Protein Synthesis in Bacteria
Development and Homeostasis Block
Thursday, September 23, 2004
PROTEIN SYNTHESIS - REGULATION OF TRANSLATION
Topics to be covered
1. Genetic code
7. Protein synthesis in mitochondria
1. Genetic code
Definition: relation between the nucleotide sequence of an mRNA and the amino acid sequence of the protein.
RNA is composed of 4 different nucleotides, adenosine (A), guanosine (G), cytidine (C), uridine (U) and there are 20 amino acids that have to be coded. Therefore, how many nucleotides are necessary to generate 20 amiino acids?
if use 1 then can only code for 4 amino acids
if use 2 then can only code for 16 amino acids
if use 3 then can code for 64 amino acids and
if use 4 can code for 256 amino acids.
Thus 1 or 2 is not enough and 3 or 4 is too many. Therefore the genetic code uses 3 nucleotides. However, since there are 4 bases but only 3 compose the code there are a number of possible formats that could be used, however a “commaless” triplet code is the one that has been universally adopted (Figure 1)
However since there are only 20 amino acids and three stop codons each amino acid (except for tryptophan and methionine) is coded by more than one codon : in other words the genetic code is degenerate which can be demonstrated in tabular form.(Figure 2A). The degree of degeneracy varies from one amino acid to another with arginine, leucine and serine being coded by 6 different nucleotide sequences. This degeneracy means that 20 amino acids can be matched to 61 codons using only 31 tRNAs (Figure 2B).
2. Overview of protein synthesis
While the genetic code itself resides in DNA, DNA is never used directly in the synthesis of proteins. DNA is “transcribed” into messenger RNA (mRNA) that carries information from DNA and it is mRNA that is then used to “translate” this information into a specific sequence of amino acids that constitute proteins.Similarly, amino acids cannot recognize codons directly, an adapter molecule is necessary, a transfer RNA (tRNA). Amino acids are incorporated into a protein in an order predetermined by the mRNA sequence. A tRNA can recognize more than one codon; very often, the first 2 nucleotides of the codon are sufficient to specify an amino acid and the third nucleotide varies. The first two nucleotides of the codon form a standard Watson-Crick pairing with the last two nucleotides of the tRNA. The third nucleotide of the codon forms a non standard Watson-Crick pairing or “wobble”.The overriding constraint in protein synthesis is that theere is little margin for error. Messenger RNA has to be translated correctly hence there are a variety of safeguards built in to the process to minimize errors. Protein synthesis only begins when all the components, appropriate mRNA, tRNAs with loaded amino acids, ribosomal subunits and other auxiliary factors come together to form a functional ribosome, the site of protein synthesis in the cell. Then, as a single mRNA moves stepwise through each ribosome, the sequence of nucleotides in the mRNA is translated into a corresponding sequence of amino acids to produce a nascent polypeptide chain.
3. Transcription (DNA > RNA)
RNA is synthesized on a DNA template in the process known as DNA transcription. Transcription generates the mRNA containing the information to synthesize a specific protein and also the other RNA molecules, ribosomal RNA, tRNA’s involved in the process. The key enzyme(s) involved in the process is RNA polymerase, an incredibly complex enzyme of molecular mass of 500,000kDa. DNA is transcribed by RNA polymerase binding to a specific start site or “promoter” on the DNA and proceeding until it reaches a termination signal.The DNA double helix is partially unwound by the polymerase and transcription always proceeds in a 3’ to 5’ direction on the DNA template so that the RNA produced is extended in a 5’ to 3’ direction.In theory any region of a DNA molecule could be transcribed into two RNA molecules (one form each strand). However only one strand is copied at any given time, although it is not always the same strand for different genes. The strand to be copied is determined by the promoter sequence.Typical mRNA is 70 – 10,000 nucleotides in length and is codeified by RNA splicing before becoming functional. Only one strand of DNA is copied at any one time. However either strand can be copied. The strand that is copied is determined by the promoter.The process is rapid and proceeds at a rate of about 30 nucleotides per second. There are 3 RNA polymerases, one makes mRNA while the other 2 make tRNA and rRNA. (Figure 3).
4. Translation (RNA > Protein)
Amino acids have first to be loaded onto specific tRNAs. The process is energetically unfavorable so amino acids are first activated by adenylation using ATP. The adenylated amino acid is then linked to the 3' end (always a CCA sequence) of the tRNA to form an aminoacyl tRNA. This reaction is catalyzed by a specific aminoacyl-tRNA synthase (Figure 4). There are distinct synthases for each of the 20 amino acids. Each synthase must be able to recognize the correct amino acid and an appropriate tRNA (Figure 5).
Simplistically, a polypeptide can be formed by the stepwise addition of new amino acids to its carboxy-terminal end. If the aminoacyl tRNAs can be correctly aligned, the only additional requirement would be a peptidyl transferase enzyme to synthesize the peptide bond between the incoming amino acid and the polypeptide.(Figure 6). The sequence of amino acids in the polypeptide would then be determined only by the tRNA that delivers the amino acid. (Figure 7).
Since alignment is determined by the mRNA, in theory no additional components are required for protein synthesis to proceed since the specificity is provided by the mRNA and the tRNA. (Figure 8).
However to ensure the absolute fidelity and to make the process much more efficient the whole process occurs on a protein/RNA complex, the ribosome(Figure 9a). Ribosomes and some detail of their structure can often be visualized by electron microscopy (Figure 9b).
The ribosome is composed of 2 subunits, the smaller of which binds to the mRNA and has two specific sites (A and P) that bind tRNA. The larger subunit contains the peptidyl transferase activity required to synthesize the peptide bond. It associates with both the tRNA and the small ribosomal subunit (Figure 10).
Together they ensure that 2 aminoacyl-tRNAs come together on the mRNA to form a peptide bond and that the complex advances smoothly along the mRNA (Figure 11).
5. Initiation, elongation and termination
In theory any mRNA can be translated in any of three different "reading frames" depending on the nucleotide sequence at which translation starts. Thus 3 different amino acid sequences can be obtained from a single mRNA (Figure 12).
However in both eucaryotic and procaryotic cells the correct reading frame is set by several initiation factors and by the fact that the start codon for protein synthesis is always AUG, which codes for methionine. Therefore in all proteins, at least as they are synthesized on the ribosome, their first amino acid is always methionine(Figure 13).
However there are a few significant differences between procaryotes and eucaryotes at this point in the process. Initiation always begins at the 5' end of the mRNA and in eucaryotes the 5' end is codeified by the addtion of a “cap”. In eucaryotes the 3' end of the mRNA is always polyadenylated. Furthermore, procaryotic mRNA is often polycistronic (one mRNA can produce several different proteins). This never occurs in eucaryiotes. Thus eucaryotic mRNA is monocystronic (Figure 14A). Initiation always begins at the 5' end of the mRNA and in eukaryotes the 5' end is codeified by the addtion of a "cap". The cap is composed of a terminal 7-methyl guanosine linked by a 5'-to-5' triphosphate bridge. The 1st and 2nd ribose of the mRNA are also often methylated at the 2' hydroxyl (Figure 14B). The end result is that the 5' end of the mRNA becomes positively charged.
Initiation factors are required in order for the ribosome to locate an initiation site. The first event is association of the small ribosomal subunit with a Met-tRNA. In procryotes IF2 is required to locate the AUG start codon and in eucaryotes eIF4 helps the small ribosomal subunit bind to the mRNA cap and search for an AUG. Once an AUG is located the large ribosomal subunit joins the complex and initiation is complete (Figure 14C). The initiation factors then dissociate from the ribosome and a second amino can be added to start the elongation process (Figure 14D).
Elongation proceeds when a second aminoacyl-tRNA is brought to the A site on the ribosme. Peptide bond formation occurs, the ribosome advances 3 bases along the mRNA, the Met-tRNA is displaced and there is a translocation of the peptidyl-tRNA from the A site to the P site (Figure 15A).
The previous diagram is an oversimplification. Several elongation factors are required to maintain the fidelity of the process. The reason relates to the degeneracy of the genetic code. Because of the similarities between certain codons (unavoidable in a 3 digit code). For example, AAC and AAU code for asparagine whereas AAA and AAG code for lysine. The anticodon of an aminoacyl tRNA loaded with asparagine would be able to bind by Watson-Crick and “wobble” pairing to AAA or AAG and asparagine could therefore become incorporated into a protein instead of lysine. Nevertheless the error rate in protein synthesis is extremely low. One incorrect amino acid is incorporated for every 10,000 correct amino acids. So that one out of every 25 proteins produced could contain an error.To maintain the low error rate an elongation factor termed EF1 (Tu in procaryotes) that is a GTP binding protein associates with any aminoacyl-tRNA that is transiently bound to the ribosomal A-site This association triggers EF1 's GTPase activity and GTP is hydrolysed to GDP and EF1 dissociates from the aminoacyl-tRNA (Figure 15B). Because this requires a finite amount of time, aminoacyl-tRNAs will dissociate from the A-site unless they have the correct anticodon. These GTP-binding/GTPase proteins are used quite often to function as biological "timers" or "switches" and function in kinetic proofreading (Figure 15C).
GTP is used as an energy source for elongation and termination. Overall 4 GTPs are hydrolyzed for each polypeptide bond. Therefore protein synthesis consumes more energy than any other biosynthetic process. Termination of the polypeptide chain occurs when any of three codons (UAA, UAG or UGA) is encountered. These three do not code for any amino acid and therefore do not recruit a tRNA (Figure 16A). Instead cytoplasmic "release factors" bind to the A site on the ribosome and causes the peptidyl transferase to add an H2O instead of forming a peptide bond, thus freeing the carboxy terminous of the polypeptide chain. Since this is the attachment to the peptidyl tRNA and the ribosome the newly synthesized polypeptide is released. The ribosomal subunits and the remaining tRNA dissociate from the mRNA (Figure 16B).
The entire process of protein assembly from individual amino acids and the participation of the various RNAs can be seen in this short animation. Animation courtesy of the Department of Biological Sciences at the University of Southern Mississippi (http://tidepool.st.usm.edu/)
Quite often several copies of a protein are produced by several ribosomes advancing sequentially along a single mRNA to form a "polyribosome" which is large enough to be visualized by electron microscopy (Figure 17).
6. Mechanism of action of antibiotics
Almost all of the widely used antibiotics are inhibitors of some component of the protein synthetic process. Those that are used therepeutically act to inhibit procaryotic protein synthesis and therefore stop bacterial growth. However there are others that inhibit both procaryotic and eucaryotic protein synthesis and some that are affective only in procaryotes (Figure 18). The latter have been useful in elucidating the cellular mechanisms of protein synthesis.The inhibition often involves an interaction between the antibiotic and the ribosome. The mechanism of inhibition of protein synthesis by many antibiotics has been elucidated. For example, the chemical structure of puromycin is similar to an aminoacyl-tRNA. This similarity allows puromycin to fool the ribosome and enables it to bind to the the A-site. The peptidyl transferase activity of the ribosome then catalyses the formation of an amide bond between the growing polypeptide chain and puromycin thereby causing premature termination of polypeptide chain elongation and release of the partially formed polypeptide from the ribosome. As might be expected puromycin inhibits protein synthesis in both procaryotes and eucaryotes.
7. Protein synthesis in mitochondria
Why do mitochondria (and chloroplasts) have their own genetic systems whereas other cytoplasmic organelles do not? There is no simple answer. The maintenance of a distinct protein synthetic system in mitochondria is an expensive proposition for the cell. Almost 100 proteins have to be made specifically for this purpose (Figure 19). It has been hypothesized that since most of the mitochondrial enzymes are associated with its membrane, these proteins are so hydrophobic that it would be impossible to get them from the cytosol to the membrane.
However, this now seems an unlikely explanation since there are many other hydrophobic proteins in the cell that can be transported from their cytoplasmic site of synthesis to a membrane without difficulty. Nonetheless, the mitochondria does have its own genome and because of its small size the entire human mitochondrial genome has been sequenced (Figure 20).There are some interesting differences between the mitochondrial and nuclear genomes. Every nucleotide in mitochondrial DNA appears to be a part of a coding sequence and some codons are unique to the mitochondrial genome (Figure 21). Interestingly, because it is acquired by non-Mendelian or cytoplasmic inheritance the mammalian mitochondrial genome is always maternal.
The End
Click here for a printable version of the lecture in PDF format. Note that this is a large file (approx 2mBytes) and the resolution of the images will depend on the quality of your printer.
For information or questions concerning this site please contact: mailto:djfranks@uottawa.ca
pour information au sujet de ce site s'adresser à:djfranks@uottawa.ca
1. Genetic code
Definition: relation between the nucleotide sequence of an mRNA and the amino acid sequence of the protein.
RNA is composed of 4 different nucleotides, adenosine (A), guanosine (G), cytidine (C), uridine (U) and there are 20 amino acids that have to be coded. Therefore, how many nucleotides are necessary to generate 20 amiino acids?
if use 1 then can only code for 4 amino acids
if use 2 then can only code for 16 amino acids
if use 3 then can code for 64 amino acids and
if use 4 can code for 256 amino acids.
Thus 1 or 2 is not enough and 3 or 4 is too many. Therefore the genetic code uses 3 nucleotides. However, since there are 4 bases but only 3 compose the code there are a number of possible formats that could be used, however a “commaless” triplet code is the one that has been universally adopted (Figure 1)
However since there are only 20 amino acids and three stop codons each amino acid (except for tryptophan and methionine) is coded by more than one codon : in other words the genetic code is degenerate which can be demonstrated in tabular form.(Figure 2A). The degree of degeneracy varies from one amino acid to another with arginine, leucine and serine being coded by 6 different nucleotide sequences. This degeneracy means that 20 amino acids can be matched to 61 codons using only 31 tRNAs (Figure 2B).
2. Overview of protein synthesis
While the genetic code itself resides in DNA, DNA is never used directly in the synthesis of proteins. DNA is “transcribed” into messenger RNA (mRNA) that carries information from DNA and it is mRNA that is then used to “translate” this information into a specific sequence of amino acids that constitute proteins.Similarly, amino acids cannot recognize codons directly, an adapter molecule is necessary, a transfer RNA (tRNA). Amino acids are incorporated into a protein in an order predetermined by the mRNA sequence. A tRNA can recognize more than one codon; very often, the first 2 nucleotides of the codon are sufficient to specify an amino acid and the third nucleotide varies. The first two nucleotides of the codon form a standard Watson-Crick pairing with the last two nucleotides of the tRNA. The third nucleotide of the codon forms a non standard Watson-Crick pairing or “wobble”.The overriding constraint in protein synthesis is that theere is little margin for error. Messenger RNA has to be translated correctly hence there are a variety of safeguards built in to the process to minimize errors. Protein synthesis only begins when all the components, appropriate mRNA, tRNAs with loaded amino acids, ribosomal subunits and other auxiliary factors come together to form a functional ribosome, the site of protein synthesis in the cell. Then, as a single mRNA moves stepwise through each ribosome, the sequence of nucleotides in the mRNA is translated into a corresponding sequence of amino acids to produce a nascent polypeptide chain.
3. Transcription (DNA > RNA)
RNA is synthesized on a DNA template in the process known as DNA transcription. Transcription generates the mRNA containing the information to synthesize a specific protein and also the other RNA molecules, ribosomal RNA, tRNA’s involved in the process. The key enzyme(s) involved in the process is RNA polymerase, an incredibly complex enzyme of molecular mass of 500,000kDa. DNA is transcribed by RNA polymerase binding to a specific start site or “promoter” on the DNA and proceeding until it reaches a termination signal.The DNA double helix is partially unwound by the polymerase and transcription always proceeds in a 3’ to 5’ direction on the DNA template so that the RNA produced is extended in a 5’ to 3’ direction.In theory any region of a DNA molecule could be transcribed into two RNA molecules (one form each strand). However only one strand is copied at any given time, although it is not always the same strand for different genes. The strand to be copied is determined by the promoter sequence.Typical mRNA is 70 – 10,000 nucleotides in length and is codeified by RNA splicing before becoming functional. Only one strand of DNA is copied at any one time. However either strand can be copied. The strand that is copied is determined by the promoter.The process is rapid and proceeds at a rate of about 30 nucleotides per second. There are 3 RNA polymerases, one makes mRNA while the other 2 make tRNA and rRNA. (Figure 3).
4. Translation (RNA > Protein)
Amino acids have first to be loaded onto specific tRNAs. The process is energetically unfavorable so amino acids are first activated by adenylation using ATP. The adenylated amino acid is then linked to the 3' end (always a CCA sequence) of the tRNA to form an aminoacyl tRNA. This reaction is catalyzed by a specific aminoacyl-tRNA synthase (Figure 4). There are distinct synthases for each of the 20 amino acids. Each synthase must be able to recognize the correct amino acid and an appropriate tRNA (Figure 5).
Simplistically, a polypeptide can be formed by the stepwise addition of new amino acids to its carboxy-terminal end. If the aminoacyl tRNAs can be correctly aligned, the only additional requirement would be a peptidyl transferase enzyme to synthesize the peptide bond between the incoming amino acid and the polypeptide.(Figure 6). The sequence of amino acids in the polypeptide would then be determined only by the tRNA that delivers the amino acid. (Figure 7).
Since alignment is determined by the mRNA, in theory no additional components are required for protein synthesis to proceed since the specificity is provided by the mRNA and the tRNA. (Figure 8).
However to ensure the absolute fidelity and to make the process much more efficient the whole process occurs on a protein/RNA complex, the ribosome(Figure 9a). Ribosomes and some detail of their structure can often be visualized by electron microscopy (Figure 9b).
The ribosome is composed of 2 subunits, the smaller of which binds to the mRNA and has two specific sites (A and P) that bind tRNA. The larger subunit contains the peptidyl transferase activity required to synthesize the peptide bond. It associates with both the tRNA and the small ribosomal subunit (Figure 10).
Together they ensure that 2 aminoacyl-tRNAs come together on the mRNA to form a peptide bond and that the complex advances smoothly along the mRNA (Figure 11).
5. Initiation, elongation and termination
In theory any mRNA can be translated in any of three different "reading frames" depending on the nucleotide sequence at which translation starts. Thus 3 different amino acid sequences can be obtained from a single mRNA (Figure 12).
However in both eucaryotic and procaryotic cells the correct reading frame is set by several initiation factors and by the fact that the start codon for protein synthesis is always AUG, which codes for methionine. Therefore in all proteins, at least as they are synthesized on the ribosome, their first amino acid is always methionine(Figure 13).
However there are a few significant differences between procaryotes and eucaryotes at this point in the process. Initiation always begins at the 5' end of the mRNA and in eucaryotes the 5' end is codeified by the addtion of a “cap”. In eucaryotes the 3' end of the mRNA is always polyadenylated. Furthermore, procaryotic mRNA is often polycistronic (one mRNA can produce several different proteins). This never occurs in eucaryiotes. Thus eucaryotic mRNA is monocystronic (Figure 14A). Initiation always begins at the 5' end of the mRNA and in eukaryotes the 5' end is codeified by the addtion of a "cap". The cap is composed of a terminal 7-methyl guanosine linked by a 5'-to-5' triphosphate bridge. The 1st and 2nd ribose of the mRNA are also often methylated at the 2' hydroxyl (Figure 14B). The end result is that the 5' end of the mRNA becomes positively charged.
Initiation factors are required in order for the ribosome to locate an initiation site. The first event is association of the small ribosomal subunit with a Met-tRNA. In procryotes IF2 is required to locate the AUG start codon and in eucaryotes eIF4 helps the small ribosomal subunit bind to the mRNA cap and search for an AUG. Once an AUG is located the large ribosomal subunit joins the complex and initiation is complete (Figure 14C). The initiation factors then dissociate from the ribosome and a second amino can be added to start the elongation process (Figure 14D).
Elongation proceeds when a second aminoacyl-tRNA is brought to the A site on the ribosme. Peptide bond formation occurs, the ribosome advances 3 bases along the mRNA, the Met-tRNA is displaced and there is a translocation of the peptidyl-tRNA from the A site to the P site (Figure 15A).
The previous diagram is an oversimplification. Several elongation factors are required to maintain the fidelity of the process. The reason relates to the degeneracy of the genetic code. Because of the similarities between certain codons (unavoidable in a 3 digit code). For example, AAC and AAU code for asparagine whereas AAA and AAG code for lysine. The anticodon of an aminoacyl tRNA loaded with asparagine would be able to bind by Watson-Crick and “wobble” pairing to AAA or AAG and asparagine could therefore become incorporated into a protein instead of lysine. Nevertheless the error rate in protein synthesis is extremely low. One incorrect amino acid is incorporated for every 10,000 correct amino acids. So that one out of every 25 proteins produced could contain an error.To maintain the low error rate an elongation factor termed EF1 (Tu in procaryotes) that is a GTP binding protein associates with any aminoacyl-tRNA that is transiently bound to the ribosomal A-site This association triggers EF1 's GTPase activity and GTP is hydrolysed to GDP and EF1 dissociates from the aminoacyl-tRNA (Figure 15B). Because this requires a finite amount of time, aminoacyl-tRNAs will dissociate from the A-site unless they have the correct anticodon. These GTP-binding/GTPase proteins are used quite often to function as biological "timers" or "switches" and function in kinetic proofreading (Figure 15C).
GTP is used as an energy source for elongation and termination. Overall 4 GTPs are hydrolyzed for each polypeptide bond. Therefore protein synthesis consumes more energy than any other biosynthetic process. Termination of the polypeptide chain occurs when any of three codons (UAA, UAG or UGA) is encountered. These three do not code for any amino acid and therefore do not recruit a tRNA (Figure 16A). Instead cytoplasmic "release factors" bind to the A site on the ribosome and causes the peptidyl transferase to add an H2O instead of forming a peptide bond, thus freeing the carboxy terminous of the polypeptide chain. Since this is the attachment to the peptidyl tRNA and the ribosome the newly synthesized polypeptide is released. The ribosomal subunits and the remaining tRNA dissociate from the mRNA (Figure 16B).
The entire process of protein assembly from individual amino acids and the participation of the various RNAs can be seen in this short animation. Animation courtesy of the Department of Biological Sciences at the University of Southern Mississippi (http://tidepool.st.usm.edu/)
Quite often several copies of a protein are produced by several ribosomes advancing sequentially along a single mRNA to form a "polyribosome" which is large enough to be visualized by electron microscopy (Figure 17).
6. Mechanism of action of antibiotics
Almost all of the widely used antibiotics are inhibitors of some component of the protein synthetic process. Those that are used therepeutically act to inhibit procaryotic protein synthesis and therefore stop bacterial growth. However there are others that inhibit both procaryotic and eucaryotic protein synthesis and some that are affective only in procaryotes (Figure 18). The latter have been useful in elucidating the cellular mechanisms of protein synthesis.The inhibition often involves an interaction between the antibiotic and the ribosome. The mechanism of inhibition of protein synthesis by many antibiotics has been elucidated. For example, the chemical structure of puromycin is similar to an aminoacyl-tRNA. This similarity allows puromycin to fool the ribosome and enables it to bind to the the A-site. The peptidyl transferase activity of the ribosome then catalyses the formation of an amide bond between the growing polypeptide chain and puromycin thereby causing premature termination of polypeptide chain elongation and release of the partially formed polypeptide from the ribosome. As might be expected puromycin inhibits protein synthesis in both procaryotes and eucaryotes.
7. Protein synthesis in mitochondria
Why do mitochondria (and chloroplasts) have their own genetic systems whereas other cytoplasmic organelles do not? There is no simple answer. The maintenance of a distinct protein synthetic system in mitochondria is an expensive proposition for the cell. Almost 100 proteins have to be made specifically for this purpose (Figure 19). It has been hypothesized that since most of the mitochondrial enzymes are associated with its membrane, these proteins are so hydrophobic that it would be impossible to get them from the cytosol to the membrane.
However, this now seems an unlikely explanation since there are many other hydrophobic proteins in the cell that can be transported from their cytoplasmic site of synthesis to a membrane without difficulty. Nonetheless, the mitochondria does have its own genome and because of its small size the entire human mitochondrial genome has been sequenced (Figure 20).There are some interesting differences between the mitochondrial and nuclear genomes. Every nucleotide in mitochondrial DNA appears to be a part of a coding sequence and some codons are unique to the mitochondrial genome (Figure 21). Interestingly, because it is acquired by non-Mendelian or cytoplasmic inheritance the mammalian mitochondrial genome is always maternal.
The End
Click here for a printable version of the lecture in PDF format. Note that this is a large file (approx 2mBytes) and the resolution of the images will depend on the quality of your printer.
For information or questions concerning this site please contact: mailto:djfranks@uottawa.ca
pour information au sujet de ce site s'adresser à:djfranks@uottawa.ca
