Protein synthesis

The information for the amino acid sequence of each of the 30—50,000 different proteins in the body is contained in the DNA in the nucleus of each cell. As required, a working copy of the information for an individual protein (the gene for that protein) is transcribed, as messenger RNA (mRNA), and this is then translated during protein synthesis on the ribosomes. Both DNA and RNA are linear polymers of nucleotides. In RNA the sugar is ribose, whereas in DNA it is deoxyribose.

9.2.1 The structure and information content of DNA

As shown in Figure 9.4, DNA is a linear polymer of nucleotides. It consists of a backbone of alternating deoxyribose and phosphate units, with the phosphate groups forming links from carbon-3 of one sugar to carbon-5 of the next. The bases of the nucleotides project from this sugar—phosphate backbone. There are of two strands of

Dna Protein Metabolism
3'end OH free 3' hydroxyl group

Figure 9.4 The structure of DNA.

deoxyribonucleotides, held together by hydrogen bonds between a purine (adenine or guanine) and a pyrimidine (thymine or cytosine): adenine forms two hydrogen bonds to thymine, and guanine forms three hydrogen bonds to cytosine.

The double strand coils into a helix, the so-called 'double helix' (Figure 9.5). The two strands of a DNA molecule run in opposite directions - one strand has a 3'-hydroxyl group at the end and on the complementary strand there is a free 5'-hydroxyl group. The information of DNA is always read from the 3' end towards the 5' end.

Only about 10% of the DNA in a human cell carries information for the 3050,000 genes which make up the human genome. The remainder is made up of:

  • Control regions, which promote or enhance the expression of individual genes, and include regions which respond to hormones and other factors which control gene expression (section 10.4), as well as sites for the initiation and termination of DNA replication.
  • Spacer regions, both between and within genes, which carry no translatable message but serve to link those regions that do carry a translatable message. When such regions occur within a gene sequence, they are called introns.
  • Pseudo-genes, which seem to be genes that have undergone mutation in our evolutionary past and are now untranslatable. Presumably, these are a reminder of evolutionary history.
Dna Protein Metabolism
Figure 9.5 The structure of DNA — hydrogen bonding between bases and coiling of the chains to form the double helix.

9.2.1.1 The genetic code

It is difficult at first sight to understand how a code made up of only four letters (A, G, C, and T) can carry the information which must be contained in the nucleus of the cell, for the 21 different amino acids that make up the 30—50,000 different proteins which are to be synthesized. The answer is that the bases are read in groups of three, not singly. Since each group of three can contain any one of the four bases in each position, there are 64 possible combinations. This means that four bases give a code consisting of 64 words. Each group of three nucleotides is a codon — a single unit of the genetic code.

While 64 codons might not seem much to carry complex information, there is a need for only 22 codons. The information which has to be coded for in DNA is the sequence of the 21 amino acids in proteins, together with a code for the end of the message.

As can be seen from the genetic code (transcribed to RNA) in Tables 9.7 and 9.8, most amino acids are coded for by more than one codon. This provides a measure of protection against mutations — in many cases a single base change in a codon will not affect the amino acid that is incorporated into the protein, and therefore will have no functional significance.

Table 9.7 The genetic code, showing the codons in mRNA

Amino acid

Abbreviation

Codon(s)

Alanine

Ala

GCU

GCC

GCA

GCG

Arginine

Arg

CGU

CGC

CGA

CGG

Asparagine

Asn

AAU

AAC

Aspartic acid

Asp

GAU

GAC

Cysteine

Cys

UGU

UGC

Glutamic acid

Glu

GAA

GAG

Glutamine

Gln

CAA

CAG

Glycine

Gly

GGU

GGC

GGA

GGG

Histidine

His

CAU

CAG

Isoleucine

Ile

AUU

AUC

AUA

Leucine

Leu

UUA

UUG

CUU

CUC

Lysine

Lys

AAA

AUG

Methionine

Met

AUG

Phenylalanine

Phe

UUU

UUC

Proline

Pro

CCU

CCC

CCA

CCG

Serine

Ser

UCU

UCC

UCA

UCG

Threonine

Thr

ACU

ACC

ACA

ACG

Tryptophan

Trp

UGG

Tyrosine

Tyr

UAU

UAC

Valine

Val

GUU

GUC

GUA

GUG

Stop

UAA

UAG

UGA*

t

  • UGA also codes for selenocysteine in a specific context.
  • UGA also codes for selenocysteine in a specific context.
Table 9.8 The genetic code, showing the codons in mRNA

First base

Second base

Third base

U

C

A

G

U

Phe

Ser

Tyr

Cys

U

U

Phe

Ser

Tyr

Cys

C

U

Leu

Ser

Stop

Stop*

A

U

Leu

Ser

Stop

Trp

G

C

Leu

Pro

His

Arg

U

C

Leu

Pro

His

Arg

C

C

Leu

Pro

Gln

Arg

A

C

Leu

Pro

Gln

Arg

C

A

Ile

Thr

Asn

Ser

U

A

Ile

Thr

Asn

Ser

C

A

Ile

Thr

Lys

Arg

A

A

Met

Thr

Lys

Arg

G

G

Val

Ala

Asp

Gly

U

G

Val

Ala

Asp

Gly

C

G

Val

Ala

Glu

Gly

A

G

Val

Ala

Glu

Gly

G

  • UGA also codes for selenocysteine in a specific context.
  • UGA also codes for selenocysteine in a specific context.

Three codons (UAA, UAG and UGA) do not code for amino acids, but act as stop signals to show the end of the message to be translated and so terminate protein synthesis.

UGA also codes for the selenium analogue of cysteine, selenocysteine (section 11.15.2.5). It is normally read as a stop codon, but in the presence of a specific sequence in the untranslated region of mRNA it is read as coding for selenocysteine.

9.2.2 Ribonucleic acid (RNA)

In RNA the sugar is ribose, rather than deoxyribose as in DNA, and RNA contains the pyrimidine uracil where DNA contains thymine. There are three main types of RNA in the cell:

  • Messenger RNA (mRNA) is synthesized in the nucleus, as a copy of one strand of DNA (the process of transcription; section 9.2.2.1). After some editing of the message, it is transferred into the cytosol, where it binds to ribosomes. The information carried by the mRNA is then translated into the amino acid sequence of the protein.
  • Ribosomal RNA (rRNA) is part of the structure of the ribosomes on which protein is synthesized (section 9.2.3.2).
  • Transfer RNA (tRNA) provides the link between mRNA and the amino acids required for protein synthesis on the ribosome (section 9.2.3.1).

9.2.2.1 Transcription to form messenger RNA (mRNA)

In the transcription of DNA to form mRNA a part of the desired region of DNA is uncoiled, and the two strands of the double helix are separated. A complementary copy of one DNA strand is then synthesized by binding the complementary nucleotide triphosphate to the each base of DNA in turn, followed by condensation to form the phosphodiester link between ribose moieties.

Transcription control sites in DNA include start and stop messages and promoter and enhancer sequences. The main promoter region for any gene is about 25 bases before (upstream of) the beginning of the gene to be transcribed. It acts as a signal that what follows is a gene to be transcribed.

Enhancer and promoter regions may be found further upstream of the message, downstream or sometimes even in the middle of the message. The function of these regions, and of hormone response elements (section 10.4), is to increase the rate at which the gene is transcribed.

The first step in the transcription of a gene is to uncoil that region of DNA from its associated proteins, so as to allow the various enzymes involved in transcription to gain access to the DNA. RNA polymerase moves along the DNA strand which is to be transcribed, and matches complementary ribonucleotide triphosphates one at a time to the bases in the DNA. Adenine in DNA is matched by guanosine triphosphate, guanine by adenosine triphosphate and thymine by cytosine triphosphate. However, cytidine in DNA is matched by uridine triphosphate — RNA contains uridine rather than thymidine as in DNA.

There are three steps in the processing of the initial transcript formed by RNA polymerase before it is exported from the nucleus as messenger RNA:

  • The 5' end of the RNA is blocked by the formation of the unusual base 7-methylguanosine triphosphate. This is called the 'cap' and has a role in the initiation of protein synthesis (section 92.3.2). The 5' end of the RNA is the first to be synthesized, and the cap is added before transcription has been completed.
  • A tail of between up to 250 adenosine residues (the poly-A tail) is added to the 3' end of the RNA, after the termination codon.

The introns that have been copied from DNA, which do not carry information for protein synthesis, are excised, and the remaining coding regions (exons) are spliced together to form messenger RNA (mRNA), which is exported into the cytosol.

9.2.3 Translation of mrna - the

PROCESS OF PROTEIN SYNTHESIS

The process of protein synthesis consists of translating the message carried by the sequence of bases on mRNA into amino acids, and then forming peptide bonds between the amino acids to form a protein. This occurs on the ribosome and requires a variety of enzymes, as well as specific transfer RNA (tRNA) molecules for each amino acid.

9.2.3.1 Transfer RNA (tRNA)

The key to translating the message carried by the codons on mRNA into amino acids is transfer RNA (tRNA). There are 56 different types (species) of tRNA in the cell. They all have the same general structure, RNA twisted into a clover-leaf shape, and consisting of some 70-90 nucleotides. About half the bases in tRNA are paired by hydrogen bonding, which maintains of the shape of the molecule. The 3' and 5' ends of the molecule are adjacent to each other as a result of this folding.

The different species of tRNA have many regions in common with each other, and all have a -CCA tail at the 3' end, which reacts with the amino acid. Two regions are important in providing the specificity of the tRNA species:

  • The anticodon, a sequence of three bases at the base of the clover-leaf. The bases in the anticodon are complementary to the bases of the codon of mRNA, and each species of tRNA binds specifically to one codon or, in some cases, two closely related codons for the same amino acid.
  • The region at the 5' end of the molecule, which again contains a base sequence specific for the amino acid, and hence repeats the information contained in the anticodon.

Amino acids bind to activating enzymes (amino acyl-tRNA synthetases), which recognize both the amino acid and the appropriate tRNA molecule. The first step is reaction between the amino acid and ATP, to form amino acyl AMP, releasing pyrophosphate. The amino acyl AMP then reacts with the -CCA tail of tRNA to form amino acyl-tRNA, releasing AMP

The specificity of these enzymes is critically important to the process of translation. Each enzyme recognizes only one amino acid but will bind and react with all the various tRNA species that carry an anticodon for that amino acid. Mistakes are extremely rare. The easiest possible mistake would be the attachment of valine to the tRNA for isoleucine, or vice versa, because of the close similarity between the structures of these two amino acids (see Figure 4.18). However, it is only about once in every 3,000 times that this mistake occurs. Amino acyl-tRNA synthetases have a second active site that checks that the correct amino acid has been attached to the tRNA and, if this is found not to be the case, hydrolyses the newly formed bond, releasing tRNA and the amino acid.

9.2.3.2 Protein synthesis on the ribosome

The subcellular organelle concerned with protein synthesis is the ribosome. This consists of two subunits, composed of RNA with a variety of associated proteins. The ribosome permits the binding of the anticodon region of amino acyl tRNA to the codon on mRNA, and aligns the amino acids for formation of peptide bonds. As shown in Figure 9.6, the ribosome binds to mRNA, and has two tRNA binding sites. One, the P site, contains the growing peptide chain, attached to tRNA, while the other, the A site, binds the next amino acyl tRNA to be incorporated into the peptide chain.

Methionine Rna Codon

initiation

oo iiiiil HIHI

iiiiil HIHI

termination

Figure 9.6 Ribosomalprotein synthesis.

The first codon of mRNA (the initiation codon) is always AUG, the codon for methionine. This means that the amino terminus of all newly synthesized proteins is methionine, although this may well be removed in post-translational modification of the protein (section 9.2.3.4).

An initiator methionine tRNA forms a complex with the small ribosomal subunit, then together with a variety of initiation factors (enzymes and other proteins) binds to the initiator codon of mRNA, and finally to a large ribosomal subunit, to form the complete ribosome. The 5' cap of mRNA is important for this process, as it marks the position of the initiator codon. AUG is the only codon for methionine, and anywhere else in mRNA it binds the normal methionine tRNA. It is only immediately adjacent to the cap that AUG binds the initiator methionine tRNA.

After the ribosome has been assembled, with the initiator tRNA bound at the P site and occupying the AUG initiator codon, the next amino acyl tRNA binds to the A site of the ribosome, with its anticodon bound to the next codon in the sequence.

The methionine is released from the initiator tRNA at the P site, and forms a peptide bond to the amino group of the amino acyl tRNA at the A site of the ribosome. The initiator tRNA is then released from the P site, and the growing peptide chain, attached to its tRNA, moves from the A site to the P site. As the peptide chain is attached to tRNA, which occupies a codon on the mRNA, this means that as the peptide chain moves from the A site to the P site, so the whole assembly moves one codon along the mRNA.

As the growing peptide chain moves from the A site to the P site, and the ribosome moves along the mRNA chain, so the next amino acyl tRNA occupies the A site, covering its codon. The growing peptide chain is transferred from the tRNA at the P site, forming a peptide bond to the amino acid at the A site. Again the free tRNA at the P site is released, and the growing peptide, attached to tRNA, moves from the A site to the P site, moving one codon along the mRNA as it does so.

The stop codons (UAA, UAG and UGA) are read not by tRNA but by protein release factors. These occupy the A site of the ribosome and hydrolyse the peptide— tRNA bond. This releases the finished protein from the ribosome. As the protein leaves, so the two subunits of the ribosome separate, and leave the mRNA; they are now available to bind another initiator tRNA and begin the process of translation over again.

Just as several molecules of RNA polymerase can transcribe the same gene at the same time, so several ribosomes translate the same molecule of mRNA at the same time. As the ribosomes travel along the ribosome, so each has a longer growing peptide chain than the one following. Such assemblies of ribosomes on a molecule of mRNA are called polysomes.

Termination and release of the protein from the ribosome requires the presence of a stop codon and the protein release factors. However, protein synthesis can also come to a halt if there is not enough of one of the amino acids bound to tRNA. In this case, the growing peptide chain is not released from the ribosome, but remains, in arrested development, until the required amino acyl tRNA is available. This means that if the intake of one of the essential amino acids is inadequate then, once supplies are exhausted, protein synthesis will come to a halt.

9.2.3.3 The energy cost of protein synthesis

The minimum estimate of the energy cost of protein synthesis is four ATP equivalents per peptide bond formed, or 2.8 kJ per gram of protein synthesized:

  • Formation of the amino acyl tRNA requires the formation of amino acyl AMP, with the release of pyrophosphate, which again breaks down to yield phosphate. Hence, for each amino acid attached to tRNA there is a cost equivalent to 2 mol of ATP being hydrolysed to ADP plus phosphate.
  • The binding of each amino acyl tRNA to the A site of the ribosome involves the hydrolysis of GTP to GDP plus phosphate, which is equivalent to the hydrolysis of ATP to ADP plus phosphate.
  • Movement of the growing peptide chain from the A site of the ribosome to the P site again involves the hydrolysis of ATP to ADP plus phosphate.

If allowance is made for the energy cost of active transport of amino acids into cells, the cost of protein synthesis is increased to 3.6 kJ/g. Allowing for the nucleoside triphosphates required for mRNA synthesis gives a total cost of 4.2 kJ per gram of protein synthesized.

In the fasting state, when the rate of protein synthesis is relatively low, about 8% of total energy expenditure (i.e. about 12% of the basal metabolic rate) is accounted for by protein synthesis. After a meal, when the rate of protein synthesis increases, it may account for 12—20% of total energy expenditure.

9.2.3.4 Post-translational modification of proteins

Proteins that are to be exported from the cell, or are to be targeted into mitochondria, are synthesized with a hydrophobic signal sequence of amino acids at the amino terminus to direct them through the membrane. This is removed in the process of post-translational modification. Many other proteins have regions removed from the amino or carboxy terminus during post-translational modification, and the initial (amino-terminal) methionine is removed from most newly synthesized proteins.

Many proteins contain carbohydrates and lipids, covalently bound to amino acid side-chains. Others contain covalently bound cofactors and prosthetic groups, such as vitamins and their derivatives, metal ions or haem. Again the attachment of these non-amino acid parts of the protein is part of the process of post-translational modification to form the active protein.

Some proteins contain unusual amino acids for which there is no codon and no tRNA. These are formed by modification of the protein after translation is complete. Such amino acids include:

  • Methylhistidine in the contractile proteins of muscle.
  • Hydroxyproline and hydroxylysine in the connective tissue proteins. The formation of hydroxyproline and hydroxylysine requires vitamin C as a cofactor. This explains why wound healing, which requires new synthesis of connective tissue, is impaired in vitamin C deficiency (section 11.14.3). See Problem 9.3 for the role of vitamin C in synthesis of hydroxyproline and hydroxylysine.
  • Interchain links in collagen and elastin, formed by the oxidation of lysine residues. This reaction is catalysed by a copper-dependent enzyme, and copper deficiency leads to fragility of bones and loss of the elasticity of connective tissues (section 11.15.2.2).
  • y-Carboxyglutamate in several of the blood clotting proteins, and in osteocalcin in bone. The formation of y-carboxyglutamate requires vitamin K (section 11.15.2). See Problem 9.2 for the role of vitamin K in synthesis of y-carboxyglutamate.
Drop The Fat Now

Drop The Fat Now

Statistics For Obesity Are Rising And The Majority Of People Are Not Getting Enough Exercise Nor Are Having Any Regard For Their Health! Will You Finally Make Good On Your Promise And Set Your Goals To Improve Your Fitness And Live The Healthy Lifestyle You Want? With A Little Bit of Motivation, You Can Set Yourself On the Correct Path To Losing Weight And Feeling Great Using Nothing But Purely Natural Means!

Get My Free Ebook


Post a comment