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Transcription and Translation Reproduction is one of the basic properties of life. It involves the transmission of information from parent to offspring. This transmission is termed heredity. A gene is a unit of heredity that consists of a sequence of DNA bases. This sequence of bases does not, in itself, give any observable characteristic in an organism. The function of most genes it so specify the sequence of amino acids in a particular polypeptide. A protein is composed of one or more polypeptides. It is protein that often directly or indirectly determine the observable characteristics of an individual. Two processes are needed to produce a specific polypeptide, using the base sequence of a gene. Transcription is the synthesis of RNA, using DNA as a template. The base sequence of the RNA is the same as one of the DNA strands, with uracil in place of thymine. Translation is the synthesis of a polypeptide, with an amino acid sequence that is determined by a molecule of RNA. The type of RNA that carries the information needed to synthesize a polypeptide is called messenger RNA (mRNA). Although most RNA is mRNA, there are two other types. Transfer RNA (tRNA) is involved in decoding the base sequence into an amino acid sequence during translation. Ribosomal RNA (rRNA) is part of the structure of the ribosome, where polypeptide synthesis by translation of mRNA occurs. In most cases, one polypeptide is synthesized using one type of mRNA that is obtained by transcribing one gene. This is referred to as the one gene – one polypeptide concept. Although there are exceptions to it, the concept helps to understand the flow of information in cells. Transcription mRNA is made in a process called transcription. There are other types of RNA such as transfer RNA which has a role in translation, and ribosomal RNA which is a structural and functional component of ribosomes. The production of these other forms of RNA also occurs through the process of transcription. Because RNA is single-stranded, transcription occurs along one strand only. The RNA that is produced has a sequence that is complementary to the DNA that is used as a template for transcription. What follows is an outline of transcription: The enzyme RNA polymerase binds to a site called the promoter on the DNA The DNA to be transcribed is separated by the RNA polymerase in the region of the gene to be transcribed. 1 RNA nucleotides pair with their complementary bases on one strand of the DNA only. There is not thymine in RNA, so uracil pairs in a complementary fashion with adenine. RNA polymerase forms covalent bonds between the nucleotides The RNA separates from the DNA and the double helix reforms. Details of transcription RNA polymerase splits DNA into two single strands during transcription. These two strands have complementary base sequences. One is referred to as the sense strand and the other as the antisense strand. The mRNA that is created by transcription has the same base sequence as the sense strand, with uracil in place of thymine. This is achieved by using the antisense strand as the template for transcription. So, ironically, although it is a copy of the base sequence of the sense strand that is required, it is the antisense strand that is transcribed by RNA polymerase. 2 Introns and Exons In eukaryotes, the immediate product of mRNA transcription is referred to as “premRNA”, as it must go through several stages of post-transcriptional modification to become mature mRNA. One of these stages is called RNA splicing, shown below. Interspersed throughout the mRNA are sequences that will not contribute to the formation of the polypeptide. They are referred to as intervening sequences, or introns. These introns must be removed. The remaining coding portions of the mRNA are called exons. These will be spliced together to form the mature mRNA. Genes in prokaryotes do not normally contain introns. Prokaryotes also lack the molecular mechanisms needed to carry out RNA splicing. There is therefore a potential problem if eukaryotic genes are transferred to prokaryotes by genetic engineering. Introns are not edited out and polypeptides are synthesized with extra sequences of amino acids that disrupt the polypeptides’ functioning. Special copies of eukaryotic genes without intros are therefore needed for transfer to prokaryotes. 3 Repetitive sequences The Human Genome Project has led us to understand that there are a number of recognizable patterns observed in DNA. It has been estimated that there are approximately 25,000 protein-coding genes in the human genome. In addition, some genes are transcribed to produce other forms of RNA other than mRNA. Most genes only occur at one position on one chromosome type, so they are referred to as unique or single-copy genes. Originally, estimates for the number of genes were much higher. This prediction was based on observed diversity of phenotypes. It has been found that the non-gene factors can have an influence on phenotype and on gene expression, and these nongene factors may be one source of the diversity. Most of the genome is not transcribed. Originally called “junk DNA”, it is being increasingly recognized that elements of this “junk” play roles in gene expression. Within this “junk” region, there are elements that affect gene expression as well as highly repetitive sequences (satellite DNA). The latter can form between 5 and 45 per cent of the genome. The repeating sequences may be duplicated as many as 105 times per genome. The genetic code The base sequence in the mRNA molecule is used as a guide for assembling the sequence of amino acids that will be a polypeptide. The process of protein production using mRNA as a guide is called translation. The “translation dictionary” that enables the cellular machinery to convert the base sequence on the mRNA into an amino acid sequence is called the genetic code. A sequence of three bases on the mRNA is called a codon. Each codon codes for a specific amino acid to be added to the polypeptide. Amino acids are carried on another kind of RNA, called tRNA. Each amino acid is carried by a specific tRNA, which has a three-base anti-codon complementary to the mRNA codon for that particular amino acid. A tRNA with the correct anti-codon attaches to the codon on the mRNA. Your codon chart list all of the 64 possible codons. The three bases of an mRNA codon are designated in the table as first, second and third position. Note that different codons can code for the same amino acid. For example, the codons GUU and GUC both code for the amino acid valine. For this reason, the code is said to be “degenerate”. Note that also three codons are “stop” codons that code for the end of translation. 4 The genetic code is universal in that it operates in the same way in nearly all life on Earth. There are some very rare exceptions. For example, in some cases, the stop codons are used to code for non-standard amino acids. Translation Translation takes place on cell structures known as ribosomes. These are in the cytoplasm, outside the cell nucleus. Each ribosome comprises a small and a large subunit. An outline of translation follows. An mRNA binds to the small subunit of the ribosome. tRNA molecules are present, each one carrying the specific amino acid corresponding to its anticodon. The tRNA binds to the ribosome at the site where its anti-codon matches the codon on the mRNA. Two tRNAs bind at once and the first one is transfers the growing polypeptide chain to the second one in. The ribosome moves along the mRNA and the process continues until a stop codon is reached when the completed polypeptide is released. Details of translation Translation always begins in the cytoplasm. If the proteins are destined eventually for lysosomes or for export, then the ribosomes bind to the ER and complete the process of translation while bound. Proteins that are for use within the cytosol are synthesized by unbound ribosomes. Ribosomes are composed of ribosomal RNA, or rRNA and a large variety of individual proteins. Each ribosome is composed of two subunits, one larger than the other. Each ribosome has three tRNA binding sites – the “E” or exit side, the “P” or peptidyl site and the “A” or aminoacyl site. 5 Often, more than one ribosome can be actively translating the same mRNA molecule at the same time. The resulting complex of ribosomes along a single mRNA is called polyribosome or polysome. Each tRNA molecule is recognized by a tRNA-activating enzyme that binds to a specific amino acid to the tRNA, using ATP for energy. The amino acid attaches at the 3’ end of the tRNA. The 3’ end of the tRNA terminates with the nucleotide sequence CCA. 6 7