Genes are functional elements on the chromosome that are capable of transmitting information from the DNA template via the production of messenger ribonucleic acid (mRNA) to the production of proteins. The human genome contains over 20 000 genes, although many of these are inactive or silenced in different cel types, reflecting the variable gene expression responsible for cel-specific characteristics. The central dogma is the pathway describing the basic steps of protein production: transcription, splicing, translation and protein modification (Fig. 1). Although this is now recognised as an over-simplification (contrary to this linear relationship, a single gene will often encode many different proteins), it remains a useful starting point to explore protein production.
Fig1. The central dogma of protein production. Double-stranded DNA as a template for single-stranded RNA, which codes for the production of a peptide chain of amino acids. Each of these chains has an orientation. For DNA and RNA, this is 5 to 3 . For peptides, this is N-terminus to C-terminus.
Transcription: DNA to messenger RNA
Transcription describes the production of ribonucleic acid (RNA) from the DNA template. For transcription to commence, an enzyme called RNA polymerase binds to a segment of DNA at the start of the gene: the promoter. Once bound, RNA polymerase moves along one strand of DNA, producing an RNA molecule complementary to the DNA template. In protein-coding genes this is known as messenger RNA (mRNA). A DNA sequence close to the end of the gene, called the polyadenylation signal, acts as a signal for termination of the RNA transcript (Fig2).
Fig2. RNA synthesis and its translation into protein. Gene transcription involves binding of RNA polymerase I to the promoter of genes being transcribed with other proteins (transcription factors) that regulate the transcription rate. The primary RNA transcript is a copy of the whole gene and includes both introns and exons, but the introns are removed within the nucleus by splicing and the exons are joined to form the messenger RNA (mRNA). Prior to export from the nucleus, a methylated guanosine nucleotide is added to the 5 end of the RNA (‘cap’) and a string of adenine nucleotides is added to the 3 (‘polyA tail’). This protects the RNA from degradation and facilitates transport into the cytoplasm. In the cytoplasm, the mRNA binds to ribosomes and forms a template for protein production. (tRNA = transfer RNA; UTR = untranslated region)
RNA differs from DNA in three main ways:
● RNA is single-stranded.
● The sugar residue within the nucleotide is ribose, rather than deoxyribose.
● It contains uracil (U) in place of thymine (T).
The activity of RNA polymerase is regulated by transcription fac tors. These proteins bind to specific DNA sequences at the promoter or to enhancer elements that may be many thousands of base pairs away from the promoter; a loop in the chromosomal DNA brings the enhancer close to the promoter, enabling the bound proteins to interact. The human genome encodes more than 1200 different transcription factors. Mutations (now more frequently referred to as gene variants) within transcription factors, promoters and enhancers can cause disease. For example, the blood disorder alpha-thalassaemia is usually caused by gene deletions. However, it can also result from a variant in an enhancer located more than 100 000 base pairs (bp) from the α-globin gene promoter, leading to greatly reduced transcription.
Gene activity, or expression, is influenced by a number of complex interacting factors, including the accessibility of the gene promoter to transcription factors. DNA can be modified by the addition of a methyl group to cytosine molecules (methylation). If DNA methylation occurs in promoter regions, transcription is silenced, as methyl cytosines are usually not available for transcription factor binding. A second mechanism determining promoter accessibility is the structural configuration of chromatin. In open chromatin, called euchromatin, gene promoters are accessible to RNA polymerase and transcription factors; therefore it is transcriptionally active. This contrasts with heterochromatin, which is densely packed and transcriptionally silent. The chromatin configuration is determined by modifications (such as methylation or acetylation) of specific amino acid residues of histone protein tails.
Modifications of DNA and histone protein tails are termed epigenetic (‘epi-’ meaning ‘above’ the genome), as they do not alter the primary sequence of the DNA code but have biological significance in chromosomal function. Abnormal epigenetic changes are increasingly recognised as important events in the progression of cancer, allowing expression of normally silenced genes that result in cancer cell de-differentiation and proliferation. They also afford therapeutic targets. For instance, the his tone deacetylase inhibitor vorinostat has been successfully used to treat cutaneous T-cell lymphoma, due to the re-expression of genes that had previously been silenced in the tumour. These genes encode transcription factors that promote T-cell differentiation as opposed to proliferation, thereby causing tumour regression.
RNA splicing, editing and degradation
Transcription produces an RNA molecule that is a copy of the whole gene, termed the primary or nascent transcript. This nascent transcript then undergoes splicing, whereby regions not required to make protein (the intronic regions) are removed while those segments that are necessary for protein production (the exonic regions) are retained and rejoined.
Splicing is a highly regulated process that is carried out by a multimeric protein complex called the spliceosome. Following splicing, the mRNA molecule is exported from the nucleus and used as a template for protein synthesis. Many genes produce more than one form of mRNA (and thus protein) by a process termed alternative splicing, in which different combinations of exons are joined together. Different proteins from the same gene can have entirely distinct functions. For example, in thyroid C cells the calcitonin gene produces mRNA encoding the osteoclast inhibitor calcitonin, but in neurons the same gene produces an mRNA with a different complement of exons via alternative splicing that encodes a neurotransmitter, calcitonin-gene-related peptide.
Translation and protein production
Following splicing, the segment of mRNA containing the code that directs synthesis of a protein product is called the open reading frame (ORF). The inclusion of a particular amino acid in the protein is specified by a codon composed of three contiguous bases. There are 64 different codons with some redundancy in the system: 61 codons encode one of the 20 amino acids, and the remaining three codons – UAA, UAG and UGA (known as stop codons) – cause termination of the growing polypeptide chain. ORFs in humans most commonly start with the amino acid methionine. All mRNA molecules have domains before and after the ORF called the 5 untranslated region (UTR) and 3 UTR, respectively. The start of the 5 UTR contains a cap structure that protects mRNA from enzymatic degradation, and other elements within the 5 UTR are required for efficient translation. The 3 UTR also contains elements that regulate efficiency of translation and mRNA stability, including a stretch of adenine bases known as a polyA tail (see Fig. 2).
The mRNAs then leave the nucleus via nuclear pores and associate with ribosomes, the sites of protein production (see Fig. 2). Each ribosome consists of two subunits (40S and 60S), which comprise non-coding rRNA molecules complexed with proteins. During translation, a different RNA molecule known as transfer RNA (tRNA) binds to the ribosome. The tRNAs deliver amino acids to the ribosome so that the newly synthesised protein can be assembled in a stepwise fashion. Individual tRNA molecules bind a specific amino acid and ‘read’ the mRNA ORF via an ‘anticodon’ of three nucleotides that is complementary to the codon in mRNA (see Fig. 2). A proportion of ribosomes is bound to the membrane of the endoplasmic reticulum (ER), a complex tubular structure that surrounds the nucleus.
Proteins synthesised on these ribosomes are translocated into the lumen of the ER, where they undergo folding and processing. From here, the protein may be transferred to the Golgi apparatus, where it under goes post-translational modifications, such as glycosylation (covalent attachment of sugar moieties), to form the mature protein that can be exported into the cytoplasm or packaged into vesicles for secretion. The clinical importance of post-translational modification of proteins is shown by the severe developmental, neurological, haemostatic and soft tissue abnormalities that are associated with the many different congenital dis orders of glycosylation. Post-translational modifications can also be disrupted by the synthesis of proteins with abnormal amino acid sequences. For example, the most common CFTR gene variant that causes cystic fibrosis (ΔF508) results in an abnormal protein that cannot be exported from the ER and Golgi.
