By the end of this session the student should be able to:
* Use the genetic code to translate coding sequences
* Calculate the number of codons and amino acids from the number of bases
* Name the main sites of non-coding DNA segments
* Name the function and features of the promoter sequence
* Distinguish between introns and exons
* Explain how gene expression is controlled
A gene is a stretch of DNA that carries a coded message for the synthesis of a specific protein. Most of the DNA of an organism does not code for proteins. A stretch of DNA is recognised as a gene coding for a protein if it is preceded by a promoter sequence. The promoter sequence is the site of attachment for RNA polymerase, which is responsible for the process of transcription of the genetic message to m-RNA. The promoter sequence is present only on the template (transcribed) strand. The sequence of bases on m-RNA is complementary to that on the transcribed strand of DNA but corresponds to that on the coding strand except that T is replaced by U (Fig. 4.1).
The genetic code is, by convention, interpreted with reference to the sequence of bases on m-RNA. The m-RNA sequence happens to correspond to the coding strand of DNA, with the exception that U on RNA corresponds to T on DNA.
The sequence of bases on m-RNA determines the exact sequence of amino acids in the protein. The bases are read in triplets. Each triplet of bases is termed a codon and corresponds to a particular amino acid. The genetic code designates how the codons correspond to the amino acids. There are four base (A, U, C and G) and 64 possible ways in which these can be combined to form codons as shown in Fig. 4.2. Each codon is specific for one amino acid. However, there are only 20 amino acids and so one amino acid may be represented by more than one codon. Because of this the genetic code is described as degenerate.
The codon AUG codes for methionine but when it occurs after a promoter sequence it also serves as a "start" signal indicating the beginning of the coded message.
The codons UAA (also called "ochre"), UAG (also called "amber") and UGA do not code for any amino acid but act as "stop" signals for the end of a gene message. Note that all the ‘stop’ codons start with U and include an A.
Every three nucleotides correspond to one codon and one amino acid. If a gene contains 1,200 nucleotides, this corresponds to 1,200/3 = 400 codons and the resulting protein contains 400 amino acids.
The size of a protein is often expressed as its molecular mass. The molecular masses of amino acids vary but it can be assumed that amino acids have an average molecular mass of approximately 100 Daltons. Thus a protein consisting of 400 amino acids will have a molecular mass of approximately 40,000 Daltons. Conversely, a protein with a molecular mass of 60,000 Daltons is expected to contain about 60,000/100 = 600 amino acids and the gene coding for this protein will consist of about 600 codons and 600 x 3 = 1,800 nucleotides.
The amount of DNA contained in a cell of a particular organism is termed the C-value. Every organism has a specific c-value. More complex organisms are expected to contain more genes than simple organisms and consequently would require more DNA. In fact, however, the C-value or DNA content for a particular organism is not proportional to the number of genes or to the complexity of the organism. Thus the frog has seven times the DNA content of Man, and the Lilly has 100 times the DNA content of Man. This is the C-value paradox.
It has been estimated that the human genome contains 3.5 billion base pairs. This amount of DNA could contain about 2 million genes. In fact, however, the total number of genes in man has been estimated to be only about 40,000 to 80,000. This means that about 3% of human genome codes for proteins, and 97 % of the human genome is non-coding DNA.
Although over 95% of the total DNA is non-coding, it may have other important functions such as the regulation of gene activity. The main examples of non-coding DNA are the following:
1. Promoter sequences
2. Intervening sequences (introns)
3. Terminator sequences
4. Sequences related to chromosome structure
6. Repetitive DNA
These are explained in the following sections.
In order to be transcribed a gene must be preceded by a promoter sequence. This is the recognition site for the attachment of RNA polymerase, the enzyme responsible for transcription. The promoter sequence is followed by an initiator sequence, which marks the site where transcription to m-RNA begins. The initiator codon or start signal on m-RNA is the sequence AUG, which is also codon 1 and corresponds to the amino acid methionine. It also marks the site where translation begins (Fig. 4.3).
The initiator sequence is not the beginning of the gene itself but the first part to be transcribed to m-RNA. The beginning of the gene itself is indicated by the sequence "AUG" on m-RNA. This corresponds to “TAC” on the transcribed strand. This is codon 1 of the gene and is translated to the amino acid methionine. Subsequent triplets of bases are read as codons and are translated according to the genetic code until a “stop” signal is encountered.
The promoter sequence has two characteristic sequences by which it is recognised (Fig. 4.4). These sequences are read as they occur on the coding strand of DNA in the 5’ to 3’ direction.
1. The "TATA box" is a characteristic sequence that is often TATAATA, or a closely similar sequence consisting of A and T bases. It occurs about 25 bases before the start (upstream) of the initiator region. This type of sequence occurs quite regularly, with little variation in most organisms and so is referred to as a highly conserved region or consensus sequence.
2. The "CAT box" is a sequence consisting of CCAAT or a similar sequence, and occurs about 75 bases upstream of the initiator region. This is also a conserved consensus sequence.
Note that DNA sequences are continuous and that there are no gaps demarcating promoter, initiator and codons. In figures 4.3 and 4.4, the boxes indicating the different sequences and the spaces between the codons have been used here only for the sake of clarity.
The coded message of DNA is usually interrupted by one or more sequences that are not part of the gene message. These intervening sequences are termed introns, while the actual coding sequences that are to be translated or "expressed" are termed exons (Fig.4.5). Introns are like blank spaces separating paragraphs in a written message. They are transcribed on to the newly formed m-RNA but are removed or "spliced" before translation of the m-RNA begins. Splicing involves cutting out the intron and re-fusion of the cut ends of the m-RNA. An intron usually begins with GU and ends with AG. These sequences are recognition sites for splicing. The number of introns varies from 0 to 60 in different genes. For a particular gene the number of introns is always the same.
A terminator sequence is one that causes RNA polymerase to stop transcription. It occurs about 30 base pairs after the end of the coding region of the gene. This region usually contains the sequence AATAA or a closely similar sequence. Distinguish this from the terminator codon (UAA, UAG or UGA) that marks the end of translation or the end of polypeptide synthesis.
These are other non-coding sequences that have the following functions:
a) Sequences marking the telomeres (chromosome ends);
b) Sequences marking the centromeres and the kinetochores for attachment of the mitotic spindle;
c) Sequences marking sites where DNA replication begins; many of these sites occur at intervals along each chromosome.
These are sequences that are similar to the sequences of normal genes, but lack a promoter region and therefore are non-functional or“false” genes. They are the result of evolution. Genes, which had been at one time functional, had lost their promoter sequence through mutations during the process of evolution, but have remained in the genome as "pseudogenes". Usually, they are situated close to normal genes having a similar sequence. Examples are seen in the globin genes of hemoglobin.
These are sequences that are repeated many times over. Some of these are trinucleotide repeats e.g. CAG CAG CAG … etc repeated many times over. They are often present in non-coding regions of the genome, but sometimes they are part of a coding region of a gene and are translated into amino-acids e.g. CAG repeats in the example above translate to “polyglutamine” repeats in the protein.
Other examples are di-nucleotide repeats, which are sometimes repeated hundreds of times. Some telomere and centromere sequences are repetitive DNA sequences.
Although all cells of an individual contain the same genes they are not all functional at the same time. The genes that are active in a particular cell depend on the cell type and its state of activity. The genes that are active in brain cells are different from those that are active in liver, kidney, muscle, fibroblasts etc. Furthermore, some genes are active only during embryonic development and are switched off in the mature tissues. How are genes switched on an off?
In any one particular cell most genes are inactive and only those relevant to the functioning of the particular cell are active. As noted above, the promoter region binds to RNA polymerase, which then transcribes m-RNA. In inactive genes the binding of RNA polymerase is prevented by means of a repressor, a molecule that attaches itself to the promoter region, blocking the RNA polymerase binding site (Fig.4.6). Activation of the gene requires removal of the repressor. This is achieved by an inducer, which combines with the repressor and prevents it from binding to DNA. This is termed negative regulation of gene activity. Most genes are normally inactive, or repressed, and are switched on or de-repressed, only when required.
Activators may also control gene activity. These molecules enhance the binding of RNA polymerase to the promoter region and therefore exert positive control on transcription.
In some cases a group of functionally related genes are situated next to one another on the same DNA strand, and share the same promoter region. Such a unit is termed an operon. An example is the lac operon in bacteria. This operon contains three genes (called Z,Y and A) that are responsible for three enzymes involved in different stages of lactose metabolism. The three genes are situated next to one another, are transcribed together and are controlled by one promoter. When the promoter region is repressed all three genes are switched off.
1. The following sequence is part of the transcribed strand of DNA:
3' G C T A A T C A G T G C G T A 5'
Write the sequences of:
a. the coding strand of DNA
c. amino acids
2. The following is a schematic diagram of DNA being transcribed:
a. DNA transcribed strand
b. DNA coding strand
The direction of transcription is shown.
Label the 5' and 3' ends of each strand.
3. The following is the sequence of amino acids of the last part of a protein molecule. Using the genetic code, write the codons on m-RNA and label the 3' and 5' ends. (Where more than one codon is possible use the first one in the table.) Then, write the corresponding codons on the template strand of DNA.
m-RNA - arg - leu - asp - phe - pro - ile - glu - gly - val -DNA template strand