The Genetic Code Was Cracked Using Artificial mRNA Templates
By the 1960s it had long been apparent that at least three nucleotide residues of DNA are necessary to en-code each amino acid. The four code letters of DNA (A, T, G, and C) in groups of two can yield only 4 2 =16 different combinations, insufficient to encode 20 amino acids. Groups of three, however, yield 4 3 = 64 different combinations.
A codon is a triplet of nucleotides that codes for a specific amino acid. Translation occurs in such a way that these nucleotide triplets are read in a successive, non-overlapping fashion. A specific first codon in the sequence establishes the reading frame, in which a new codon begins every three nucleotide residues. There is no punctuation between codons for successive amino acid residues. The amino acid sequence of a pro-tein is defined by a linear sequence of contiguous triplets. In principle, any given single-stranded DNA or mRNA sequence has three possible reading frames. Each reading frame gives a different sequence of codons (Fig. 27–5), but only one is likely to encode a given pro-tein.
In 1961 Marshall Nirenberg and Heinrich Matthaei re-ported the first breakthrough. They incubated synthetic polyuridylate, poly (U), with an E. coli extract, GTP, ATP, and a mixture of the 20 amino acids in 20 different tubes, each tube containing a different radioactively labeled amino acid. Because poly (U) mRNA is made up of many successive UUU triplets, it should promote the synthesis of a polypeptide containing only the amino acid encoded by the triplet UUU. A radioactive polypeptide was indeed formed in only one of the 20 tubes, the one containing radioactive phenylalanine. Niren-berg and Matthaei therefore concluded that the triplet codon UUU encodes phenyl-alanine. The same approach re-vealed that polycytidylate, poly(C), encodes a polypep-tide containing only proline (polyproline), and polyadeny-late, poly (A), encodes polylysine. Polyguanylate did not generate any polypeptide in this experiment because it spontaneously forms tetraplexes that can-not be bound by ribosomes
The synthetic polynucleotides used in such experiments were prepared with polynucleotide phosphory-lase, which catalyzes the formation of RNA polymers starting from ADP, UDP, CDP, and GDP. This enzyme requires no template and makes polymers with a base composition that directly reflects the relative concentrations of the nucleoside 5′-diphosphate pre-cursors in the medium. If polynucleotide phosphorylase is presented with UDP only, it makes only poly(U). If it is presented with a mixture of five parts ADP and one part CDP, it makes a polymer in which about five-sixths of the residues are adenylate and one-sixth are cytidy-late. This random polymer is likely to have many triplets of the sequence AAA, smaller numbers of AAC, ACA, and CAA triplets, relatively few ACC, CCA, and CAC triplets, and very few CCC triplets (Table 27–1). Using a variety of artificial mRNAs made by polynucleotide phosphorylase from different starting mixtures of ADP, GDP, UDP, and CDP, investigators soon identified the base compositions of the triplets coding for almost all the amino acids. Although these experiments revealed the base composition of the coding triplets, they could not reveal the sequence of the bases
In 1964 Nirenberg and Philip Leder achieved an-other experimental breakthrough. Isolated E. coli ribo-somes would bind a specific aminoacyl-tRNA in the presence of the corresponding synthetic polynucleotide messenger. For example, ribosomes incu-bated with poly(U) and phenylalanyl-tRNA Phe (Phe-tRNA Phe ) bind both RNAs, but if the ribosomes are incubated with poly(U) and some other aminoacyl-tRNA, the aminoacyl-tRNA is not bound, because it does not recognize the UUU triplets in poly(U). Even trinucleotides could promote specific binding of appropriate tRNAs, so these experiments could be car-ried out with chemically synthesized small oligonu-cleotides. With this technique researchers determined which aminoacyl-tRNA bound to about 50 of the 64 possible triplet codons. For some codons, either no amino-acyl-tRNA or more than one would bind. Another method was needed to complete and confirm the entire genetic code
H. Gobind Khorana, developed chemical methods to synthesize poly-ribonucleotides with defined, repeating sequences of two to four bases. The polypeptides produced by these mRNAs had one or a few amino acids in repeating patterns. These patterns, when combined with information from the random polymers used by Nirenberg and colleagues, permitted unambiguous codon assignments. The copolymer (AC)n, for example, has alternating ACA and CAC codons: ACACACACACACACA. The polypeptide syn-thesized on this messenger contained equal amounts of threonine and histidine. Given that a histidine codon has one A and two Cs (Table 27–1), CAC must code for his-tidine and ACA for threonine.
Consolidation of the results from many experiments permitted the assignment of 61 of the 64 possible codons. The other three were identified as termination codons, in part because they disrupted amino acid coding patterns when they occurred in a synthetic RNA polymer (Fig. 27–6). Meanings for all the triplet codons (tabulated in Fig. 27–7) were established by 1966 and have been verified in many different ways. The cracking of the genetic code is regarded as one of the most important scientific discoveries of the twentieth century.
Several codons serve special functions. The initiation codon AUG is the most common signal for the beginning of a polypeptide in all cells, in addition to coding for Met residues in internal positions of polypep-tides. The termination codons (UAA, UAG, and UGA), also called stop codons or nonsense codons, normally signal the end of polypeptide synthesis and do not code for any known amino acids In a random sequence of nucleotides, 1 in every 20 codons in each reading frame is, on average, a termination codon. In general, a reading frame without a ter-mination codon among 50 or more codons is referred to as an open reading frame (ORF). Long open reading frames usually correspond to genes that encode pro-teins. In the analysis of sequence databases, sophisticated programs are used to search for open reading frames in order to find genes among the often huge background of nongenic DNA. An uninterrupted gene coding for a typical protein with a molecular weight of 60,000 would require an open reading frame with 500 or more codons.
A striking feature of the genetic code is that an amino acid may be specified by more than one codon, so the code is described as degenerate. The degeneracy of the code is not uni-form. Whereas methionine and tryptophan have single codons, for example, three amino acids (Leu, Ser, Arg) have six codons, five amino acids have four, isoleucine has three, and nine amino acids have two (Table 27–3).
The genetic code is universal i.e. prokaryotic and eukaryotic organisms use same codon to specify each amino acid. Rare exception is codon use in yeast mitochondria and Mycoplasma. Thus all life forms have common evolutionary ancestor,whose genetic code has been preserved through biological evolution.
Wobble Allows Some tRNAs to Recognize More than One Codon
When several different codons specify one amino acid, the difference between them usually lies at the third base position (at the 3′ end). For example, alanine is coded by the triplets GCU, GCC, GCA, and GCG. The codons for most amino acids can be symbolized by XYAG or XYUC. The first two letters of each codon are the primary determinants of specificity, a feature that has some interesting consequences. Transfer RNAs base-pair with mRNA codons at a three-base sequence on the tRNA called the anticodon. The first base of the codon in mRNA (read in the 5′—3′ direction) pairs with the third base of the anticodon. If the anticodon triplet of a tRNA recognized only one codon triplet through Watson-Crick base pairing at all three positions, cells would have a different tRNA for each amino acid codon. This is not the case, however, because the anticodons in some tRNAs include the nucleotide inosinate (designated I), which contains the uncommon base hypoxanthine. Inosinate can form hydrogen bonds with three different nucleotides (U, C, and A) althoughthese pairings are much weaker than the hydrogen bonds of Watson-Crick base pairs (GmC and AUU). In yeast, one tRNAArg has the anticodon (5′) ICG, which recognizes three arginine codons: (5′)CGA, (5′)CGU,and (5′)CGC. The first two bases are identical (CG) and form strong Watson-Crick base pairs with the corresponding bases of the anticodon, but the third base (A, U, or C) forms rather weak hydrogen bonds with the I residue at the first position of the anticodon. Examination of these and other codon-anticodon pairings led Crick to conclude that the third base of most codons pairs rather loosely with the corresponding base of its anticodon; to use his picturesque word, the third base of such codons (and the first base of their corre-sponding anticodons) “wobbles.” Crick proposed a set of four relationships called the wobble hypothesis:
1. The first two bases of an mRNA codon always form strong Watson-Crick base pairs with the corresponding bases of the tRNA anticodon and confer most of the coding specificity.
2.The first base of the anticodon (reading in the 5’__3’direction; this pairs with the third base of the codon) determines the number of codons recognized by the tRNA. When the first base of the anticodonis C or A, base pairing is specific and only one codon is recognized by that tRNA.
3.When the first base is U or G, binding is less specific and two different codons may be read. When inosine (I) is the first (wobble) nucleotide of an anticodon, three different codons can be recognized—the maximum number for any tRNA. When an amino acid is specified by several different codons, the codons that differ in either of the first two bases require different tRNAs.
4.A minimum of 32 tRNAs are required to translate all 61 codons (31 to encode the amino acids and 1 for initiation). The wobble (or third) base of the codon con-tributes to specificity, but, because it pairs only loosely with its corresponding base in the anticodon, it per-mits rapid dissociation of the tRNA from its codon dur-ing protein synthesis. If all three bases of a codon engaged in strong Watson-Crick pairing with the three bases of the anticodon, tRNAs would dissociate too slowly and this would severely limit the rate of protein synthesis. Codon-anticodon interactions balance the requirements for accuracy and speed.The genetic code tells us how protein sequence in-formation is stored in nucleic acids and provides some clues about how that information is translated into protein.
Breif generalizations about Genetic code:-
- The genetic code is universal i.e. prokaryotic and eukaryotic organisms use same codon to specify each amino acid. Rare exception is codon use in yeast mitochondria and Mycoplasma.Thus all life forms have common evolutionary ancestor,whose genetic code has been preserved through biological evolution.
- The code is degenerate i.e more than one arrangement of nucleotide triplet specify same aminoacid. Thus UUA, UUG, CUU, CUC, CUA and CUG all code for leucine.
- For a given amino acid, first two bases are limited to one or two combination, but third base can have as many as more. This suggests that a change in third base by mutation may still allow the correct translation of a given aminoacid into protein.
- The degeneracy of codon is not uniform. Whereas methionine and tryptophan has single codon, Leu, Ser and Arg have six codons, isoleucine has three and nine amino acids have two, five amino acids have four codons.
- The code is non-overlapping i.e adjacent codons do not overlap.
- The code is commaless i.e there are no specific signals or commas between codons.
- Of 64 codons, 61 are employed for encoding aminoacids. Three UAA, UAG and UGA are called termination codons.
- AUG serve as initiator codon as well as internal methionine codons.
- In general amino acid with hydrocarbon side chain have U or C as second base, those with branched methyl groups have U as the second base. Basic and acidic amino acids have A or G as the second base.
- Polarity: The genetic code has polarity, that is, the code is always read in a fixed direction, i.e., in the 5′ →3′direction. It is apparent that if the code is read in opposite direction (i.e., 3′ → 5′), it would specify 2 different proteins, since the codon would have reversed base sequence :
Codon : UUG AUC GUC UCG CCA ACA AGG
Polypeptide :→Leu Ile Val Ser Pro Thr Arg
Val Leu Leu Ala Thr Thr Gly ←
- Non-ambiguity While the same amino acid can be coded by more than one codon (the code is degenerate), the same codon shall not code for two or more different amino acids (non-ambiguous).
A newly synthesized RNA molecule is called a pri-mary transcript. The most extensive process-ing of primary transcripts occurs in eukaryotic mRNAs and in tRNAs of both bacteria and eukaryotes. The primary transcript for a eukaryotic mRNA typ-ically contains sequences encompassing one gene, al-though the sequences encoding the polypeptide may not be contiguous. Noncoding tracts that break up the coding region of the transcript are called introns, and the coding segments are called exons. In a process called splicing,the introns are removed from the pri-mary transcript and the exons are joined to form a con-tinuous sequence that specifies a functional polypep-tide. Eukaryotic mRNAs are also modified at each end. A modified residue called a 5cap (p. 1008) is added at the 5 end. The 3 end is cleaved, and 80 to 250 A residues are added to create a poly(A) “tail.” The some-times elaborate protein complexes that carry out each of these three mRNA-processing reactions do not oper-ate independently. They appear to be organized in as-sociation with each other and with the phosphorylated CTD of Pol II; each complex affects the function of the others.
The 5′ cap (red) is added before synthesis of the primary transcript is complete. A non coding sequence following the last exon is shown in orange. Splicing can occur either before or after the cleavage and polyadenylation steps. All the processes shown here take place within the nucleus.
Eukaryotic mRNAs Are Capped at the 5’End
Most eukaryotic mRNAs have a 5′ cap, a residue of 7-methylguanosine linked to the 5′-terminal residue of the mRNA through an unusual 5′, 5’-triphosphate linkage. The 5′ cap helps protect mRNA from ribonucleases. The cap also binds to a specific cap-binding complex of proteins and participates in binding of the mRNA to the ribosome to initiate translation
The 5cap is formed by condensation of a molecule of GTP with the triphosphate at the 5’end of the tran-script. The guanine is subsequently methylated at N-7, and additional methyl groups are often added at the 2′ hydroxyls of the first and second nucleotides adjacent to the cap The methyl groups are derived from S-adenosylmethionine. All these reactions occur very early in transcription, after the first 20 to 30 nu-cleotides of the transcript have been added.
All three of the capping enzymes, and through them the 5′ end of the transcript itself, are associated with the RNA poly-merase II CTD until the cap is synthesized. The capped 5’end is then released from the capping enzymes and bound by the cap-binding complex.
RNA Catalyzes the Splicing of Introns
There are four classes of introns. The first two, the group I and group II introns, differ in the details of their splicing mechanisms but share one surprising charac-teristic: they are self-splicing—no protein enzymes are involved. Group I introns are found in some nuclear, mi-tochondrial, and chloroplast genes coding for rRNAs, mRNAs, and tRNAs. Group II introns are generally found in the Primary transcripts of mitochondrial or chloro-plast mRNAs in fungi, algae, and plants. Group I and group II introns are also found among the rarer exam-ples of introns in bacteria. Neither class requires a high-energy cofactor (such as ATP) for splicing. The splicing mechanisms in both groups involve two transesterifica-tion reaction steps
The group I splicing reaction requires a guanine nucleoside or nucleotide cofactor, but the cofactor is not used as a source of energy; instead, the 3′-hydroxyl group of guanosine is used as a nucleophile in the first step of the splicing pathway. The guanosine 3′-hydroxyl group forms a normal 3′,5′-phosphodiester bond with the 5′ end of the intron. The 3′ hydroxyl of the exon that is displaced in this step then acts as a nucleophile in a similar reaction at the 3’end of the intron. The result is precise excision of the intron and ligation of the exons.
In group II introns the reaction pattern is similar ex-cept for the nucleophile in the first step, which in this case is the 2-hydroxyl group of an A residue within the intron (Fig. 26–15). A branched lariat structure is formed as an intermediate.
Most introns are notself-splicing, and these types are not designatede with a group number. The third and largest class of introns includes those found in nuclear mRNA primary transcripts. These are called spliceo-somal introns, because their removal occurs within and is catalyzed by a large protein complex called a spliceosome. Within the spliceosome, the introns undergo splicing by the same lariat-forming mechanism as the group II introns. The spliceosome is made up of spe-cialized RNA-protein complexes, small nuclear ribonucleoproteins (snRNPs, often pronounced “snurps”). Each snRNP contains one of a class of eukaryotic RNAs, 100 to 200 nucleotides long, known as small nuclear RNAs (snRNAs). Five snRNAs (U1, U2, U4, U5, and U6) involved in splicing reactions are generally found in abundance in eukaryotic nuclei. The RNAs and proteins in snRNPs are highly conserved in eukaryotes from yeasts to humans. Spliceosomal introns generally have the dinu-cleotide sequence GU and AG at the 5’and 3’ends, respectively and these sequences mark the sites where splicing occurs. The U1 snRNA contains a sequence complementary to sequences near the 5′ splice site of nuclear mRNA introns and the U1 snRNP binds to this region in the primary transcript. Addition of the U2, U4, U5, and U6 snRNPs leads to formation of the spliceosome. The snRNPs together contribute five RNAs and about 50 proteins to the spliceosome, a supramolecular assembly nearly as complex as the ribosome. ATP is required for assembly of the spliceosome, but the RNA cleavage-ligation reactions do not seem to require ATP. Some mRNA introns are spliced by a less common type of spliceosome, in which the U1 and U2 snRNPs are re-placed by the U11 and U12 snRNPs. Whereas U1- and U2-containing spliceosomes remove introns with (5′) GU and AG(3′) terminal sequences whereas the U11- and U12-containing spliceosomes remove a rare class of introns that have (5′)AU and AC(3′) terminal sequences to mark the intronic splice sites. The spliceosomes used in nuclear RNA splicing may have evolved from more ancient group II introns, with the snRNPs replacing the catalytic domains of their self-splicing ancestors.
The U1 snRNA has a sequence near its 5′ end that is complementary to the splice site at the 5′ end of the intron. Base pairing of U1 to this region of the primary transcript helps define the 5′ splice site during spliceosome assembly. U2 is paired to the intron at a position encompassing the A residue that becomes the nucleophile during the splicing reaction. Base pairing of U2 snRNA causes a bulge that displaces and helps to activate the adenylate, whose 2′ OH will form the lariat structure through a 2′,5′-posphodiester bond. The U1 and U2 snRNPs bind, then the remaining snRNPs (the U4/U6 complex and U5) bind to form an inactive spliceosome. Internal rearrangements convert this species to an active spliceosome in which U1 and U4 have been expelled and U6 is paired with both the 5′ splice site and U2. This is followed by the catalytic steps, which parallel those of the splicing of group II introns.
The fourth class of introns, found in certain tRNAs, is distinguished from the group I and II introns in that the splicing reaction requires ATP and an endonuclease. The splicing endonuclease cleaves the phosphodiester bonds at both ends of the intron, and the two exons are joined by a mechanism similar to the DNA ligase reaction.
Mechanism of the DNA ligase reaction. In each of the three steps, one phosphodiester bond is formed at the expense of another. Steps 1 and 2 lead to a ctivation of the phosphate in the nick. An AMP group is transferred first to a Lys residue on the enzyme and then to the phosphate in the nick. In step 3 , the 3-hydroxyl group attacks this phosphate and displaces AMP, producing a phosphodiester bond to seal the nick. In the E. coli DNA ligase reaction, AMP is derived from NAD. The DNA ligases isolated from a number of viral and eukaryotic sources use ATP rather than NAD and they release pyrophosphate rather than nicotinamide mononu-cleotide (NMN) in step
Eukaryotic mRNAs have a Distinctive 3’End Structure
At their 3′ end, most eukaryotic mRNAs have a string of 80 to 250 A residues, making up the poly(A) tail. This tail serves as a binding site for one or more specific proteins. The poly (A) tail and its associated pro-teins probably help protect mRNA from enzymatic de-struction. Many prokaryotic mRNAs also acquire poly (A) tails, but these tails stimulate decay of mRNA rather than protecting it from degradation.
Pol II synthesizes RNA beyond the segment of the transcript containing the cleavage signal sequences, including the highly conserved upstream sequence (5′) AAUAAA. The cleavage signal sequence is bound by an enzyme complex that includes an endonuclease, a polyadenylate polymerase, and several other multisub-unit proteins involved in sequence recognition, stimulation of cleav-age, and regulation of the length of the poly (A) tail. 2 The RNA is cleaved by the endonuclease at a point 10 to 30 nucleotides 3 to (downstream of) the sequence AAUAAA. 3 The polyadenylate poly-merase synthesizes a poly(A) tail 80 to 250 nucleotides long, begin-ning at the cleavage site.
Overview of the processing of a eukaryotic mRNA. The ovalbumin gene, shown here, has introns A to G and exons 1 to 7 and L (L encodes a signal peptide sequence that targets the protein for export from the cell. About three-quarters of the RNA is removed during processing. Pol II extends the primary tran-script well beyond the cleavage and polyadenylation site (“extra RNA”) before terminating transcription. Termination signals for Pol II have not yet been defined.
Processing of pre-rRNA transcripts in bacteria
Before cleavage, the 30S RNA precursor is methylated at specific bases. The cleavage liberates precursors of rRNAs and tRNA(s). Cleavage at the points labeled 1, 2, and 3 is carried out by the enzymes RNase III, RNase P, and RNase E, respectively. RNase P is a ribozyme. The final 16S, 23S, and 5S rRNA products result from the action of a variety of specific nucleases. The seven copies of the gene for pre-rRNA in the E. coli chromosome differ in the number, location, and identity of tRNAs included in the primary transcript. Some copies of the gene have additional tRNA gene segments between the 16S and 23S rRNA segments and at the far 3′ end of the primary transcript.
Processing of tRNAs in bacteria and eukaryotes
The yeast tRNATyr(the tRNA specific for tyrosine binding) is used to illustrate the important steps. The nucleotide sequences shown in yellow are removed from the primary transcript. The ends are processed first, the 5′ end before the 3′ end. CCA is then added to the 3′ end, a necessary step in processing eukaryotic tRNAs and those bacterial tRNAs that lack this sequence in the primary transcript. While the ends are being processed, specific bases in the rest of the transcript are modified. For the eukaryotic tRNA shown here, the final step is splicing of the 14-nucleotide intron. In-trons are found in some eukaryotic tRNAs but not in bacterial tRNAs
Introduction To Applied Microbiology
Course No.:- Micro 304
[Question paper Download link below ⇓ ]
Micro 304 Paper
Topic:- Food borne Microbial Diseases
Maximum Marks: 35
Part A ( Objective)
Q-1 Mark the correct answer from the following Multiple choice questions (MCQ) (1×5=5)
- A food borne illness is a disease transmitted to people who eat _______ food.
- High proteins
- Which of the following causative agents is the most common cause of food borne disease outbreaks in the United States?
- Which of the following food borne diseases is different from the others?
- Typhoid Fever
- Which of the following is a food infection?
- Staphylococcal intoxication
- None of these
- A bacterial food intoxication refers to
- illness caused by presence of pathogens
- food borne illness caused by the presence of a bacterial toxin formed in food
- both (a) and (b)
- none of the above
Q-2 Fill in the Blanks (1×5=5)
- Microorganisms capable of causing food borne illness include: virus, fungi, parasites and _________
- Write full form of FERG __________________________________________.
- The action of monitoring food to ensure that it will not cause food borne illness is known as_________________________.
- ____________ Viruses are Cause the most common food borne illness.
- The delay between the consumption of contaminated food and the appearance of the first symptoms of illness is called the________________.
Part –B (Subjective)
Q-3 Attempt the following Questions (2×5=10)
- What are food borne diseases?
- What are some examples of food borne diseases?
- What is the WHO Initiative to Estimate the Global Burden of Food borne Diseases?
- Who are the members of the Food borne Diseases Burden Epidemiology Reference Group (FERG)?
- What do anthrax and tapeworm infection have in common?
Q-4 Attempt the following Questions (5×3=15)
- How widespread are food borne diseases?
- What is the situation regarding food borne diseases in developed and developing countries?
- How do food borne diseases challenge public health security?
◊ Answer Key OF This Question Paper ⇓ [ Answer key Download link Below]
Answer Key To Micro 304 Paper
Part A ( Objective)
- iii) Contaminated
- i) Viruses
- i) Typhoid fever, shigellosis and cholera are all bacterial foodborne infections. Amoebiasis is a parasitic infection.
- i) Salmonellois
- ii) food borne illness caused by the presence of a bacterial toxin formed in food
Q-2 Fill in the Blanks
- Foodborne Diseases Burden Epidemiology Reference Group
- food safety
- incubation period
Part –B (Subjective)
Q-3 (2 marks Questions )
- Foodborne diseases encompass a wide spectrum of illnesses and are a significant cause of morbidity and mortality worldwide. They are illnesses associated with the ingestion of food contaminated by bacteria, viruses, parasites and chemicals as well as bio-toxins.
- Foodborne diseases can be caused:
- by micro-organisms (e.g. salmonella, campylobacter, enterohaemorrhagic E. Coli, listeria, cholera);
- by parasites (e.g. fasciola, echinococcus, taenia solium);
- by chemical agents and bio-toxins such as: – naturally-occurring toxins (e.g. mycotoxins, which are toxins in fungi), – persistant organic pollutants (i.e. pollutants that accumulate in the environment and human bodies) ; – metals (which accumulate in food e.g. lead, mercury, cadmium); and – unconventional agents (e.g. the agent that caused bovine spongiform encephalopathy – also known as “mad cow disease”).
- The Initiative to Estimate the Global Burden of Foodborne Diseases was launched by the Department of Food Safety, Zoonoses and Foodborne Diseases (FOS) of the World Health Organization (WHO) at an international consultation in September 2006. The participants in this consultation included representatives from Ministries of Health, governmental food safety and communicable diseases organizations, and academic institutions. All six WHO regions were represented at the meeting. The consultation participants recommended that WHO establish a Foodborne Diseases Burden Epidemiology Reference Group (FERG), and provided the strategic framework for a comprehensive assessment of global foodborne diseases burden arising from microbiological and chemical contaminants in food.
- The FERG members were appointed by the WHO following a public call for advisers in the scientific press, and include world experts in the areas of burden of disease, enteric pathogens, parasites, and chemicals and toxins.
- Raw meat consumption from sick and dying animals (like ox, cow, sheep, goat, camel) is responsible for transmitting anthrax, and raw beef and pork are the source of tapeworm infection
Q-4 ( 5 marks Questions )
- Foodborne diseases are a widespread and growing public health problem, both in developed and developing countries. In 2000 the World Health Assembly recognized that the prevention and control of foodborne diseases is an important public health issue (resolution WHA53.15). While most foodborne diseases are sporadic and often not reported, foodborne disease outbreaks may take on massive proportions. For example, the current Salmonella Saintpaul outbreak in the United States affected 43 out of 50 states in the country. The melaminecontaminated dairy products in China affected over 54,000 children. The WHO Global Burden of Disease: Update 2004 has estimated that 2.16 million children die every year from diarrhoeal diseases as a result of exposure to unsafe water, food, and poor sanitation and hygiene. However the proportion of these deaths that is attributable to eating unsafe food is not currently known. Additionally, diarrhoea is a major cause of malnutrition in infants and young children.
- In some industrialized countries, the percentage of the population suffering from foodborne diseases each year is estimated to be up to 30%. While less well documented, developing countries bear the brunt of the problem. People living in developing countries are more likely to be exposed to unhealthy environments through:
a) Poor access to clean water to adequately wash food items
b) Unsafe transportation and/or inadequate storage of foods,
c) Insufficient knowledge of safe food processing and handling practices,
d) Compromised immune responses to foodborne infections, particularly in populations where malnutrition and HIV/AIDS are prevalentThis is compounded by poor countries’ limited capacity to enforce effective food safety measures, including:
a) Efficient foodborne disease surveillance and monitoring systems,
b) Food safety regulations and functioning inspection systems,
c) Food safety education programs, and
d) Effective emergency planning and relief.
The high prevalence of diarrhoeal diseases in many developing countries, including those caused by parasites, points out the urgent need to prioritize foodborne disease prevention and control in national health and development plans.
The full picture of the impact and costs of foodborne diseases – in industrial just as in developing countries – is to date unknown. Many foodborne disease outbreaks go unrecognized, unreported or uninvestigated and may only be visible if connected to situations that have a major public health or economic impact. In order to fill this current data vacuum, the WHO Department of Food Safety, Zoonoses and Foodborne Diseases (FOS) together with its partners launched the Initiative to Estimate the Global Burden of Foodborne Diseases.
3. Many recent developments have accelerated the spread of foodborne diseases worldwide:
- In today’s interconnected and interdependent world, local foodborne disease outbreaks have become a potential threat to the entire globe.
- Both accidentally and deliberately contaminated food products can affect the health of people in many countries at the same time, as well as causing considerable economic losses from lost production and trade embargoes, and damage to a country’s tourist industry.
- Foodborne diseases are not only spreading faster, they also appear to be emerging more rapidly than ever before1 and are able to circumvent conventional control measures.
- The growing industrialization of food production catalyses the appearance and spread of new pathogens, as was the case for prions associated with bovine spongiform encephalopathy (BSE) which led to new variant Creutzfeldt-Jakob disease (vCJD) in humans in the United Kingdom during the 1990s. The increasing resistance of pathogens to antibiotics is also a major problem.
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Microbiology Sample Question Papers
Microbiology Definition:- Microbiology is the study of microscopic organisms, such as bacteria, viruses, archaea, fungi and protozoa. This discipline includes fundamental research on the biochemistry, physiology, cell biology, ecology, evolution and clinical aspects of microorganisms, including the host response to these agents. To Download Microbiology Sample question papers Please proceed reading, all links are given below.
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Definition:- Transcription is the first step of gene expression, in which a particular segment of DNA is copied into RNA (especially mRNA) by the enzyme RNA polymerase
RNA:- Expression of the information in a gene generally involves production of an RNA molecule transcribed from a DNA template. Strands of RNA and DNA may seem quite similar at first glance, differing only in that RNA has a hydroxyl group at the 2’position of the aldopentose and uracil instead of thymine. However, unlike DNA, most RNAs carry out their functions as single strands, strands that fold back on themselves and have the potential for much greater structural diversity than DNA. RNA is thus suited to a variety of cellular functions. RNA is the only macromolecule known to have a role both in the storage and transmission of information and in catalysis. The discovery of catalytic RNAs, or ribozymes, has changed the very definition of an enzyme, extending it beyond the domain of proteins. In the modern cell, all nucleic acids, including RNAs, are complexed with proteins. Some of these complexes are quite elaborate; and RNA can assume both structural and catalytic roles within complicated biochemical machines.
All RNA molecules except the RNA genomes of certain viruses are derived from information permanently stored in DNA. During transcription, an enzyme system converts the genetic information in a segment of double-stranded DNA into an RNA strand with a base sequence complementary to one of the DNA strands.
• Three major kinds of RNA are produced.
- Messenger RNAs (mRNAs) encode the amino acid sequence of one or more polypeptides specified by a gene or set of genes
- Transfer RNAs (tRNAs) read the information encoded in the mRNA and transfer the appropriate amino acid to a growing polypeptide chain during protein synthesis.
- Ribosomal RNAs (rRNAs) are constituents of ribosomes, the intricate cellular machines that synthesize proteins.
Many additional specialized RNAs have regulatory or catalytic functions or are precursors to the three main classes of RNA.
• DNA-Dependent Synthesis of RNA
Transcription resembles replication in its fundamental chemical mechanism, its polarity (direction of synthesis), and its use of a template. And like replication, transcription has initiation, elongation, and termination phases and initiation is further divided into discrete phases of DNA binding and initiation of RNA synthesis.
During replication the entire chromosome is usually copied, but transcription is more selective. Transcription differs from replication in that it does not require a primer and, generally, involves only limited segments of a DNA molecule. Additionally, within transcribed segments only one DNA strand serves as a template. Specific regulatory sequences mark the beginning and end of the DNA segments to be transcribed and designate which strand in duplex DNA is to be used as the template.
• RNA Is Synthesized by RNA Polymerases
By 1960, four research groups had independently detected an enzyme in cellular extracts that could form an RNA polymer from ribonucleoside 5-triphosphates. Subsequent work on the purified Escherichia coli RNA polymerase helped to define the fundamental properties of transcription. DNA-dependent RNA polymerase requires, in addition to a DNA template, all four ribonucleoside 5ʹ-triphosphates (ATP, GTP, UTP, and CTP) as precursors of the nucleotide units of RNA, as well as Mg2+. The protein also binds one Zn2+.
• Catalytic mechanism of RNA synthesis by RNA polymerase
RNA polymerase elongates an RNA strand by adding ribonucleotide units to the 3′-hydroxyl end, building RNA in the 5′–3′ direction. The 3′-hydroxyl group acts as a nucleophile, attacking the phosphate of the incoming ribonucleoside triphosphate and releasing pyrophosphate. The reaction involves two Mg2+ ions, coordinated to the phosphate groups of the incoming NTP and to three Asp residues which are highly conserved in the RNA polymerases of all species. One Mg2+ ion facilitates attack by the 3′-hydroxyl group on the α- phosphate of the NTP and the other Mg2+ ion facilitates displacement of the pyrophosphate and both metal ions stabilize the pentacovalent transition state.RNA polymerase requires DNA for activity and is most active when bound to a double-stranded DNA and only one of the two DNA strand serves as a template. The template DNA strand is copied in the 3′—5’direction (antiparallel to the new RNA strand), just as in DNA replication. Each nucleotide in the newly formed RNA is selected by Watson-Crick base-pairing interactions; U residues are inserted in the RNA to pair with A residues in the DNA template, G residues are inserted to pair with C residues, and so on. Base-pair geometry may also play a role in base selection.
• Transcription by RNA polymerase in E. coli.
RNA polymerase does not require a primer to initiate synthesis. Initiation occurs when RNA polymerase binds at specific DNA sequences called promoters. For synthesis of an RNA strand complementary to one of two DNA strands in a double helix, the DNA is transiently unwound over a short distance, forming a transcription “bubble.”About 17 bp are unwound at any given time. RNA polymerase and the bound transcription bubble move from left to right along the DNA thus facilitating RNA synthesis. The 8 bp RNA-DNA hybrid occurs in this unwound region. The DNA is unwound ahead and rewound behind as RNA is transcribed. As the DNA is rewound, the RNA-DNA hybrid is displaced and the RNA strand extruded. The RNA polymerase is in close contact with the DNA ahead of the transcription bubble, as well as with the separated DNA strands and the RNA within and immediately behind the bubble. A channel in the protein funnels new nucleoside triphosphates (NTPs) to the polymerase active site. The polymerase footprint encompasses about 35 bp of DNA during elongation.
The two complementary DNA strands have different roles in transcription. The strand that serves as template for RNA synthesis is called the template strand.
The DNA strand complementary to the template, the non template strand, or coding strand, is identical in base sequence to the RNA transcribed from the gene, with U in the RNA in place of T in the DNA. The coding strand for a particular gene may be located in either strand of a given chromosome. The regulatory sequences that control transcription are by convention designated by the sequences in the coding strand.
Template and nontemplate DNA strands
• RNA polymerase of E. coli
The RNA polymerase of E. coli is a large, complex enzyme with five core subunits (α2ββ’ω; Mr 390,000) and a sixth subunit, one of a group designated σ, with variants designated by size (molecular weight). The σ-subunit binds transiently to the core and directs the enzyme to specific binding sites on the DNA. These six subunits constitute the RNA polymerase holoenzyme.
The RNA polymerase holoenzyme of E. coli exists in several forms, depending on the type of σ subunit. The three σ subunits with molecular weight 70,000 (σ70), 32,000(σ32) and 60,000 (σ60) have been observed. While major holoenzyme transcribes majority of E.coli genes, σ32 holoenzyme has special function of transcribing the heat shock protein genes and σ60 holoenzyme controls the expression of glutamine synthetase gene and other nitrogen metabolism genes.
RNA polymerases lack a separate proofreading 3′—5′ exonuclease active site (like many DNA polymerases), and the error rate for transcription is higher than that for chromosomal DNA replication- approximately one error for every 104 to 105 ribonucleotides incorporated into RNA. Because many copies of RNA are generally produced from a single gene and all RNAs are eventually degraded and replaced, a mistake in an RNA molecule is of less harmful to the cell than a mistake in the permanent information stored in DNA. Many RNA polymerases, including bacterial RNA polymerase and the eukaryotic RNA polymerase II, do pause when a mispaired base is added during transcription, and they can remove mismatched nucleotides from the 3′ end of a transcript by direct reversal of the polymerase reaction.
• RNA Synthesis Begins at Promoters
RNA polymerase binds to specific sequences in the DNA called promoters, which direct the transcription of adjacent segments of DNA (genes). The sequences where RNA polymerases bind can be quite variable. In E. coli, RNA polymerase binding occurs within a region stretching from about 70 bp before the transcription start site to about 30 bp beyond it. The promoter region thus extends between positions -70 and +30. The nucleotides preceeding the +1 are given –ve numbers and are upstream and nucleotides succeeding +1 are given +ve numbers and are downstream.
Analyses and comparisons of the most common class of bacterial promoters (those recognized by an RNA polymerase holoenzyme containing σ70) have revealed similarities in two short sequences centered about positions -10 and -35. These sequences are important interaction sites for the σ70 subunit. Although the sequences are not identical for all bacterial promoters in this class, certain nucleotides that are particularly common at each position form a consensus sequence. The consensus sequence at the -10 region is (5′) TATAAT(3′); the consensus sequence at the -35 region is (5′)TTGACA(3′). A third AT-rich recognition element, called the UP (upstream promoter) element, occurs between positions -40 and -60 in the promoters of certain highly expressed genes. The UP element is bound by α subunit of RNA polymerase. The efficiency with which an RNA polymerase binds to a promoter and initiates transcription is determined in large measure by these sequences, the spacing between them, and their distance from the transcription start site. Variations in the consensus sequence also affect the efficiency of RNA polymerase binding and transcription initiation. A change in only one base pair can decrease the rate of binding by several orders of magnitude.
Typical E. coli promoters recognized by an RNA polymerase holoenzyme containing σ 70.
Sequences of the nontemplate strand are shown, read in the 5′—-3′ direction, as is the convention for representations of this kind. The sequences vary from one promoter to the next, but comparisons of many promoters reveal similarities, particularly in the -10 and -35 regions. The sequence element UP, not present in all E. coli promoters, is shown in the P1 promoter for the highly expressed rRNA gene rrnB. UP elements, generally occurring the region between -40 and -60, strongly stimulate transcription at the promoters that contain them. The UP element in the rrnB P1 promoter encompasses the region between -38 and -59.
The consensus sequence for E. coli promoters recognized by σ70 is shown second from the top. Spacer regions contain slightly variable numbers of nucleotides (N). Only the first nucleotide coding the RNA transcript (at position +1) is the transcription start site.
•Transcription initiation, elongation and terminationby E. coli RNA polymerase.
The pathway of transcription initiation is becoming much better defined. It consists of two major parts, binding and initiation, each with multiple steps. Binding :
First, the polymerase binds to the promoter, forming, in succession, a closed complex (in which the bound DNA is intact) and an open complex (in which the bound DNA is intact and partially unwound near the -10 sequence). A 12 to 15 bp region of DNA-from within the -10 region to position +2 or +3 -is then unwound to form an open complex.
The transcription is initiated within the complex and RNAP holoenzyme (core + one of multiple sigma factors) catalyzes the coupling of the first base (usually ATP or GTP) to a second ribonucleoside triphosphate to form a dinucleotide. This leads to a conformational change that converts the complex to the elongation form, followed by movement of the transcription complex away from the promoter called promoter clearance. Rifampicin inhibits transcription initiation.
The elongation of molecule from the 5′ to its 3′ end continues cyclically, antiparallel to its template. The enzyme polymerizes the ribonucleotides in a specific sequence dictated by the template strand and interpreted by Watson-Crick base pairing rules. Pyrophosphate is released in the polymerization reaction. This pyrophosphate (PPi) is rapidly degraded to 2 molecules of inorganic phosphate (Pi) by ubiquitous pyrophosphatases, thereby providing irreversibility on the overall synthetic reaction. In both prokaryotes and eukaryotes, a purine ribonucleotide is usually the first to be polymerized into the RNA molecule. As with eukaryotes, 5′ triphosphate of this first nucleotide is maintained in prokaryotic mRNA. As the elongation complex containing the core RNA polymerase progresses along the DNA molecule, DNA unwinding must occur in order to provide access for the appropriate base pairing to the nucleotides of the coding strand. The extent of this transcription bubble (i.e, DNA unwinding) is constant throughout transcription and has been estimated to be about 20 base pairs per polymerase molecule. Thus, it appears that the size of the unwound DNA region is dictated by the polymerase and is independent of the DNA sequence in the complex. This suggests that RNA polymerase has associated with it an “unwindase” activity that opens the DNA helix. Topoisomerase both precedes and follows the progressing RNAP to prevent the formation of superhelical complexes. The transcription occurs processively and rapidly. The in vivo rate of transcription is 20 to 70 nucleotides per second. Once an RNAP molecule has initiated transcription and moved away from the promoter, another RNAP can follow suit. The synthesis of RNA
that are needed in large quantities, ribosomal RNAs, for example, is initiated as often as is sterically possible, about once per second
• Rho independent termination:-
Around half the transcriptional termination sites in E. coli are intrinsic or spontaneous terminators, that is, they induce termination without assistance. The sequences of these terminators share two common features:
A tract of 7 to 10 consecutive A.T’s with the A’s on the template strand, sometimes interrupted by one or more different base pairs. The transcribed RNA is terminated in or just past this sequence.
- A G.C-rich segment with a palindromic (2-fold symmetric) sequence that is immediately upstream of the series of A. T’s. The RNA transcript of this region can therefore form a self-complementary “hairpin” structure that is terminated by several U residues. The stability of a terminator’s G.C-rich hairpin and the weak base pairing of its oligo (U) tail to template DNA are important factors in ensuring proper chain termination.
• Rho dependent termination
Termination of the synthesis of the RNA molecule in bacteria is signaled by a sequence in the template strand of the DNA molecule-a signal that is recognized by a termination protein, the rho (ρ) factor. Rho is an ATP-dependent RNA-stimulated helicase that disrupts the nascent RNA-DNA complex. After termination of synthesis of the RNA molecule, the enzyme separates from the DNA template and probably dissociates to free core enzyme and free σ factor. With the assistance of another σ factor, the core enzyme then recognizes a promoter at which the synthesis of a new RNA molecule commences.
The transcription cycle in bacteria. Bacterial RNA transcription is described in four steps:
(1) Template binding: RNA polymerase (RNAP) binds to DNA and locates a promoter (P), melts the two DNA strands to form a pre initiation complex (PIC)
(2) Chain initiation: RNAP holoenzyme (core + one of multiple sigma factors) catalyzes the coupling of the first base (usually ATP or GTP) to a second ribonucleoside triphosphate to form a dinucleotide.
(3) Chain elongation: Successive residues are added to the 3′-OH terminus of the nascent RNA molecule.
(4) Chain termination and release: The completed RNA chain and RNAP are released from the template. The RNAP holoenzyme re-forms finds a promoter, and the cycle is repeated.
• Eukaryotic Cells Have Three Kinds of Nuclear RNA Polymerases
The transcriptional machinery in the nucleus of a eukaryotic cell is much more complex than that in bacteria. Eukaryotes have three RNA polymerases, designated I, II, and III, which are distinct complexes but have certain subunits in common. Each polymerase has a specific function and is recruited to a specific promoter sequence.
RNA polymerase I (Pol I) is responsible for the synthesis of only one type of RNA, a transcript called pre ribosomal RNA (or pre-rRNA), which contains the precursor for the 18S, 5.8S, and 28S rRNAs. Pol I promoters vary greatly in sequence from one species to another.
RNA polymerase II (Pol II) is involved in synthesis of mRNAs and some specialized RNAs. This enzyme can recognize thousands of promoters that vary greatly in sequence. Many Pol II promoters have a few sequence features in common, including a TATA box (eukaryotic consensus sequence TATAAA) near base pair -30 and an Inr sequence (initiator) near the RNA start site at +1.
RNA polymerase III (Pol III) makes tRNAs, the 5S rRNA, and some other small specialized RNAs. The promoters recognized by Pol III are well characterized. Interestingly, some of the sequences required for the regulated initiation of transcription by Pol III are located within the gene itself, whereas others are in more conventional locations upstream of the RNA start site
• RNA Polymerase II Requires Many Other Protein Factors for Its Activity
RNA polymerase II is central to eukaryotic gene expression. Pol II is a huge enzyme with 12 subunits. The largest subunit (RBP1) exhibits a high degree of homology to the β’ subunit of bacterial RNA polymerase. Another subunit (RBP2) is structurally similar to the bacterial β subunit, and two others (RBP3 and RBP11) show some structural homology to the two bacterial α subunits. Pol II must function with genomes that are more complex and with DNA molecules more elaborately packaged than in bacteria.
RNA polymerase II requires an array of other proteins, called transcription factors, in order to form the active transcription complex. The general transcription factors required at every Pol II promoter (factors usually designated TFII with an additional identifier) are highly conserved in all eukaryotes. The process of transcription by Pol II can be described in terms of several phases—assembly, initiation, elongation, termination—each associated with characteristic proteins.
Assembly of RNA polymerase and Transcription Factors at a Promoter
The formation of a closed complex begins when the TATA-binding protein (TBP) binds to the TATA box. TBP is bound in turn by the transcription factor TFIIB, which also binds to DNA on either side of TBP. TFIIA binding, although not always essential, can stabilize the TFIIB-TBP complex on the DNA and can be important at non consensus promoters where TBP binding is relatively weak. The TFIIB-TBP complex is next bound by another complex consisting of TFIIF and Pol II. TFIIF helps target Pol II to its promoters, both by interacting with TFIIB and by reducing the binding of the polymerase to nonspecific sites on the DNA. Finally, TFIIE and TFIIH bind to create the closed complex. TFIIH has DNA helicase activity that promotes the unwinding of DNA near the RNA start site (a process requiring the hydrolysis of ATP), thereby creating an open complex. Counting all the subunits of the various essential factors (excluding TFIIA), this minimal active assembly has more than 30 polypeptides.
• RNA Strand Initiation and Promoter Clearance
TFIIH has an additional function during the initiation phase. A kinase activity in one of its subunits phosphorylates Pol II at many places in the CTD. Several other protein kinases, including CDK9 (cyclin-dependent kinase 9), which is part of the complex pTEFb (positive transcription elongation factor b), also phosphorylate the CTD. This causes a conformational change in the overall complex, initiating transcription. Phosphorylation of the CTD is also important during the subsequent elongation phase, and it affects the interactions between the transcription complex and other enzymes involved in processing the transcript. During synthesis of the initial 60 to 70 nucleotides of RNA, first TFIIE and then TFIIH is released, and Pol II enters the elongation phase of transcription.
• Elongation, Termination, and Release
TFIIF remains associated with Pol II throughout elongation. During this stage, the activity of the polymerase is greatly enhanced by proteins called elongation factors. The elongation factors suppress pausing during transcription and also coordinate interactions between proteins complexes involved in the posttranscriptional processing of mRNAs. Once the RNA transcript is completed, transcription is terminated. Pol II is dephosphorylated and recycled, ready to initiate another transcript.