TRANSLATION AND

MICROBIAL PROTEIN PRODUCTION IN BACTERIA

Submitted by: 3373

Submitted to:

Madam Sana

Govt. Degree College for Women, GRW

TRANSLATION IN BACTERIA

Initiation: Initiation of translation in prokaryotes involves the assembly of the components of the translation system, which are: the two ribosomal subunits (50S and 30Ssubunits); the mature mRNA to be translated; the tRNA charged with N-formylmethionine (the first amino acid in the nascent peptide); guanosine triphosphate(GTP) as a source of energy; the prokaryotic elongation factor EF-P and the three prokaryotic initiation factors IF1, IF2, and IF3, which help the assembly of the initiation complex. Variations in the mechanism can be anticipated.

The ribosome has three active sites: the A site, the P site, and the E site. The A site is the point of entry for the aminoacyl tRNA (except for the first aminoacyl tRNA, which enters at the P site). The P site is where the peptidyl tRNA is formed in the ribosome. And the E site which is the exit site of the now uncharged tRNA after it gives its amino acid to the growing peptide chain.

The selection of an initiation site (usually an AUG codon) depends on the interaction between the 30S subunit and the mRNA template. The 30S subunit binds to the mRNA template at a purine-rich region (the Shine-Dalgarno sequence) upstream of the AUG initiation codon. The Shine-Dalgarno sequence is complementary to a pyrimidine rich region on the 16S rRNA component of the 30S subunit. During the formation of the initiation complex, these complementary nucleotide

sequences pair to form a double stranded RNA structure that binds the mRNA to the ribosome in such a way that the initiation codon is placed at the P site.

Elongation: Elongation of the polypeptide chain involves addition of amino acids to the carboxyl end of the growing chain. The growing protein exits the ribosome through the polypeptide exit tunnel in the large subunit.

Elongation starts when the fMet-tRNA enters the P site, causing a conformational change which opens the A site for the new aminoacyl-tRNA to bind. This binding is facilitated by elongation factor-Tu (EF-Tu), a small GTPase. For fast and accurate recognition of the appropriate tRNA, the ribosome utilizes large conformational changes (conformational proofreading). Now the P site contains the beginning of the peptide chain of the protein to be encoded and the A site has the next amino acid to be added to the peptide chain. The growing polypeptide connected to the tRNA in the P site is detached from the tRNA in the P site and a peptide bond is formed between the last amino acids of the polypeptide and the amino acid still attached to the tRNA in the A site. This process, known as peptide bond formation, is catalyzed by a ribozyme (the 23S ribosomal RNA in the 50S ribosomal subunit). Now, the A site has the newly formed peptide, while the P site has an uncharged tRNA (tRNA with no amino acids). The newly formed peptide in the A site tRNA is known as dipeptide and the whole assembly is called dipeptidyl-tRNA. The tRNA in the P site minus the amino acid is known to be deacylated. In the final stage of elongation, called translocation, the deacylated tRNA (in the P site) and thedipeptidyl-tRNA (in the A site) along with its corresponding codons move to the E and P sites, respectively, and a new codon moves into the A site. This process is catalyzed by elongation factor G (EF-G). The deacylated tRNA at the E site is released from the ribosome during the next A-site occupation by an aminoacyl-tRNA again facilitated by EF-Tu.

The ribosome continues to translate the remaining codons on the mRNA as more aminoacyl-tRNA bind to the A site, until the ribosome reaches a stop codon on mRNA(UAA, UGA, or UAG).

The translation machinery works relatively slowly compared to the enzyme systems that catalyze DNA replication. Proteins in prokaryotes are synthesized at a rate of only 18 amino acid residues per second, whereas bacterial replisomes synthesize DNA at a rate of 1000 nucleotides per second. This difference in rate reflects, in part, the difference between polymerizing four types of nucleotides to make nucleic acids and polymerizing 20 types of amino acids to make proteins. Testing and rejecting incorrect aminoacyl-tRNA molecules takes time and slows protein synthesis. In bacteria, translation initiation occurs as soon as the 5' end of an mRNA is synthesized, and translation and transcription are coupled. This is not possible in eukaryotes because transcription and translation are carried out in separate compartments of the cell (the nucleus and cytoplasm).

Termination:

Termination occurs when one of the three termination codons moves into the A site. These codons are not recognized by any tRNAs. Instead, they are recognized by proteins called release factors, namely RF1 (recognizing the UAA and UAG stop codons) or RF2 (recognizing the UAA and UGA stop codons). These factors trigger the hydrolysis of the ester bond in peptidyl-tRNA and the release of the newly synthesized protein from the ribosome. A third release factor RF-3 catalyzes the release of RF-1 and RF-2 at the end of the termination process.

Recycling:

The post-termination complex formed by the end of the termination step consists of mRNA with the termination codon at the A-site, an uncharged tRNA in the P site, and the intact 70S ribosome. Ribosome recycling step is responsible for the disassembly of the post-termination ribosomal complex. Once the nascent protein is released in termination, Ribosome Recycling Factor and Elongation Factor G (EF-G) function to release mRNA and tRNAs from ribosomes and dissociate the 70S ribosome into the 30S and 50S subunits. IF3 then replaces the deacylated tRNA releasing the mRNA. All translational components are now free

for additional rounds of translation.

Polysomes:

Translation is carried out by more than one ribosome simultaneously. Because of the relatively large size of ribosomes, they can only attach to sites on mRNA 35 nucleotides apart. The complex of one mRNA and a number of ribosomes is

called a polysome or polyribosome

Regulation of translation:

When bacterial cells run out of nutrients, they enter stationary phase and down regulate protein synthesis. Several processes mediate this transition. For instance, in E. coli, 70S ribosomes form 90S dimers upon binding with a small 6.5 kDa protein, ribosome modulation factor RMF. These intermediate ribosome dimers can subsequently bind a hibernation promotion factor protein, HPFmolecule to form a mature 100S ribosomal particle, in which the dimerization interface is made by the two 30S subunits of the two participating ribosomes. The ribosome dimers represent a hibernation state and are translationally inactive. A third protein that can bind to ribosomes when E. coli cells enter the stationary phase is YfiA (previously known as RaiA). HPF and YfiA are structurally similar, and both proteins can bind to the catalytic A- and P-sites of the ribosome. RMF blocks ribosome binding to mRNA by preventing interaction of the messenger with 16S rRNA.When bound to the ribosomes the C-terminal tail of E. coli YfiA interferes

with the binding of RMF, thus preventing dimerization and resulting in the formation of translationally inactive monomeric 70S ribosomes.

Mechanism of ribosomal subunit dissociation by RsfS (= RsfA)

In addition to ribosome dimerization, the joining of the two ribosomal subunits can be blocked by RsfS (formerly called RsfA or YbeB). RsfS binds to L14, a protein of the large ribosomal subunit, and thereby blocks joining of the small subunit to form a functional 70S ribosome, slowing down or blocking translation entirely. RsfS proteins are found in almost all eubacteria (but not archaea) and homologs are present in mitochondria and chloroplasts . However, it is not known yet how the expression or activity of RsfS is regulated.

Effect of antibiotics:

Several antibiotics exert their action by targeting the translation process in bacteria. They exploit the differences to selectively inhibit protein synthesis in bacteria

without affecting between prokaryotic and eukaryotic translationmechanis the host.

Microbial protein production in bacteria

Special vectors for expression of foreign genes in E.coli:If a foreign (i.e., non-bacterial) gene is simply ligated into a standard vector and cloned in E. coli, it is very unlikely that a significant amount of recombinant protein will be synthesized. This is because expression is dependent on the gene being surrounded by a collection of signals that can be recognized by the bacterium. These signals, which are short sequences of nucleotides, advertise the presence of the gene and provide instructions for the transcriptional and translational apparatus of the cell. The three most important signals for E. coli genes are as follows the promoter, which marks the point at which transcription of

the gene should start. In E. coli, the promoter is recognized by the subunit of the transcribing enzyme RNA polymerase.

The terminator, which marks the point at the end of the gene where transcription should stop. A terminator is usually a nucleotide sequence that can base pair with itself to form a stem–loop structure.The ribosome binding site, a short nucleotide sequence recognized by the ribosome as the point at which it should attach to the mRNA molecule. The initiation codon of the gene is always a few nucleotides downstream of this site. The genes of higher organisms are also surrounded by expression signals, but their nucleotide sequences are not the same as theE. coli versions. This is illustrated by comparing the promoters of E. coli and human genes . There are similarities,but it is unlikely that an E. coli RNA polymerase would be able to attach to a human promoter. A foreign gene is inactive in E. coli, simply because the bacterium does not recognize its expression signals. A solution to this problem would be to

insert the foreign gene into the vector in such a way that it is placed under control of a set of E. coli expression signals. If this can be achieved, then the gene should be transcribed and translated . Cloning vectors that provide these signals, and can therefore be used in the production of recombinant protein, are called expression vectors.The promoter is the critical component of an expression vector

The promoter is the most important component of an expression vector. This is because the promoter controls the very first stage of gene expression (attachment of an RNA polymerase enzyme to the DNA) and determines the rate at which mRNA is synthesized. The amount of recombinant protein obtained therefore depends to a great extent on the nature of the promoter carried by the expression vector. The promoter must be chosen with care. The two sequences shown in

are consensus sequences, averages of all the E. coli promoter sequences that are known. Although most E. coli promoters do notdiffer much from these consensus sequences (e.g., TTTACA instead of TTGACA), a small variation may have a major effect on the efficiency with which the promoter can direct transcription. Strong promoters are those that can sustain a high rate of transcription;strong promoters usually control genes whose translation products arerequired in large amounts by the cell . In contrast, weak promoters, which are relatively inefficient, direct transcription of genes whose products are needed in only small amounts . Clearly an expression vector should carry a strong promoter, so that the cloned gene is transcribed at the highest possible rate.A second factor to be considered when constructing an expression vector is whether

it will be possible to regulate the promoter in any way. Two major types of gene regulation are recognized in E. coli—induction and repression. An inducible gene is one whose transcription is switched on by addition of a chemical to the growth medium; Often this chemical is one of the substrates for the enzyme coded by the inducible gene. In contrast, a repressible gene is switched off by addition of the regulatory chemical .Gene regulation is a complex process that only indirectly involves the promoter itself.However, many of the sequences important for induction and repression lie in the region surrounding the promoter and are therefore also present in an expression vector.It may therefore be possible to extend the regulation to the expression vector, so that the chemical that induces or represses the gene normally controlled by the promoter is also able to regulate expression of the cloned gene. This can be a distinct advantage in the production of recombinant protein.Examples of promoters used in expression vectorsSeveral E. coli promoters combine the desired features of strength and ease of regulation.Those most frequently used in expression vectors are as follows:l The lac promoter is the sequence that controls transcription ofthe lacZ gene coding for b-galactosidase (and also the lacZ gene ′fragment carried by the pUC and M13mp vectors; p. 79). The lac promoter is induced by isopropylthiogalactoside (IPTG, p. 80), so addition of this chemical into the growth medium switches on transcription of a gene inserted downstream of the lac promoter carried by an expression vector.The trp promoter is normally upstream of the cluster of genes coding for several of the enzymes involved in biosynthesis of the amino acid tryptophan. The trp promoter is repressed by tryptophan, but is more easily induced by 3-b-indoleacrylic acid.l The tac promoter is a hybrid between the trp and lac promoters.It is stronger than either, but still induced by IPTG.

The EPL promoter is one of the promoters responsible for transcription of the e DNA molecule. ePL is a very strong promoter that is recognized by the E. coli RNA polymerase, which is subverted by e into transcribing the bacteriophage DNA. The promoter is repressed by the product of the ecI gene. Expression vectors that carry the ePL promoter are used with a mutant E. coli host that synthesizes a temperature-sensitive form of the cI protein (p. 40). At a low temperature (less than 30°C) this mutant cI protein is able to repress the ePL promoter, but at higher temperatures the protein is inactivated, resulting in transcription of the cloned gene.Cassettes and gene fusions An efficient expression vector requires not only a strong, regulatable promoter, but also an E. coli ribosome binding sequence and a terminator. In most vectors these expression signals form a cassette, so-called because the foreign gene is inserted into a unique restriction site present in the middle of the expression signal clusters. Ligation of the foreign gene into the cassette therefore places it in the ideal position relative to the expression signals.With some cassette vectors the cloning site is not immediately adjacent to the ribosome binding sequence, but instead is preceded by a segment from the beginning of an E.coli gene. Insertion of the foreign gene into this restriction site must be performed in such a way as to fuse the two reading frames, producing a hybrid gene that starts with the E. coli segment and progresses without a break into the codonsof the foreign gene. The product of gene expression is therefore a hybrid or fusion protein, consisting of the short peptide coded by the E. coli reading frame fused to the amino-terminus of the foreign protein. This fusion system has four advantagesEfficient translation of the mRNA produced from the cloned gene depends not only on the presence of a ribosome binding site, but is also affected by the nucleotide sequence at the start of the coding region. This is probably because secondary structures resulting from

intrastrand base pairs could interfere with attachment of the ribosome to its binding site.

General problems with the production of recombinant proteins in E.coli

Despite the development of sophisticated expression vectors, there are still numerous difficulties associated with the production of protein E.coli. These problems can be grouped into two categories: those that are due to the sequence of the foreign gene, and those that are due to the limitations of E. coli as a host for recombinant protein synthesis.

Problems resulting from the sequence of foreign gene

The foreign gene might contain introns. This would be a major problem, as E. coli genes do not contain introns and therefore the bacterium does not possess the necessary machinery for removing introns from transcripts. The foreign gene might contain sequences that act as termination signals in E. coli.These sequences are perfectly innocuous in the normal host cell,but in the bacterium result in premature termination and a loss of gene expression

Problems caused by E. coli

E. coli might not process the recombinant protein correctly. The proteins of most organisms are processed after translation by

chemical modification of amino acids within the polypeptide. Often these processing events are essential for the correctbiological activity of the protein. Unfortunately, the proteins of bacteria and higher organisms are not processed identically. In particular, some animal proteins are glycosylated, meaning that they have sugar groups attached to them after translation. Glycosylation is extremely uncommon in bacteria and recombinant glycosylated correctly. E. coli might not fold the recombinant protein correctly, and generally is unable to synthesize the disulphide bonds present in many animal proteins. If the protein does not take up its correctly folded tertiary structure, then usually it is insoluble and forms an inclusion body within the bacterium. Recovery of the protein from the inclusion body is not a problem, but converting the protein into its correctly folded form can be difficult or impossible in the test tube. Under these circumstances the protein is, of course, inactive. E. coli might degrade the recombinant protein. Exactly how E. coli can recognize the foreign protein, and thereby subject it to preferential turnover, is not known.