Proteins
4.5 Protein turnover
Similar to covalent regulation, allosteric regula- tion of enzymes is highly important for sport and exercise metabolism, in order to regulate the rate of energy provision according to the intensity and duration of the exercise. In such circumstances, products of energy-producing reactions such as ADP, AMP, Pi and H+, etc. can all act as a feedback loop mechanism to fine tune the rate of enzymatic reactions during exercise (Parolin et al., 1999). This will be highlighted in much more detail in future chapters, where we will examine the regulation of metabolism during different types of exercise.
DNA
mRNA
Protein
Translation Amino Acids
Degradation
Transcription
Figure 4.15 The pathway from DNA to protein
of a sugar (deoxyribose), a phosphate and four organic bases, two of which are pyrimidine bases (cytosine and thymine) and two of which are purine bases (adenine and guanine). The bases are often abbreviated using the capital of the first letter in their name i.e. C, T, A and G, respectively. A chain of sugar and phosphate essentially provides the backbone for which the bases to attach, as shown in Figure 4.16. In this example, the bases are ordered as A, G, T and C, although it is important to note that the precise ordering of bases will vary throughout the stretch of the DNA molecule. Indeed, a DNA molecule is typically millions of these units long, and if extended (as opposed to existing in its double helix format) would be around 2 m long!
We have already alluded to the double helix structure of DNA in that DNA is essentially structured as a double-stranded molecule which is held together by hydrogen bonds between bases.
The bases therefore exist asbase pairs, where the adenine in one strand is always joined to thymine in the other strand. Similarly, the guanine in one strand is always bonded to cytosine in the other strand. Each strand differs in polarity from one another, as one begins with a free phosphate
group attached to deoxyribose (known as the 5’
end) and one strand ends with a free OH group attached to deoxyribose (known as the 3’ end). In essence, the two strands are thereforeantiparallel, as one runs from 5’ to 3’ and the other runs from 3’ to 5’ (see Figure 4.17).
4.5.3 Transcription
During the process of transcription, a specific segment of DNA (i.e. a gene) is copied to make a newly formed molecule known as messenger ribonucleic acid (mRNA). The order of bases in the DNA therefore serves as a template for which to produce mRNA. The bases in the newly formed mRNA have the same base pairing as they do in DNA, with the exception that thymine in mRNA is actually replaced by uracil (U).
Transcription begins when an enzyme known as RNA polymerase II binds to thepromoter region of a gene, which is an approximate 100-base pair DNA sequence. RNA polymerase II is, in turn, first attracted to the promoter region of the gene following the binding of a regulatory protein known as a transcription factor (TF) to the promoter. In this way, effective binding of the transcription factor protein subsequently recruits RNA polymerase II to the promoter region. RNA polymerase then ‘scans’ the entire length of the gene, transcribing the base sequence in one strand of DNA into its complementary strand of mRNA (see Figure 4.18).
4.5.4 The genetic code
The sequence of the bases in the mRNA molecule can now determine the exact sequence of the amino acids in the primary structure of the protein to be made. For this to occur, the bases in the mRNA molecule are read in groups of three known as codons. It is the specific sequence of bases within the codons which underpins the genetic code, a code which is used to translate the three-base sequence into a corresponding amino acid. The genetic code is shown in Table 4.4,
N
N N
N
CH2 O O P O
O− O
NH2
HN
N N
N
CH2 O O P O
O− O
H2N O
N
CH2 O O P O
O− O
N
CH2 O O P O
O− O
HN O
CH3
O
N NH2
O
O P O
O O− Adenine (A)
Guanine (G)
Thymine (T)
Cytosine (C)
Figure 4.16 Example of a strand from a DNA molecule, showing the attachment of the bases (indicated by black text) to the sugar-phosphate backbone (indicated by red text)
where the amino acid (symbolized by its three- letter abbreviation) corresponding to a particular codon is written adjacent to the base sequence.
Given that there are four bases present in mRNA and that they are read in combinations of three, theoretically this gives rise to 43 codons (i.e. 64) and hence 64 amino acids. However, there are, of course, only 20 amino acids used to make proteins, so multiple codons can therefore code for the same amino acid. (see Table 4.4). Of the 64 codons, 61 code for amino acids and three are ‘stop’ or
‘termination’ signals (UAA, UGA and UAG). The latter codons signal the end of translation of the information contained in mRNA into a polypeptide chain. The start codon or initiation codon is always
AUG, which also corresponds to the amino acid methionine. For this reason, methionine is always the first amino acid used for protein synthesis.
Returning to Figure 4.18, we can see that only one strand of DNA is used for transcription, and this is known as the template strand. The template strand is read in the 3’ to 5’ direction.
The DNA strand which is not copied is known as the sense strand and has the same base sequence as the mRNA, with the exception that U now replaces T. In this way, the polarity of the sense strand and mRNA are the same, but are oppo- site to that of the template strand. The mRNA strand is therefore read in the 5’ to 3’ direction, which is often referred to as from ‘upstream to
S A T S OH
P P
P
S T A S
P P
S G C S
P S P
S C G
P HO
3′ End 5′ End
5′ End 3′ End
Figure 4.17 Illustration of double strands of DNA, showing base pairing between complementary strands.
S-P denotes sugar-phosphate backbone (as indicated by red text)
downstream’. To facilitate your understanding of the genetic code, the base sequences in the mRNA strand shown are separated into codons and the corresponding amino acid for which they will eventually code is also shown.
4.5.5 Translation
Having formed the mRNA molecule in the cell nucleus, the next stage in the process of protein synthesis is to translate the base sequence in mRNA into its corresponding amino acid. As discussed above, the process of translation is underpinned by the genetic code. However, for translation to occur, the mRNA has first to exit the pores in the nuclear membrane and travel to theribosomes(the
‘factories’ where proteins are made) – the cellular location where translation occurs.
As the strand of mRNA emerges along the ribosome, each codon is recognized by an anticodon which is bound to a transfer RNA (tRNA) molecule. The other binding site on tRNA molecules is that which binds the corresponding amino acid.
We have already mentioned how the first amino acid translated is always methionine. As each codon is paired with an anticodon on tRNA, the corre- sponding amino acid forms a peptide bond with the previous amino acid which was translated and thus the length of the peptide chain continues to grow.
Finally, translation will stop when one of the stop codons on the mRNA strand is reached. A simplified overview of translation is shown in Figure 4.19. When the complete polypeptide has been formed and is present in the cytoplasm, it then folds into its three-dimensional structure so as to gain full biological function.
Although we have presented an overview of the process involved in transcription and translation, the regulation of protein synthesis is extremely complex and involves the coordinated interplay between a multitude of regulatory proteins which have not even been discussed in the above text.
The precise molecular mechanisms underpinning these processes (and, indeed, protein degradation) are beyond the scope of the present text; inter- ested readers are directed to more focused texts (Spurway & Wackerhage, 2006; Houston, 2006) and reviews (Reid, 2005; Drummondet al., 2009;
Rose & Richter, 2009) which are especially rele- vant to skeletal muscle.
In the context of exercise and skeletal muscle adaptation, transcription of genes is thought to occur during and/or in the hours following an exercise session, such that changes in mRNA content for a specific gene can be detected within this timescale. However, up-regulation of the actual protein content is usually only detected within hours to days after the exercise bout. More often, it usually takes weeks of repetitive exercise sessions (i.e. training) before changes in protein content are observed.
It is this repetitive and transient change in gene expression which is thought to form the molecular basis for training adaptation (Coffey & Hawley, 2007). Understanding how differences in exercise intensity, duration and mode can affect the transcriptional responses to exercise is therefore one of the major challenges facing the exercise scientist in the coming decades. Such research is not only important for helping to optimize athletic
T T C A C T A A G T G A
A U G C G U
A G U A A G U G A T A C G C A T
C G A T G C G T A G C
RNA Polymerase II
Promoter Region
Sense strand
template strand
mRNA strand
stop codon codon n
codon 3 codon 2
codon 1
Arg Ser Met
Will eventuall y code fo
r
Lys
Figure 4.18 Schematic illustration of the process of transcription. Note that in order for transcription to occur, the DNA strands first have to be unravelled from the double helix structure. The sequence of bases in the mRNA strand is identical to those in the sense strand, with the exception that U replaces T
Table 4.4 The genetic code, detailing how the combinations of bases in the 1st, 2nd and 3rd position code for different amino acids
First position Second position Third position
(5’ end) U C A G (3’ end)
U UUU Phe
UUC Phe UUA Leu UUG Leu
UCU Ser UCC Ser UCA Ser UCG Ser
UAU Tyr UAC Tyr UAA Stop* UAG Stop*
UGU Cys UGC Cys UGA Stop* UGG Trp
U C A G
C CUU Leu
CUC Leu CUA Leu CUG Leu
CCU Pro CCC Pro CCA Pro CCG Pro
CAU His CAC His CAA Gln CAG Gln
CGU Arg CGC Arg CGA Arg CGG Arg
U C A G
A AUU Ile
AUC Ile AUA Ile AUG Met**
ACU Thr ACC Thr ACA Thr ACG Thr
AAU Asn AAC Asn AAA Lys AAG Lys
AGU Ser AGC Ser AGA Arg AGG Arg
U C A G
G GUU Val
GUC Val GUA Val GUG Val
GCU Ala GCC Ala GCA Ala GCG Ala
GAU Asp GAC Asp GAA Glu GAG Glu
GGU Gly GGC Gly GGA Gly GGG Gly
U C A G
*Stop codons do not have an amino acid assigned to them.
**Codes for the amino acid methionine but also is the start or initiation codon.
A U
A U
C A C
G AAA
anticodon
Ribosome
Direction of translation mRNA
Incoming t RNA Growing peptide chain
in cytoplasm
UUU A
U G U
Lys Trp
Phe
Figure 4.19 Schematic illustration of the process of translation. tRNA molecules originating from the cytoplasm enter the ribosome. Codons on the mRNA strand are recognized by specific anticodons on tRNA and hence by the corresponding amino acid. With continuing codon-anticodon binding, amino acids are joined by peptide bonding and are later folded into their three-dimensional structure in the cytoplasm
performance, but also in optimizing exercise training protocols which may improve health and well-being and offer protection against metabolic related diseases such as diabetes and obesity, etc.
(Booth & Laye, 2009).