Structure and Function of the Genome

Transcription

‘Transcription’ is the process of the information encoded in DNA being transferred into a strand of mRNA. During transcription the RNA polymerase, which constructs the complementary mRNA, reads from the DNA strand complementary to the RNA molecule. This is known as the antisense strand, while the opposite strand, which has the same base pair composition as the RNA molecule (with thymidine (T) in place of uracil (U) as mentioned previously), is the sense strand. Gene sequences are expressed as the sequence of the sense strand of DNA, although it is in fact the antisense strand which is read ( Fig. 1.4 ). The vast majority of genes consist of a 5′ untranslated region (UTR) containing response elements to which proteins may bind that influence transcription. The 5′ regions of genes are frequently characterised by elements such as the TATA and CAAT boxes (see Fig. 1.3 ) and are often richer in GC pairs than elsewhere in the genome. This is frequently the case around the 5′ ends of ‘housekeeping’ genes that are constitutively expressed in the majority of tissues. There then follows the transcribed sequence. The expressed coding parts of the gene are known as the exons, while the intervening sequences are known as introns. The coding portion of the gene is often interrupted by one or more noncoding intervening sequences, although numerous examples of single exon genes exist. Initially, the RNA molecule transcribes both introns and exons and is known as heavy nuclear RNA (hnRNA). The exons are perfectly spliced out (as marked by the splice boundary sequences) and a protective cap added before the now mature mRNA exits the nucleus. Hence cytoplasmic mRNA consists only of coding regions flanked by UTRs at the two ends. A polyadenine (poly A) tail is added to most mRNA molecules at their 3′ end, facilitated by the polyadenylation signal found past the stop codon in the coding sequence. This tail, found on the great majority of expressed mRNAs, serves to protect the RNA from degradation prior to translation by the ribosome (see later).

Translation

The term ‘translation’ describes the process whereby the cellular machinery reads the mRNA code and creates a chain of polypeptides (i.e. a protein). Once in the cytoplasm, the mRNA message is translated into protein by a ribosome. Ribosomes, consisting of a complex bundle of proteins and ribosomal RNA, attach to mRNA at the 5′ end. Protein synthesis begins at the amino terminal, and amino acids are sequentially added at the freshly made carboxyl end. Amino acids are brought into the reaction by specific transfer RNA (tRNA) molecules. Each tRNA is a single-stranded molecule which folds in a way that allows complementary base pairing between parts of the same strand. The specific configuration allows the tRNA molecule to bind to its specific amino acid. There remains, unpaired, at one end of the molecule, three bases which are complementary to the codon coding for the amino acid. This anticodon binds to the codon of the mRNA and places the amino acid in the correct sequence of the protein (see Fig. 1.4 ). Usually, several ribosomes translate a single mRNA molecule at any one time.

Replication

‘Replication’ is the process whereby DNA is copied or replicated to permit transmission of genetic information to offspring. DNA replication is performed prior to cell division, when an identical copy must be made for each daughter cell resulting from division. Replication occurs before mitosis, the normal form of cellular division where resulting cells have identical DNA to the original. Meiosis, the second form of cellular division, occurs during gametogenesis and results in haploid cells (i.e. cells with half the usual complement of DNA). In meiosis the resulting cells (gametes) are haploid (i.e. carry only a single copy of the genomic sequence).

It is important to note that since this dogma was first established in 1958 by Crick, a number of exceptions have been identified. For example, retroviruses (e.g. human immunodeficiency virus (HIV)-1) can cause information to flow from RNA to DNA by integrating their genome (carried as RNA) into that of the host. A second example is ribozymes, which are functional enzymes composed solely of RNA and hence have no need to be translated into protein.

Regulation of Gene Expression

When a gene is actively being transcribed into mRNA and then translated into a protein, it is said to be ‘expressed’. Gene expression can be controlled at several levels. Transcription of DNA into mRNA is generally regulated by the binding of specific proteins, known as transcription factors, to the region of DNA just upstream, or 5′, of the coding sequence itself. Other proteins can bind enhancer sequences that may be within the gene or a long way upstream or downstream.

The promoter contains specific DNA sequence motifs which bind transcription factors. In general, transcription factors become active when the cells receive some form of signal and then translocate to the nucleus, where they bind to specific sequences in the promoters of specific genes and activate transcription. Other genes, often known as housekeeping genes, have a constant level of expression and are not induced in this way.

Many different types of transcription factor exist with different modes of action. Typical examples of two types will be considered here, namely intracellular nuclear hormone receptors (which are transcription factors) and cell surface receptors, which are capable of activating transcription factors.

Members of the nuclear hormone receptor superfamily, such as the progesterone receptor and the thyroid hormone receptor, are present mainly in the cytoplasm of the cell. When a steroid hormone crosses the lipid bilayer of the cell membrane, it binds to the receptor which is usually dimerised to form pairs of receptor molecules. The receptor/hormone dimer complex then translocates to the nucleus and binds to response elements in the promoters of target genes, where it activates (or indeed represses) transcription. This process also involves the recruitment of many other cofactors to the dimer complex which are also involved in regulation of the expression of the target gene.

Cell surface receptors, subsequent to binding of ligands, can activate pathways leading to the formation of active transcription factors. For example, activation of tyrosine kinase–linked receptors on the cell surface may lead to a series of phosphorylation events within the cell, culminating in the phosphorylation of the protein Jun. Jun will then combine with the protein Fos to form a dimer transcription factor called AP-1, which can bind to specific AP-1 binding sites in the promoters of responsive genes.

In another example of cell surface receptor action, the ‘inflammatory’ transcription factor NF-κB exists in the cytoplasm of cells as dimers bound to an inhibitory protein IκB. Mediators of inflammation, such as the inflammatory cytokine interleukin-1β, bind to cell surface receptors and activate a chain of biochemical events that result in the phosphorylation and subsequent breakdown of IκB. Uninhibited NF-κB dimers then translocate to the nucleus to activate genes whose promoters contain NF-κB DNA-binding motifs.

Gene expression can also be controlled by regulation of the stability of the transcript. Most mRNA molecules are protected from degradation by the presence of their poly-A tail. Degradation of mRNA is controlled by specific destabilising elements within the sequence of the molecule. One type of destabilising element has been well characterised. The Shaw–Kayman or AU-rich sequence (ARE) is a region of RNA, usually within the 3′ UTR, in which the motif AUUUA is repeated several times. Rapid response genes, whose expression is rapidly switched on and then off again in response to some signal, often contain an ARE within their 3′ UTR. Binding of specific proteins to the ARE leads to removal of the mRNA’s poly-A tails and then to degradation of the molecule.