11.1 The Functions of Genetic Material
- DNA serves two important cellular functions: It is the genetic material passed from parent to offspring and it serves as the information to direct and regulate the construction of the proteins necessary for the cell to perform all of its functions.
- The central dogma states that DNA organized into genes specifies the sequences of messenger RNA (mRNA), which, in turn, specifies the amino acid sequence of proteins.
- The genotype of a cell is the full collection of genes a cell contains. Not all genes are used to make proteins simultaneously. The phenotype is a cell’s observable characteristics resulting from the proteins it is producing at a given time under specific environmental conditions.
11.2 DNA Replication
- The DNA replication process is semiconservative, which results in two DNA molecules, each having one parental strand of DNA and one newly synthesized strand.
- In bacteria, the initiation of replication occurs at the origin of replication, where supercoiled DNA is unwound by DNA gyrase, made single-stranded by helicase, and bound by single-stranded binding protein to maintain its single-stranded state. Primase synthesizes a short RNA primer, providing a free 3’-OH group to which DNA polymerase III can add DNA nucleotides.
- During elongation, the leading strand of DNA is synthesized continuously from a single primer. The lagging strand is synthesized discontinuously in short Okazaki fragments, each requiring its own primer. The RNA primers are removed and replaced with DNA nucleotides by bacterial DNA polymerase I, and DNA ligase seals the gaps between these fragments.
- Termination of replication in bacteria involves the resolution of circular DNA concatemers by topoisomerase IV to release the two copies of the circular chromosome.
- Eukaryotes typically have multiple linear chromosomes, each with multiple origins of replication. Overall, replication in eukaryotes is similar to that in prokaryotes.
- The linear nature of eukaryotic chromosomes necessitates telomeres to protect genes near the end of the chromosomes. Telomerase extends telomeres, preventing their degradation, in some cell types.
- Rolling circle replication is a type of rapid unidirectional DNA synthesis of a circular DNA molecule used for the replication of some plasmids.
11.3 RNA Transcription
- During transcription, the information encoded in DNA is used to make RNA.
- RNA polymerase synthesizes RNA, using the antisense strand of the DNA as template by adding complementary RNA nucleotides to the 3’ end of the growing strand.
- RNA polymerase binds to DNA at a sequence called a promoter during the initiation of transcription.
- Genes encoding proteins of related functions are frequently transcribed under the control of a single promoter in prokaryotes, resulting in the formation of a polycistronic mRNA molecule that encodes multiple polypeptides.
- Unlike DNA polymerase, RNA polymerase does not require a 3’-OH group to add nucleotides, so a primer is not needed during initiation.
- Termination of transcription in bacteria occurs when the RNA polymerase encounters specific DNA sequences that lead to stalling of the polymerase. This results in release of RNA polymerase from the DNA template strand, freeing the RNA transcript.
- Eukaryotes have three different RNA polymerases. Eukaryotes also have monocistronic mRNA, each encoding only a single polypeptide.
- Eukaryotic primary transcripts are processed in several ways, including the addition of a 5’ cap and a 3′-poly-A tail, as well as splicing, to generate a mature mRNA molecule that can be transported out of the nucleus and that is protected from degradation.
11.4 Protein Synthesis (Translation)
- In translation, polypeptides are synthesized using mRNA sequences and cellular machinery, including tRNAs that match mRNA codons to specific amino acids and ribosomes composed of RNA and proteins that catalyze the reaction.
- The genetic code is degenerate in that several mRNA codons code for the same amino acids. The genetic code is almost universal among living organisms.
- Prokaryotic (70S) and cytoplasmic eukaryotic (80S) ribosomes are each composed of a large subunit and a small subunit of differing sizes between the two groups. Each subunit is composed of rRNA and protein. Organelle ribosomes in eukaryotic cells resemble prokaryotic ribosomes.
- Some 60 to 90 species of tRNA exist in bacteria. Each tRNA has a three-nucleotide anticodon as well as a binding site for a cognate amino acid. All tRNAs with a specific anticodon will carry the same amino acid.
- Initiation of translation occurs when the small ribosomal subunit binds with initiation factors and an initiator tRNA at the start codon of an mRNA, followed by the binding to the initiation complex of the large ribosomal subunit.
- In prokaryotic cells, the start codon codes for N-formyl-methionine carried by a special initiator tRNA. In eukaryotic cells, the start codon codes for methionine carried by a special initiator tRNA. In addition, whereas ribosomal binding of the mRNA in prokaryotes is facilitated by the Shine-Dalgarno sequence within the mRNA, eukaryotic ribosomes bind to the 5’ cap of the mRNA.
- During the elongation stage of translation, a charged tRNA binds to mRNA in the A site of the ribosome; a peptide bond is catalyzed between the two adjacent amino acids, breaking the bond between the first amino acid and its tRNA; the ribosome moves one codon along the mRNA; and the first tRNA is moved from the P site of the ribosome to the E site and leaves the ribosomal complex.
- Termination of translation occurs when the ribosome encounters a stop codon, which does not code for a tRNA. Release factors cause the polypeptide to be released, and the ribosomal complex dissociates.
- In prokaryotes, transcription and translation may be coupled, with translation of an mRNA molecule beginning as soon as transcription allows enough mRNA exposure for the binding of a ribosome, prior to transcription termination. Transcription and translation are not coupled in eukaryotes because transcription occurs in the nucleus, whereas translation occurs in the cytoplasm or in association with the rough endoplasmic reticulum.
- Polypeptides often require one or more post-translational modifications to become biologically active.
- A mutation is a heritable change in DNA. A mutation may lead to a change in the amino-acid sequence of a protein, possibly affecting its function.
- A point mutation affects a single base pair. A point mutation may cause a silent mutation if the mRNA codon codes for the same amino acid, a missense mutation if the mRNA codon codes for a different amino acid, or a nonsense mutation if the mRNA codon becomes a stop codon.
- Missense mutations may retain function, depending on the chemistry of the new amino acid and its location in the protein. Nonsense mutations produce truncated and frequently nonfunctional proteins.
- A frameshift mutation results from an insertion or deletion of a number of nucleotides that is not a multiple of three. The change in reading frame alters every amino acid after the point of the mutation and results in a nonfunctional protein.
- Spontaneous mutations occur through DNA replication errors, whereas induced mutations occur through exposure to a mutagen.
- Mutagenic agents are frequently carcinogenic but not always. However, nearly all carcinogens are mutagenic.
- Chemical mutagens include base analogs and chemicals that modify existing bases. In both cases, mutations are introduced after several rounds of DNA replication.
- Ionizing radiation, such as X-rays and γ-rays, leads to breakage of the phosphodiester backbone of DNA and can also chemically modify bases to alter their base-pairing rules.
- Nonionizing radiation like ultraviolet light may introduce pyrimidine (thymine) dimers, which, during DNA replication and transcription, may introduce frameshift or point mutations.
- Cells have mechanisms to repair naturally occurring mutations. DNA polymerase has proofreading activity. Mismatch repair is a process to repair incorrectly incorporated bases after DNA replication has been completed.
- Pyrimidine dimers can also be repaired. In nucleotide excision repair (dark repair), enzymes recognize the distortion introduced by the pyrimidine dimer and replace the damaged strand with the correct bases, using the undamaged DNA strand as a template. Bacteria and other organisms may also use direct repair, in which the photolyase enzyme, in the presence of visible light, breaks apart the pyrimidines.
- Through comparison of growth on the complete plate and lack of growth on media lacking specific nutrients, specific loss-of-function mutants called auxotrophs can be identified.
- The Ames test is an inexpensive method that uses auxotrophic bacteria to measure mutagenicity of a chemical compound. Mutagenicity is an indicator of carcinogenic potential.
11.6 How Asexual Prokaryotes Achieve Genetic Diversity
- Horizontal gene transfer is an important way for asexually reproducing organisms like prokaryotes to acquire new traits.
- There are three mechanisms of horizontal gene transfer typically used by bacteria: transformation, transduction, and conjugation.
- Transformation allows for competent cells to take up naked DNA, released from other cells on their death, into their cytoplasm, where it may recombine with the host genome.
- In generalized transduction, any piece of chromosomal DNA may be transferred by accidental packaging of the degraded host chromosome into a phage head. In specialized transduction, only chromosomal DNA adjacent to the integration site of a lysogenic phage may be transferred as a result of imprecise excision of the prophage.
- Conjugation is mediated by the F plasmid, which encodes a conjugation pilus that brings an F plasmid-containing F+ cell into contact with an F- cell.
- The rare integration of the F plasmid into the bacterial chromosome, generating an Hfr cell, allows for transfer of chromosomal DNA from the donor to the recipient. Additionally, imprecise excision of the F plasmid from the chromosome may generate an F’ plasmid that may be transferred to a recipient by conjugation.
- Conjugation transfer of R plasmids is an important mechanism for the spread of antibiotic resistance in bacterial communities.
- Transposons are molecules of DNA with inverted repeats at their ends that also encode the enzyme transposase, allowing for their movement from one location in DNA to another. Although found in both prokaryotes and eukaryotes, transposons are clinically relevant in bacterial pathogens for the movement of virulence factors, including antibiotic resistance genes.
11.7 Gene Regulation: Operon Theory
- Gene expression is a tightly regulated process.
- Gene expression in prokaryotes is largely regulated at the point of transcription. Gene expression in eukaryotes is additionally regulated post-transcriptionally.
- Prokaryotic structural genes of related function are often organized into operons, all controlled by transcription from a single promoter. The regulatory region of an operon includes the promoter itself and the region surrounding the promoter to which transcription factors can bind to influence transcription.
- Although some operons are constitutively expressed, most are subject to regulation through the use of transcription factors (repressors and activators). A repressor binds to an operator, a DNA sequence within the regulatory region between the RNA polymerase binding site in the promoter and first structural gene, thereby physically blocking transcription of these operons. An activator binds within the regulatory region of an operon, helping RNA polymerase bind to the promoter, thereby enhancing the transcription of this operon. An inducer influences transcription through interacting with a repressor or activator.
- The trp operon is a classic example of a repressible operon. When tryptophan accumulates, tryptophan binds to a repressor, which then binds to the operator, preventing further transcription.
- The lac operon is a classic example an inducible operon. When lactose is present in the cell, it is converted to allolactose. Allolactose acts as an inducer, binding to the repressor and preventing the repressor from binding to the operator. This allows transcription of the structural genes.
- The lac operon is also subject to activation. When glucose levels are depleted, some cellular ATP is converted into cAMP, which binds to the catabolite activator protein (CAP). The cAMP-CAP complex activates transcription of the lac operon. When glucose levels are high, its presence prevents transcription of the lac operon and other operons by catabolite repression.
- Small intracellular molecules called alarmones are made in response to various environmental stresses, allowing bacteria to control the transcription of a group of operons, called a regulon.
- Bacteria have the ability to change which σ factor of RNA polymerase they use in response to environmental conditions to quickly and globally change which regulons are transcribed.
- Prokaryotes have regulatory mechanisms, including attenuation and the use of riboswitches, to simultaneously control the completion of transcription and translation from that transcript. These mechanisms work through the formation of stem loops in the 5’ end of an mRNA molecule currently being synthesized.
- There are additional points of regulation of gene expression in prokaryotes and eukaryotes. In eukaryotes, epigenetic regulation by chemical modification of DNA or histones, and regulation of RNA processing are two methods.