fig. Inhibition of hydrolysis of lipids in lipid droplets affects the development of poliovirus replication organelles.
HeLa cells were infected with 10 PFU/cell of poliovirus and incubated with 400μM of DEUP for 4 h p.i. A. Transmission EM image, arrows indicated scattered clusters of replication organelles in DEUP-treated cells. B. Distribution of the viral antigen 2B visualized in DEUP-treated and control cells after Triton X-100 permeabilization.
A. Degradation of IκB in infected cells does not depend on activation of membrane synthesis. HeLa cells were pre-incubated in choline-free medium for
72h and were infected with poliovirus at an MOI of 10 PFU/cell and incubated in either a choline-free- or a choline-supplemented medium for 6 h. B. Differential expression of anti-viral response genes in choline-deprived and choline-supplemented poliovirus-infected cells. HeLa cells were pre-incubated in choline-free medium for
72h and were infected with poliovirus at an MOI of 10 PFU/cell and incubated in either a choline-free- or a choline-supplemented medium after infection. At 6 h p.i., the cellular RNA was isolated and analyzed with a qPCR panel profiling 84 human genes involved in anti-viral response (Qiagen). The genes whose expression demonstrated statistically significant difference in expression more than 1.5x are shown. IL6, interleukin 6 (GenBank ID: NM_000600), a cytokine involved in inflammation and the maturation of B cells . NFKBIA, NFKB inhibitor alpha (GenBank ID: NM_020529), encodes a member of the NF-kappa-B inhibitor family which is involved in the control of inflammation . JUN, Jun proto-oncogene, AP-1 transcription factor subunit (GenBank ID: NM_002228), involved in the TLR signaling and control of inflammation . CYLD, CYLD lysine 63 deubiquitinase, (GenBank ID: NM_015247), a negative regulator of multiple signaling pathways . FOS, Fos proto-oncogene, AP-1 transcription factor subunit subunit (GenBank ID: NM_005252), involved in the TLR signaling and control of inflammation . IL8, interleukin 8 (GenBank ID: NM_000584), a major mediator of the inflammatory response . C. Interferon-stimulated genes are expressed similarly in non-infected cells in choline-free and choline-supplemented media. HeLa cells were incubated for 60 h without choline and then incubated overnight with 20 units of universal type 1 interferon also in choline-free medium. After that the IFN-containing medium was removed and the cells were incubated in either choline-free or choline-supplemented medium for additional 6 or 24h.
S6 Fig. A list of genes involved in the anti-viral response whose expression was reliably detected in choline-deprived and choline-supplemented poliovirus-infected cells in a representative experiment.
HeLa cells were pre-incubated in choline-free medium for
72 h and were infected with poliovirus at an MOI of 10 PFU/cell and incubated in either choline-free- or choline-supplemented medium after infection. At 6 h p.i., the cellular RNA was isolated and analyzed with a qPCR panel profiling 84 human genes involved in anti-viral response (Qiagen.