DNA
DNA's double helical structure is deceptively simple, yet the rules encoded in this structure specify the form and function of all cells within an organism (Fig. 1). DNA consists of two long strands of polydeoxyribonucleotides that twist around each other clockwise to form an unbroken double helix. Alternating deoxyribose phosphate groups form the backbone of the helix, with the phosphate group making a 5′-3′ phosphodiester bond between the fifth carbon of one pentose ring and the third carbon of the next pentose ring (Fig. 2). Nucleic acid bases attached to the sugar groups of each strand face each other within the helix, perpendicular to the strand axis. The order of the nucleic acids specifies the eventual sequence of the protein product of the gene. There are four bases, the purines adenine and guanine (A and G) and the pyrimidines cytosine and thymine (C and T). During assembly of the double helix, a purine can pair only with a pyrimidine, and a pyrimidine with a purine. Each base pair (bp) forms one of the rungs in the twisted ladder of the DNA molecule, which can be millions of bases long. The two strands of DNA, which are held together by hydrogen bonds between complementary base pairs, have opposite chemical polarities. One strand is oriented in a 5′ to 3′ direction, whereas the other is in a 3′ to 5′ direction. Enzymes that recognize specific DNA sequences also recognize the polarity of the strand. An enzyme “reads” the nucleotide sequences on the two strands in opposite directions. Because the structure of the helical backbone is invariant, enzymes responsible for DNA copying, cleavage, and repairing strand breaks can act anywhere along the length of the DNA strand.
FIGURE 1 Depiction of the storage of genetic information in homologous chromosomes, which contain genes made up of DNA. Genetic expression involves transcription of DNA into RNA, which is translated on a ribosome into protein.
FIGURE 2 Schematic representation of the DNA double helix. The specificity of genetic information is carried in the four bases—guanine, adenine, thymine, and cytosine—that extend inward from a sugar phosphate backbone and form pairs with complementary bases on the opposing strand.
An important consequence of the A-T and G-C pairing is that the sequence of nucleotides on one strand of the double helix determines the sequence on the complementary strand. This base pairing rule is critical for the storage, retrieval, and transfer of genetic material, whether it be for duplication of DNA into a daughter cell, repair of a damaged DNA strand, or reading as a template for RNA transcription.
Chromosomes are long double helical strands of DNA tightly coiled into compacted, discrete lengths by nuclear proteins. Each chromosome varies in length and base pair composition. In human cells, the nucleus contains 23 different pairs of chromosomes, each with a specific length and base pair sequence. The combined DNA sequences (approximately 3 X 109) on all the chromosomes within a cell comprise the genome. The information carried in the genome is identical in all cells of an organism and varies little among members of a species. Indeed, the genome of humans is approximately 99% identical among individuals.
During cell division, enzymes called polymerases unwind the DNA helix in each chromosome and copy each of the two strands separately along their entire length. Each daughter cell inherits a DNA molecule containing one old and one new strand. Each of these strands can in turn generate a new strand that faithfully reproduces the original template. This fidelity of DNA replication is essential for accurate transfer of genetic information. Errors in this process are a common source of gene mutations, which are inherited in successive rounds of cell division.
A gene is a section of base sequences used as a template for the copying process of transcription, and therefore is the fundamental unit of inherited DNA information. Genes comprise only a small fraction of the DNA carried on a chromosome. Only 1% to 2% of its bases encode proteins, and the full complement of protein-coding sequences still remains to be established. The human genome contains an estimated 30,000 distinct genes. The protein coding information contained within a single gene is not continuous, but instead is encoded in multiple discontinuous packets called exons. Between these exons are variably sized stretches of DNA called introns. The function of these introns is not known. They probably contain the bulk of the regulatory information controlling the expression of the approximately 30,000 protein-coding genes, and other functional elements such as non–protein-coding genes and the sequence determinants of chromosome dynamics. Even less is known about the function of the roughly half of the genome consisting of highly repetitive sequences or of the remaining noncoding, nonrepetitive DNA.
Transcription, the first step in the expression of genetic information, serves to carry the genetic information out of the nucleus and into the cytoplasm, where the synthesis of proteins occurs. In this process, transcription of DNA to RNA requires the assembly of a template called messenger RNA (mRNA) in the nucleus (Fig. 3). A specialized enzyme called RNA polymerase copies one of the two DNA strands (the antisense strand), creating a complementary stretch of sequence that is an exact copy of the sense strand. RNA structure differs slightly from that of DNA. One of the RNA bases, uracil, replaces the DNA base thymine, and the RNA sugar phosphate component ribose replaces DNA deoxyribose. Ribose renders the RNA molecule much more susceptible to degradation than the more stable deoxyribose, which allows RNA to respond more rapidly to shifts in cellular signaling and move quickly to the cytoplasm for protein production.
FIGURE 3 The flow of genetic information. Transcription in the nucleus creates a complementary ribonucleic acid copy from one of the DNA strands in the double helix. mRNA is transported into the cytoplasm, where it is translated into protein.
The complex and highly regulated process of converting a gene to a protein involves two major steps, transcription of the DNA by RNA in the nucleus and translation of RNA into protein in the cytoplasm. Transcription begins in the nucleus by copying the DNA sequence of the gene into mRNA (see Fig. 3). The single-stranded RNA is modified at both ends. At the 5′ end, a nucleotide structure called a cap is added to increase translation efficiency by allowing ribosomes to bind to RNA. At the 3′ end, a nucleotide recognizes an A/T-rich sequence in a noncoding region and trims the transcript downstream by about 20 bp. An enzyme that adds a stretch of adenosine to form a polyA tail, which stabilizes the transcript, modifies the newly cleaved 3′ end. The transcript then undergoes splicing to remove intronic sequences. This is a highly regulated process, because unspliced transcripts are highly unstable and are cleared rapidly from the cell. Splicing is an important control point in gene expression; it must be absolutely precise, because the deletion or addition of a single nucleotide at the splice junction would throw the subsequent three-base codon translation of the RNA out of frame. The full significance of RNA splicing is not completely understood, but it must represent a critical point in the regulation of gene expression because of the large expanses of intron sequences and the inability of transcripts to leave the nucleus until their introns are removed.
Once in the cytoplasm, mRNA provides a template for translation or protein synthesis. Translation occurs on a macromolecular complex, like an assembly line, composed of ribosomes. The ribosomes read and translate the nucleotide sequence in mRNA into an amino acid sequence; that is, the four-base mRNA code is translated into the 20–amino acid alphabet of proteins. This genetic code is remarkably simple and has been conserved in most organisms. Every three RNA nucleotides encode for a single amino acid; the codon therefore is a triplet of bases. Permutations of the four RNA nucleotides result in 64 different triplets (4 x 4 x4), so that any of the 20 amino acids can be specified by more than one codon. One of the triplets, AUG, specifies methionine, the amino acid that starts each protein. Three other triplets, UAA, UGA, and UAG, program the ribosome to end translation and are therefore called stop codons.
The conversion of a codon into an amino acid requires an adapter molecule, called transfer RNA (tRNA), to decode mRNA. Each tRNA uses a unique three-base sequence or anticodon to line up with the complementary codon in mRNA (see Fig. 3). Ribosomal enzymes link adjoining amino acids, which frees them from the tRNA adapters and adds them to the growing amino acid chain. The order of the amino acids is specified by the order of the codons on the corresponding mRNA template. Translation then completes the transfer of information from DNA in the nucleus to a unique protein structure.
Because the genetic code is preserved across species, human genetic sequences can be transferred into bacteria, yeast, or insect cells, where the sequences will be replicated and decoded into RNA and protein. This principle constitutes the basis of recombinant DNA technology, which is used to produce recombinant proteins for research and therapeutic purposes (e.g., tissue plasminogen activator).
The process of gene expression requires controlled and precise regulation at multiple steps. Only a small number of genes are expressed within a cell at a given time. One set of genes is constitutively expressed in most cells, referred to colloquially as housekeeping genes. These genes are necessary for cell replication, energy generation, and survival functions. A second set of genes is expressed in a lineage-specific manner (i.e., within certain cells); they are required for cell-specific functions such as contractility. The precise regulation of lineage-specific genes determines the unique identity and function of a particular cell. Another set of genes is induced in response to environmental stimuli. These are required to produce the complex and dynamic patterns of gene expression, which allow an organism to respond to internal and external signals.
Recently, post-transcriptional regulation by small noncoding RNAs, such as microRNAs (miRNAs), has emerged as a central regulator of many cardiovascular processes. miRNAs are a large class of evolutionarily conserved, small, noncoding RNAs, typically 20 to 26 nucleotides in length, that primarily function post-transcriptionally by interacting with the 3′ untranslated region (UTR) of specific target mRNAs in a sequence-specific manner.[10] The first animal miRNA was described in 1993 as a regulator of developmental timing in Caenorhabditis elegans (a roundworm). Recognition that miRNAs are widespread in all eukaryotes, including vertebrates, did not occur until 10 years later. More than 650 miRNAs are encoded in the human genome, and each is thought to target more than 100 mRNAs, resulting in mRNA degradation or translational inhibition. Interactions between miRNAs and mRNAs are thought to require sequence homology in the 5′ region of the miRNA, but significant variance in the degree of complementation in the remaining sequences allows a single miRNA to target a wide range of mRNAs, often regulating multiple genes within a common pathway. As a result, more than one third of mRNAs in the mammalian genome are thought to be regulated by one or more miRNAs. miRNAs regulate gene expression at the post-translational level by mRNA degradation, translation repression, or miRNA-mediated mRNA decay (Fig. 4). The transcription of miRNA genes is mediated by RNA polymerase II (pol II). Inside the nucleus, the pre-miRNA has a stem loop structure that is cleaved by the ribonuclease endonuclease Drosha, leaving a cleaved pre-miRNA that is exported from the nucleus. In the cytoplasm, the ribonuclease endonuclease Dicer further cleaves the pre-miRNA into a mature double-stranded miRNA, which is incorporated into the RNA-induced silencing complex (RISC), allowing preferential strand separation of the mature miRNA to repress mRNA translation or destabilize mRNA transcripts through cleavage or deadenylation.
FIGURE 4 miRNA biogenesis and function. The process of miRNA within the nucleus and cytoplasm is shown (see text for details).
Despite advances in miRNA discovery, the role of miRNAs in physiologic and pathophysiologic processes is just emerging. miRNAs play diverse roles in fundamental biologic processes, such as lineage development, cell proliferation, differentiation, apoptosis, stress response, and tumorigenesis. For example, identification of miRNAs expressing specific cardiac cell types has led to the discovery of important regulatory roles for these small RNAs during cardiomyocyte differentiation, cell cycle, and stages of cardiac hypertrophy in the adult, suggesting that miRNAs may be almost as important as transcription factors in regulating gene expression. A similar story is developing for miRNA regulation of smooth-muscle cell fate and plasticity.
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