MOLECULAR BIOLOGY. Translation. Kolluru. V. A. Ramaiah Professor Department of Biochemistry University of Hyderabad. (Revised 30-Oct-2007)

Transcription

1 MOLECULAR BIOLOGY Translation Kolluru. V. A. Ramaiah Professor Department of Biochemistry University of Hyderabad (Revised 30-Oct-2007) CONTENTS Introduction Messenger RNA (mrna) Splicing Addition of 5 Cap Addition of poly A tail RNA editing Ribosomes Subunits, composition, morphology Processing of rrna Functions of ribosomal subunits Polysomes Transfer RNA (trna) Processing Modified bases Charging or aminoacylation Genetic Code Cell-free translational systems Synthetic mrna templates Amino acid analyses of polypeptides produced by synthetic templates Codon- charged trna- ribosome complexes Wobble and degeneracy Mutations Gene density and overlapping genes Protein synthesis Initiation Elongation Termination Ribosome recycling Differences in the initiation in eukaryotes and prokaryotes Inhibitors Translational regulation Different RNAs and functions Co- and Post-translational Modifications of proteins

2 Keywords Messenger RNA (mrna); Ribosomes; Polysomes; Transfer RNA (trna); Aminoacylation; Genetic code; Translational system; Wobble; Mutations; Gene density; Protein synthesis; Translational regulation. 2

3 Introduction Proteins are biological polymers like the nucleic acids. The alphabets of protein language are however different from that of nucleic acids. The monomeric units of proteins are the alphaamino acids. The twenty naturally occurring amino acids are like the alphabets of a language that go into the composition of different proteins. A typical α-amino acid consists of an amino group (-NH2), carboxyl group (-COOH) and an R group. It is the R group that gives specificity to an amino acid. The carboxyl group is attached to the α-carbon, the carbon next to the carboxyl group (Fig. 1). Amino acids in a protein chain are linked by a peptide bond ( ). Usually, proteins have an N-terminal end carrying a free amino group and C-terminus with COOH. The linear sequence of amino acids in each protein is specific like the alphabets in a word. Proteins are vital for life and mediate a variety of functions: in storage, structure, catalysis, signaling, transport, defense, transporting ions, blood clotting, and muscle contraction (Fig.2). Proteins like prions even mediate transmissible diseases called spongiform encephalopathies and the structure of a protein determines its function. Hence the study of biological synthesis of proteins and their modifications to attain a proper 3-dimensional structure are important. Approximately 35-45% genes make products devoted for translation, and 35-40% of the total energy generated, for example by E. coli, is consumed for protein synthesis. Hence the synthetic process and also the modification process require close monitoring and regulation. The information for the synthesis of a protein is stored in the nucleotide sequence of messenger RNA (mrna) that in turn is synthesized from the corresponding DNA. The process of RNA synthesis from a DNA template is called transcription and the synthesis of proteins from an mrna template is called translation. The information flow also occurs from an RNA template to a DNA molecule through a process called reverse transcription where DNA is synthesized from an RNA transcript in the presence of an enzyme called reverse transcriptase (Fig. 3). In prokaryotes that lack defined organelles or nucleus, the process of transcription and translation are coupled whereas in eukaryotes, the RNA synthesis occurs in the nucleus and the protein synthesis takes place in the cytosol, organelles, or on the surface of endoplasmic reticulum (ER). Organelle protein synthesis resembles prokaryotic protein synthesis. Proteins made on the surface of ER membrane are called secretory proteins (eg: serum albumin, immunoglobulins, digestive enzymes, egg white proteins etc.,) that are destined to reach various locations/organelles in the cell. Secretory/membrane proteins carry an N-terminal signal sequence that is rich in hydrophobic amino acids. Such a signal sequence is absent in cytosolic proteins. The rate of translation of eukaryotic mrnas is slower than in prokaryotes because of 3

4 the spatial and temporal separation of transcription and translational events, processing of the premessenger RNAs (mrnas) that remove the non-coding regions, and their ability to undergo distinct posttranscriptional modifications at the 5 and 3 ends. Several complex regulatory mechanisms control the gene expression in eukaryotes and they occur at different levels: transcription, post transcription, transport of RNAs into the cytosol, translation, and the degradation or stability of mrna. Like the synthesis of DNA (replication) and RNA (transcription), specific cellular machinery directs protein synthesis (translation). While differences exist, one can also see some common pattern directing the biological synthesis of these polymers viz., proteins, RNA and DNA. The syntheses of all these molecules require a template strand that contains the necessary information with start and stop signals. The addition of monomers occurs on the template. These monomers come one after another based on the sequence information coded in the template and attach to the template molecule. A covalent bond like phosphodiester bond joins the adjacent nucleotides in DNA and RNA synthesis, whereas a peptide bond brings together adjacent amino acids in protein synthesis. The primary products, or the newly made RNA and protein molecules are processed where certain nucleotides or amino acids that are non-coding or non-essential for function or structure are selectively removed to yield active molecules. Processing of RNAs and pre-proproteins yield biologically active RNA and protein molecules that are devoid of introns as in RNA, or prepro-amino acid sequences or inteins as in proteins. In addition, eukaryotic RNAs and the amino acids in proteins are also modified in several other ways during or after their synthesis and these are called post-transcriptional or post-translational modifications respectively that play important role in their functions (see latter). The expression of genes i.e., the synthesis of RNA and proteins is regulated or controlled by a wide variety of ways. While the DNA of a genome is replicated completely preceding the cell division but only portions of DNA are transcribed and or translated at different periods in the development or in different cell types. The genetic content between men and mice is 97.5% similar and the 2.5% difference separates mice from men. Among organisms, like human beings, the difference in the genetic content is 0.1% and that small difference distinguishes one individual from the other for their appearance, behavior and predisposition to certain diseases. Although different cell types like muscle cells, red blood cells and cells of pancreas differ in their functions, their genetic content is 100% similar because they are all derived from the same parent cell. Similarly a normal cell differs from an abnormal, or aged, stressed, diseased or virusinfected cell. An unfertilized egg differs from a fertilized egg. A dormant seed is different from germinating seed. This is because the gene expression or the types of RNAs and proteins made by these cells are different. Hence the gene expression critically regulates the development, differentiation of cell types and plays a role in health and disease. 4

5 In brief, the protein synthesis machinery consists of different types of RNAs (Box-1), ribosomes, enzymes such as aminoacyl synthetases that catalyze the transfer of amino acids to corresponding trnas, and a variety of protein factors that are required during the substeps in initiation, elongation, termination of translation and in ribosome recycling. The process of protein synthesis consumes a lot of ATP and GTP. While ATP hydrolysis is used in driving the protein / peptide synthesis and in unwinding any secondary structures in mrna, GTP hydrolysis facilitates conformational changes in ribosomes that are required in the joining of small subunit to the large ribosomal subunit, the translocation/movement of trna from one codon to another and also in the detachment of the factors from ribosomes. Ribosomes, and other components of protein synthesis that are involved in the decoding of information in mrna are relatively stable compared to the template mrna. In addition to the three RNAs mentioned in Box-1, several other RNAs with a variety of functions play a role in the synthesis of DNA, RNA and proteins and also in controlling gene expression (see latter). Messsenger RNA (mrna) One of the DNA strands serves as a template for the synthesis of messenger RNA (mrna). Messenger RNAs are protein-coding RNAs containing typically three regions: a 5 UTR (untranslated region), a protein coding sequence (open reading frame or ORF) and 3 UTR (Fig. 4B). The protein coding sequence of mrna has a start site at the 5 side (ribosome binding site or RBS and is about 7-10 nucleotides in length) and stop site at the 3 end. Eukaryotic mrnas are mostly monocistronic coding for a single polypeptide whereas prokaryotic mrnas are polycistronic with multiple starts and stop signals and coding for more than one protein. In prokaryotes that lack a defined nucleus, the process of RNA and protein syntheses are coupled. This means, as and when the mrna is being transcribed from a DNA template, the RNA transcript is translated by ribosomes to the corresponding protein. Further, prokaryotic mrnas lack additional features such as a cap structure at the 5 end and poly A tail at the 3 end which are found in most eukaryotic mrnas. In eukaryotes, the transcription or the synthesis of mrnas occurs in the nucleus. The pre-mrnas are called heterogenous nuclear RNA (hnrna). The hnmrna or pre-mrna molecule is bound by a spliceosome that contains several proteins and uracil-rich small nuclear RNA molecules. The primary transcripts of eukaryotes are 5

6 unusually long and contain coding and non-coding sequences called exons and introns respectively. Because of this reason, the eukaryotic genes are called split genes. This can be demonstrated by imperfect hybridization of the DNA template with its mature or processed mrna transcript. Part of the regions in DNA (corresponding to introns) cannot be base paired with its mature mrna and form single stranded loops. This can be detected using an electron micrscope or by using S1 nuclease that attacks preferentially the single stranded unpaired region in DNA. The fragments of DNA generated by S1 nuclease digestion can be separated on an acrylamide/ agarose gel electrophoresis. Thus the mature RNAs do not have certain sequences that are complimentary to their template DNA molecules and these are called introns or noncoding sequences. Splicing Splicing or joining of the exons (Fig. 4A) and removal of the introns involve two successive transestrification reactions in which the phosphodiester linkages within the mrna are broken and reformed as shown below (see figure). Introns are incidentally ancient and are found in bacterial trnas, but not in their mrnas. Archaebacteria too have introns both in their trnas and in the ribosomal RNAs. Eukaryotes have introns in many of their pre-rnas including mrnas. This suggests that probably they are lost in bacterial mrnas due to evolutionary constraints. An analysis of eukaryotic DNA sequences at the boundaries of exons and introns revealed that introns mostly have GU sequence at the beginning (on their 5 side) and AG sequence at the end (on the 3 side). In addition to these sequences, splicing of introns requires a pyrimidine rich sequence called the polypyrimidine tract (U/C) 11 preceding the AG nucleotides at the 3 side of intron and a branch point sequence consisting of 5 - YNYYRAY (Y represents any pyrimidine, N refers to any nucleotide and R means a purine A or G, and A is an adenine) that is present residues upstream (that is towards the 5 end of the intron) to the above polypyrimidine tract. Fig. 4 A and B Based on the mechanism of splicing, three groups of introns are identified. Removal of group I introns, for example present in rrnas, can proceed in the absence of any proteins. However, the splicing reaction of group I introns require the presence of guanosine and Mg 2+. The concept of 6

7 RNA enzymes has come from such splicing reactions. The RNA enzymes like the protein enzymes increase the rate of reaction by several fold and require typical 3-D structure of their substrates. However unlike most protein enzymes, RNA enzymes act on themselves (selfsplicing) and are modified at the end of reaction. Essentially RNA enzymes have nuclease and polymerase activities. They can remove nucleotides and can also add nucleotides through a transesterification reaction. The removal of group II and III introns (premrnas) are similar to each other except that the removal of group III introns requires the participation of a spliceosome, a complex of several RNAs and proteins. Unlike group I introns, the splicing of group II and III introns does not require guanosine. In both cases, the OH-group of adenine nucleotide that is part of the intron branch point sequence attacks the 3 end of the exon present at the 5 end of the mrna. Afterwards, the free 5 end of intron joins the adenine nucleotide in intron to give a lariat structure, whereas the -OH group that is generated at the 3 end of exon will attack the 3 end of intron thereby releasing the intron. At the same time, two exons present at the 5 and 3 ends of the intron are joined with each other (Fig. 4C). Alternative Splicing Alternative Splicing produces multiple mrnas that code for different proteins where as normal splicing includes all exons and elimination of introns. The processing of pre-mrnas is not always uniform. Some times the processing facilitates the joining of different combinations of exons of an mrna. Also, the cleavage and polyadenylation of pre-mrnas (Fig. 6) at different sites in the 3 end would produce different lengths of mrnas. For example, splicing and 3 cleavage sites of the calcitonin mrna that produces calcitonin hormone in the cells of thyroid gland and brain differ to produce different proteins. The mature transcript of the calcitonin mrna is found long in brain cells than in thyroid cells. This is because the thyroid calcitonin mrna contains four of the six exons (1-4) without any introns. However in brain cells, the cleavage and polyadenylation of pre-mrna occurs at the end of sixth exon. The processed or mature mrna in brain cells has exons 1, 2, 3, 5 and 6 and does not have any of the introns. Exon 4 is not included. Alternative splicing may explain the diversity of proteins produced in eukaryotes that apparently do not correspond to their estimated number of functional genes. In humans, for example, approximately 30,000-35,000 functional genes code for millions of proteins. The processing of heavy chain pre-mrnas of IgM class immunoglobulins of B cells and plasma cells, and the α-tropomysin gene in different muscle cells are some of the other examples of alternative splicing. This may be possible because of the presence /absence of specific cellular splicing factors. The plasma cell that secretes immunoglobulins into the blood produces a mature IgM transcript that retains one of the exons which produces a stretch of hydrophilic amino acids and is consistent with the ability of plasma cell to secrete the protein. In contrast, the B-cell produces a mature mrna transcript that encodes a stretch of hydrophobic amino acids that facilitates the protein to be anchored in the plasma membrane. Analysis of some proteins like LDL (low density lipoprotein) receptor indicates that the various domains of this protein come from different exons probably by a process called exon shuffling. These domains in LDL protein are strikingly similar to other proteins like epidermal growth factor receptor, blood clotting factors and C9 complement factor. 7

8 Fig. 4C: Processing of different introns Many of the examples cited above suggest that introns of pre-mrnas contain non-coding regions and are eliminated during splicing. The joining of different combinations of exons in a pre-mrna transcript produces proteins of different kinds. However this is not true always. DNA sequences that become part of intron sequences of some pre-mrnas are also shown to code for proteins suggesting that genes are embedded in genes. Examples include the pupal cuticle gene of Drosophila is an intron of another gene that codes an enzyme important in the synthesis of purines (adenine and guanine). While the sequence is excluded in the mature RNA transcript of the purine synthesizing enzyme, it however produces pupal cuticle mrna of 0.9 kilobase when it is transcribed separately from the DNA sequence. Introns are longer than exons generally, and may a play a role in the transport of processed mrnas from nucleus to cytoplasm. Mutations in an intron can disrupt the correct splicing and can enhance the transforming activity of certain oncogenes (ex: ras). Introns mark the functional protein regions while exons encode well-defined structural domains in proteins. Addition of 5 Cap Most of the eukaryotic mrnas contain a cap structure consisting of 7-methyl guanine, an additional nucleotide at the 5 end. The guanine nucleotide is added soon after the initiation of 8

9 transcription (even before the completion of the transcription of the mrna) and is a cotranscriptional event as illustrated (Fig. 5). The guanine nucleotide joins the 5 -end of the mrna by a 5-5 bond instead of a 5-3 phosphodiester bond that joins all the other nucleotides. Afterwards, a methyl transferase enzyme adds a methyl group at position 7 of the newly added guanine nucleotide. Also the 2 OH group of sugars joined to the second and third nucleotides of mrnas is methylated. The 5 cap may offer stability to eukaryotic mrnas and regulates translation by providing a binding site for several factors. Addition of Poly A tail In addition to the removal of introns, the pre-mrnas in eukaryotes are processed, at a position from nucleotides downstream of an AAUAAA consensus sequence, in their 3 untranslated regions. The cleavage is aided by polyadenylation specificity factor and cleavage stimulation factor. These factors bind to the RNA polymerase during transcription when the polymerase reaches the end of the transcribing gene. After the cleavage, a number of adenine nucleotides are added by the enzyme polya polymerase in the presence of ATP to the 3 end of a cleaved mrna (Fig. 6). The presence of poly A tail may confer stability to mrna and also helps in the circularisation of mrna during translation due to an interaction between proteins bound to the 5 and 3 ends of mrna. The pseudo-circularisation process may facilitate an efficient reinitiation of protein synthesis in eukaryotes (see latter). RNA editing Fig. 5: Steps in the addition of 5 Cap in eukaryotic mrnas This is yet another way of modifying the transcribed RNA and is different from splicing. Here nucleotides are added, deleted, or both events can happen. It can occur by chemical modifications as in the conversion of cytosine to uracil by a process called deamination or by a guide RNA. In the editing process, the RNA transcript is modified but not the DNA. For example, apolipoprotein mrna in some cells produces a full protein with a higher molecular mass. However, the same mrna has a stop codon in some cells and produces a truncated protein 9

10 with a lower molecular mass, approximately half of full form of the protein. In the latter, one of the mrna codons, CAA, that codes for the amino acid glutamine is modified to UAA, a stop codon and produces a truncated form of apolipoprotein. The C to A conversion occurs by cytidine deaminase enzyme. This modification occurs only in certain cell types. For example the editing of apolipo mrna occurs in intestinal cells and does not happen in liver cells. In some mrnas, for example, the mrna that encodes Cox II protein in trypanosomes, four Us are inserted in a specific region that alters the reading frame of the message and the type of protein produced by the mrna. The editing in this case occurs by a special RNA called guide RNA that consists of nucleotides and with the help of other proteins like an endonuclease and a ligase. Ribosomes Subunits, Composition and Morphology Fig. 6: Addition of Poly A tail to a eukaryotic mrna Free ribosomes are found in the cytoplasm and are involved in the synthesis of cytosolic proteins, whereas the membrane bound ribosomes are associated with the endoplasmic reticulum and are involved in the synthesis of secretory proteins that are destined to reach various organelles. In addition, ribosomes are found in the organelles like mitochondria and choloroplasts. Organelle ribosomes resemble prokaryotic ribosomes. The subunits differ in their size, length, width, composition, and in molecular mass. Protein synthesis occurs on the surface of ribsomes. Ribosomes are RNA-protein complexes consisting of two subunits: a small (30S in prokaryotes and 40S in eukaryotes) and large subunit (50S in prokaryotes or 60S n eukaryotes). Together these subunits form monosomes (70S or 80S in prokaryotes and eukaryotes respectively). When subjected to centrifugal force, the sedimentation value or Svedberg coefficient (S) of the monosomes and their subunits are different. S Values are dependent not only on mass but also the by shape and density. Although S values increase with molecular mass of the ribosomal subunits, the S values of a monosome are not exactly add to its constituent subunits. The S value of prokaryotic monosome is 70S and its constituent subunits are 30S and 50S. The small prokaryotic 30S subunit is divided into head, body and side bulge or platform (Fig. 8). A distinct groove separates the head from the body. This subunit is composed of 10

11 approximately 21 proteins (S1-S21, S refers to small subunit and they are named serially based on their migration in gel electrophoresis), and 16S ribosomal RNA that play a critical role in the recognition of start codon in prokaryotic mrnas. The large 50S subunit is 160 kda approximately and it consists of 34 proteins (L1-L34) and two ribosomal RNAs: 23S (2900 bases) and 5S (120 bases) (Fig. 7). The large subunit is more isometric than the small subunit with a linear size being equal to 200 to 230A 0 in all directions. At the periphery, it has three protuberances; the lateral is called the L7/L12 stalk, a central protuberance and a side lobe or L1 ridge (Fig. 8). Electron microscopic observations indicate that the appearance of eukaryotic and prokaryotic ribosomal subunits is identical except that the 40S subunit has a protuberance located on the head and appears to be bifurcated at the end of the body distal to the head. Ribosome s are not only involved in the actual peptide bond formation but a) provide the necessary platform for the correct positioning of the tuna molecules carrying amino acids or decollated tunas, b) facilitate the movement of trna on the ribosome and c) guide the accuracy of the movement. Processing of rrna Further, the size of the precursor of rrna molecule is very long and is processed. The ribosomal DNA of E. coli is transcribed as a 30S RNA precursor that is processed to yield 23S, 16S and 5S RNA species. The 23S and 5S transcripts go into the composition of the 50S subunit, whereas the 16S transcript is with the 30S small subunit (Fig. 7). The size of the precursor RNA transcript in mammalian cells is 45S and is cleaved to give rise 28S, 18S and 5.8S RNA molecules. The 28S and 5.8S rrna eventually become a part of the 60S subunit, and the 18S rrna transcript becomes part of the 40S subunit. The small ribosomal subunit of eukaryotes also contains 5S rrna but it is transcribed as a separate entity from a ribosomal RNA gene. The 5S rrna is present both in pro- and eukaryotic ribosomes and is conserved through evolution. Figs. 7 and 8: Composition and morphology of eukaryotic and prokaryotic ribosomes 11

12 Functions of ribosomal subunits The small subunit plays a role in the initiation of protein synthesis process, and regulates the fidelity of interaction between the three bases in the mrna codon- trna anticodon. In contrast, the large subunit is the center for the formation of peptide bonds between adjacent amino acids and provides the pathway for the release of nascent or emerging proteins. Ribosomes consist of three sites called aminoacyl (A), peptidyl (P) and the exit (E)- sites that interact with the amino acylated trnas, peptidyl trna (trna carrying growing polypeptide), or with deacylated trnas respectively. The distance between these sites is 20A 0-50A 0. Further, crystallographic analyses of prokaryotic 30S ribosomal subunit reveal that 16S rrna of 30S subunit modulates the movement of mrna-trna, and the ribosome dynamically changes its shape during different functional states. These facts may explain why the structural features in trnas (see latter) are conserved during evolution. Channels in the small subunit of ribosomes facilitate the entry and exit of mrnas and a channel in the large subunit facilitates the exit of nascent or newly made polypeptide. Thus ribosome is not a static component. It is a dynamic molecular machine with moving parts and a very complicated mechanism of action. Ribosomal RNA, present at the interface between ribosomal subunits, not only plays a role in the structure but also plays a role in two of the most important functions: a) decoding of mrna and b) addition of peptide bond between adjacent amino acids. Various studies suggest that the 16S rrna of the small ribosomal subunit scrutinizes the correctness of the codon-anticodon interaction and is involved in decoding the mrna sequence. The interaction between the first two base pairs of codon-anticodon is checked more rigorously by the nucleotides in 16S rrna than the interaction between the third base pair. This fact relates to the wobble hypothesis (see genetic code). 16S rrna plays an important role in identifying the start codon in mrna in prokaryotes (see latter). In contrast, the peptidyl transferase center in the 50S subunit that catalyzes peptide bond formation between amino acids contains 23S rrna. To perform these functions (decoding and peptide bond formation), the crystal structure data of ribosomes suggest that rrna is located in the center or at the interface between large and small subunits of the ribosome where as proteins are located mostly peripherally. Peptide bond formation does not consume any additional energy or require ATP hydrolysis. However it uses the energy stored in the acyl bond that was created during the charging of trna (see latter). Some of the ribosomal proteins also shield the negatively charged RNA molecules. Polysomes A given messenger RNA may be translated by one or more than one ribosome. Accordingly the number of copies of proteins produced will vary. A messenger RNA that is translated efficiently by several ribosomes can give rise to a polyribosome complex. The total ribosomes in a cell are distributed as small and large subunits (30s and 50S or 40S and 60S), monosomes (70S or 80S i.e., single ribosome bound to mrna), and polysomes (an mrna bound by two to several ribosomes). Isolation and analyses of the relative proportion of small subunits, large subunits, monosomes and polyribosomes of total ribosomes reveal to some extent the protein synthetic activity of the cell. Ribosomes isolated from a cell carrying active protein synthesis will have very few subunits and monosomes but will have more polysomes. This is due to an increased rate in the initiation of protein synthesis. Sometimes cells that are defective in protein synthesis particularly at the level of elongation may also show up an increased proportion of polysomes relative to their monosomes. Here the ribosomes pile up on mrna and are released at a slow 12

13 rate. Under such conditions, the polysome analyses can be correlated with protein synthesis in vivo. One can estimate protein synthesis in cultured cells (control and treated cells) by monitoring the incorporation of labeled amino acid like [S- 35 ]- methionine or [C 14 ] leucine into the acid precipitable protein based on the total uptake of the radioactive amino acid. In contrast, a defect in the initiation step of protein synthesis in cells leads to the formation of a high proportion of monosomes relative to polysomes. Transfer RNA (trna) Transfer RNAs are small RNAs compared to mrnas and ribosomal RNAs. The trnas act like adaptor molecules recognizing an amino acid on one side and the corresponding sequence information in the mrna sequence on the other side. Cells thus contain a minimum of twenty trnas: one specifying each amino acid. However analyses of trnas and the genetic code (see latter) have shown that cells generally contain more than twenty trnas and sometimes more than one trna specifying an amino acid. Transfer RNAs contain nucleotides. However the precursor or the primary transcripts of trnas consist of around 125 nucleotides that are processed to yield mature trnas. In both prokaryotes and eukryotes, the primary transcripts are longer and contains introns that are processed out to give rise a functional trna. The various regions of a typical mature trna are shown in Fig. 9. Processing Fig. 9: Transfer RNA with 5 Phosphate and 3 -OH group It includes cleavage of the primary transcripts, splicing (joining of exons and removal of introns) and addition of certain bases and modifications of certain bases. The processing is accomplished by enzymes like RNAses P, D and E. As and when the primary transcript is made, it folds itself into the stem-loop like structure. This folding is required and plays a critical role for the nucleases to act upon the precursor molecule to remove certain nucleotides in the 5 and 3 ends of trnas. The processing occurs in an orderly manner and is somewhat different in prokaryotes and eukaryotes. The pre-trna molecule in eukaryotes contains a 5 leader sequence of about nucleotides, an intron of nucleotides and two additional nucleotides at the 3 end. Removal of these nucleotides yields mature trna. The two most important features between the 13

14 primary and processed transcripts between prokaryotic and eukaryotic trnas are a) the absence of intron sequence near the anticodon, and b) the presence of CCA at the 3 end of pre-trna in prokaryotes. In eukaryotes, the CCA nucleotides to which the cognate amino acid is attached are added at the 3 end of mature trna by the action of nucleotidyl transferase enzyme. Modified bases Both pro- and eukaryotic trnas contain unusually rare and modified bases such as ribothymidine, dihydrouridine, pseudouridine, 4-thiouridine, I-inosine, 1-methyl guanosine and N6- isopentenyl adenosine (Fig. 10). The modified bases arise due to the activity of special trna-modifying enzymes. Thymidine is generally present in DNA but it is also found in trna. Uracil is modified to ribothymidine or pesudouridine by the addition of a methyl group or an amino group respectively. Modifications of trna are required to affect the speed and accuracy of protein synthesis. The modifications also play a critical role in maintaining proper reading frame and for the movement of trnas from A-site to P-site (see latter). For example lysyl trna (trnalys with an anticodon UUU) recognizes AAA codon in mrna and undergoes a modification of N6-threonylcarbamoyladenosine at position 37 adjacent and 3 to the anticodon to bind AAA in the A-site of ribosomal 30S subunit. Figs 10: Modified nucleotides in trnas Fig. 11: 3-D structure of trna The structures, but not the exact composition of the nucleotide sequences of trnas, are similar. The secondary structure or the two-dimensional structure of trnas resembles to clover leaf (Fig. 9). Many of the nucleotides are complimentary to each other and form intermolecular hydrogen bonds. As a result the trnas assume a typical structure which is critical for their function. The tertiary or 3-D structure of trna resembles to L-shaped structure and is a result of nine hydrogen bonds involving base pairing between several invariant residues (Fig. 11). The interactions among bases in the T- and D- arms of trna facilitate the folding of the molecule into an L-shaped structure with the anticodon at one end, and the amino acid acceptor at the other end. 14

15 Charging of trnas or Aminoacylation The joining of an appropriate amino acid to the CCA containing 3 end of amino acid acceptor stem of the trna represents aminoacylation or charging of trnas. The reaction is catalyzed by an aminoacyl synthetase and requires 2 high-energy bonds of ATP. The amino acylation of trna is very specific and occurs in two steps. In the first step, the -COOH or carboxyl group of an amino acid reacts with ATP producing aminoacyl AMP (adenylated amino acid, see Fig. 12) and inorganic pyrophosphate (PPi). In step 2, the activated adenylated amino acid is transferred to the 2 or 3 OH group of a sugar linked to an adenine base present at the 3 end of an appropriate trna. In the final reaction, the carboxyl group of the amino acid is linked to the 2 or 3 OH sugar to a base present at the 3 end of a trna. This base is always an adenine (Fig.12). Fig. 12: Aminoacylation of trna Each aminoacyl synthetase recognizes specific amino acid and a trna. Cells have twenty different synthetases, one for each of the 20 amino acids. The number of trnas present in a cell may vary from and thus exceed the number of amino acids. This suggests that there may be more than one trna for some of the amino acids. These are called isoaccepting trnas with different anticodons that accept the same amino acid (see below on wobble). The enzyme aminoacyl synthetase recognizes specific sequences in trna particularly present in the D-loop, anticodon loop and in the acceptor stem. For example, if the G3: U70 nucleotides of trna ala are used to replace the 3:70 base pair of trna cys or trna phe, then these modified trnas are recognized by alanyl trna synthestase and charged with alanine suggesting that G3:U70 base pair is a critical identity element in trna ala for its specific synthetase. Genetic Code It refers to the relation between the four-letter nucleotide sequence information in mrna to the amino acid sequence information in proteins. The cracking of the genetic code is a history now. 15

16 Several theoretical considerations and elegant experimental results supported that a group of three nucleotides, called a codon, specifies an amino acid. An mrna that contains all of its information in 4 letter nucleotides will have then 64 codons (4 3 = 64) that specify the twenty naturally occurring amino acids. In contrast, a single nucleotide or groups of two or four nucleotides would provide a maximum of 4, 16 or 256 codons respectively. While the single and double letter codons cannot represent all of the twenty amino acids, the codon containing four nucleotides was also not supported experimentally and theoretically as it uses maximally of the four letters to specify twenty amino acids compared to the triplet code. In fact, the very early mutagenesis experiments provided evidence to support the triplet code. It was shown that bacteriophage T4 was unable to tolerate genetic changes that probably have altered one or two bases. However, the phage was able to tolerate an alteration (deletion or insertion) of three bases. Based on such genetic experiments, it is suggested that the genetic code is a triplet code. In a triplet code, a change in one or two bases (insertion or deletion) alters the reading frame of the mrna and all the subsequent amino acids that are incorporated into the protein to the right side of deletion (-) or insertion (+) (that is on the COOH side of protein). However insertion of three nucleotides into an mrna sequence would change the coding ability of the message only of those triplets at, and between the insertion of bases but not the amino acid sequence downstream of the three inserted bases (see latter frame-shift mutations). Cell-free translational systems The genetic code was in fact deciphered before the cellular messenger RNAs were purified. However by then cell-free translational systems were shown to support protein synthesis of the endogenous mrnas in vitro. The proteins prepared by such cell- free extracts can be labeled by supplementing one radioactively labelled amino acid along with the other nineteen unlabelled amino acids. The protein synthesis of the extracts carried out by endogenous mrnas, or by exogenously supplied mrnas can be monitored by the incorporation of radioactively labeled amino acid into the acid precipitable protein. This is possible because the cell-free protein synthesizing systems contain all the machinery viz., the ribosomes, transfer RNAs, aminoacyl synthetases that catalyze the joining of amino acids to trnas and other soluble factors (initiation, elongation and termination factors) required for the protein synthesis. Additionally the extracts are supplemented with ATP, GTP, and energy generating enzymes along with salts like K + and Mg 2+. Such cell-free translational systems derived from bacterial extracts eventually provided a means to determine the amino acid sequence of proteins coded by artificial and natural templates. Subsequently these cell-free translational systems have been used as a source for the preparation of reconstituted lysates to identify defective components of translational machinery, for the purification of protein factors and enzymes, to study the mechanisms of antibiotic actions, and to determine the mechanics and regulation of cytosolic and secretory protein synthesis. Since many proteins are processed in physiological conditions and is difficult to know the sequence if any in prepro-proteins (unprocessed proteins), translation of such mrnas in vitro systems devoid of the peptidases or proteases has provided a means to determine the sequence of full length proteins. In addition, these translational systems are found useful to evaluate the effects of antibiotics, toxins, and a host of other novel compounds for their ability to affect protein synthesis at specific steps. While bacterial systems and rat liver cell-free translational systems are used initially, subsequently the preparation of heme-deficient rabbit reticulocyte lystates and cell-free 16

17 translational systems derived from wheat embryos have unraveled some of the crucial regulatory mechanisms involved in the initiation and elongation of mammalian cytosolic and secretory protein synthesis. Wheat germ lysates, that do not have any endogenous mrnas, were used to determine the full length nature of a secretary protein, the role of a signal peptidase that processes the signal sequence of a secretory protein, and also the role of other regulatory proteins like signal recognition particle (SRP) that inhibits secretory protein synthesis, and docking protein that relives the inhibition mediated by SRP. Similarly heme-deficient rabbit reticulocyte lysates that carry mainly translational apparatus and mostly endogenous globin mrna were used to determine the importance of heme in the regulation of globin protein synthesis and to elucidate the mechanism of inhibition of general translation in reticulocyte lysates in hemedeficient lysates. Micrococcal nuclease-treated reticulocyte lysates can also be used to determine the translational products and activity of exogenously supplemented mrnas. Micrococcal nuclease is activated in the presence of calcium and can degrade the endogenous mrna. Before supplementing the exogenous mrna, these nuclease treated lysates have to be treated with EGTA that chelates calcium and inactivates the enzyme. While many animal systems can yield cell free translational systems with varying efficiencies, plant cell-free translational systems are difficult to obtain because of the presence of vacuoles that contain hydrolytic enzymes that can damage RNA. Synthetic mrna templates The preparation of artificial template mrnas is yet another important milestone in understanding the genetic code. This has become possible because of the discovery of polynucleotide phosphorylase enzyme. This enzyme is found to catalyze in fact the degradation of RNAs to the corresponding ribonucleotides and inorganic phosphate in vivo. But, interestingly, the purified enzyme has been observed to catalyze the formation of ribonucleoside triphosphates (rntps) in the presence of high concentration of ribonucleoside diphosphates in vitro. The incorporation of a ribonucleotide (A, U, G or C) is however dependent on the relative amounts of the nucleotides used in the reaction mixture. For example, the chances of the incorporation of nucleotide A (adenosine) only once in the template molecule in a reaction mixture consisting of A and C in the ratio of 1: 4 are 1/5 whereas the chances for the incorporation of C are 4/5, the highest. In other words, a template molecule with AAA will be synthesized least and CCC will be the most abundant. Templates with two C s and one A (CCA, ACC and CAC) will be the next abundant compared to two As and one C (AAC, CAA and ACA). The codon sequence of these templates containing a mixture of nucleotides could determine to some extent based on the relative abundance of the proteins produced by these templates in cell-free translational systems, and then identify the amino acid sequences of the protein (see below). However this method alone cannot give a precise sequence of information of codons in mrnas until additional information is available from other methods. Amino acid analyses of polypeptides produced by synthetic templates Synthetic mrnas with single nucleotide or mrnas (such as AAA, CCC, UUU, and GGG.), with alternating nucleotides (ACACACACACAAC), and with repeating sequences (AACAACAACAACAACAAC) yielded a good amount of information to relate the nucleotide sequence to the amino acid sequence in proteins. For example, translation of synthetic templates containing single nucleotides (homopolymers) such as A, C, U or G in cell-free translational 17

18 systems yielded polypeptides that contained a single amino acid sequence like lysine in the case of polya, phenylalanine in the case of poly U and proline in the case of poly C. Translation of poly G is however found to be weak and produces polyglycine. The reason appears to be that the poly G mrnas lack proper folding. Translation of templates containing alternating nucleotides like ACACACACAAC, yielded a polypeptide sequence that contains threonine and histidine amino acids alternatively. To determine whether the codon sequence ACA or CAC is the cause for the incorporation of threonine or histidine into the sequence, templates with repeating trinucleotides such as AACAACAACAACAAC have been made and translated in cell-free translational systems. Such sequences produced three types of polypeptides containing only asparagine (Asn), threonine or glutamine. This is possible if the start site in the mrna in each of these cases is different. Now we know most of the natural mrnas unlike synthetic mrnas have a start site and stop site. It is likely that if the synthetic mrna sequence in the above case is read as AAC AAC AAC AAC AAC, it may give a polypeptide that contains only Asn. But if the first A is skipped, then the message is read as ACA ACA ACA ACA AC, and if the first two As are skipped then the message will have a different sequence like CAA CAA CAA CAA C. The common amino acid that is incorporated when the mrnas containing AC repeats and AAC repeats is threonine. Based on these results it is suggested that probably the ACA codon is responsible for the incorporation of the amino acid threonine. However these results required further validation. The reading frame in natural mrnas is set by start codon which is usually AUG and the code is generally nonoverlapping or in other words each nucleotide in an mrna belongs to a single reading frame. Codon, charged trna and ribosome complexes can be captured on a filter Researchers who were in the race to crack the genetic code had yet another interesting observation wherein they found that a small triplet codon (like AUG, AAA or CUG etc.,) can attract a complementary anticodon of a trna carrying the amino acid. The codon and anticodon together can bind to ribosomes. Such codon-charged trna-ribosome complexes can be captured on a filter paper. The complex can be identified because the amino acid bound to trna in the complex is radioactive. This technique is found to be relatively more informative in deciphering the genetic code because it is easy to prepare and handle small triplet nucleotide sequences than long RNA templates. The synthetic triplet codons were incubated with a mixture of trnas each carrying a radioactive amino acid. Small molecules like the trnas carrying amino acids whose anticodons are not complementary to the codons cannot bind to the codon in the reaction and will pass through the filter. This technique really facilitated to identify the recognition of 61 triplet codons that are now known to recognize the twenty naturally occurring amino acids. Wobble and degeneracy Out of the total 64 codons, 61 codons specify 20 amino acids, and three codons UAA, UAG and UGA specify stop codons. Since 61 codons specify the 20 naturally occurring amino acids, it suggests that there are many amino acids that are specified by more than one codon. Hence it is suggested that the genetic code is degenerate. Based on the number of sense codons, it is suggested that there should be 61 trnas recognizing the 61 sense codons. However purification and characterization of trnas revealed that many purified trnas are also found to recognize a) more than one amino acid, and b) trnas contain some modified bases like inosine (derived from adenine) in the anticodon that is different from the regular four bases present in RNAs. Transfer RNAs with different anticodons that accept the same acid are called isosccepting trnas. Based 18

19 on these observations, it is suggested that the first base in the 5 -anticodon of trna does not follow strict Watson-Crick base-pair rule position while pairing with the 3 end of codon. This imperfect or nonstandard base pairing called wobble occurs between the third position of the codon in mrna and the first position of the anticodon in the trna (Fig. 13). Indeed analyses of all the codons through the filter binding assays did show that 18 of the twenty amino acids have more than one codon. Two of the amino acids, viz., methionine and tryptophan are specified by one codon each. Among the others, three amino acids (serine, leucine and arginine) have 6 codons each accounting 18 of the 61 codons. Five of the amino acids (glycine, proline, alanine, valine and threonine) have 4 codons each, and nine other amino acids (phenylalanine, tyrosine, cysteine, histidine, glutamine, glutamic acid, asparagines, asparatic acid and lysine) are specified by two codons. Isoleucine has three codons, and three codons UAA, UAG and UGA specify stop codons thus accounting the 64 codons ( = 64) in the genetic code (Fig. 14). Fig. 13: Wobble Indeed, analyses of many of the codons that specify the same amino acid have shown that these codons differ in their third position. For example the codons specifying glycine are GGG, GGC, GGA and GGU. In these cases, it is the last nucleotide residue or the nucleotide residue in the third position of the codon is different. The degeneracy of the code or wobble may be economical because it reduces the number of trnas from 61 to a minimum of 30 that are required to recognize all the 61 triplet codons. The estimated number of trna species to be present in bacteria is and the number may be somewhat higher in some eukaryotes. The code is mostly universal with the exception of few of the mitochondrial and protozoan codons that behave slightly differently. For example mammalian mitochondrial UGA codes for 19

20 trp instead of a stop signal. AUG codon is used in the cytoplasm to specify methionine during initiation and elongation steps of protein synthesis. However AUA that specifies isoleucine in the cytoplasm is used to specify internal methionine or methionine during the elongation step in mitochondria. AGA and AGG codons that specify Arg amino acid in the cytoplasmic code however specifies a stop signal in mitochondria. In fruit fly, AGA and AGG specify a serine codon but not a stop codon or arginine. Again an analysis of the changes in coding specificity of different codons reveals that a change in the base occurs primarily at the third position of the codon in mrna. Such changes may facilitate to decrease the number of trnas. Mutations Fig. 14: Genetic Code Changes in the nucleotide sequences alter the genetic code. Point mutations arise because of changes in a single nucleotide which are different from other kinds of drastic changes that occur in DNA due to extensive insertions and deletions. Missense mutation / Substitution mutation A change in one codon (trinucleotide sequence) that specifies a particular amino acid to another codon that specifies a different amino acid can occur by a single base change. The best example is sickle cell anemia, a human genetic disease. Here the glutamic acid at position six in the human β-globin is replaced by valine. The change is the result of a base change at the second position of the codon GAA that codes for glutamic acid to GUA that codes for valine. 20

21 Nonsense mutation A change in the base of a codon specifying an amino acid to another base that results in a stop codon. If it happens in the middle of a genetic message, it results in premature termination and incomplete polypeptide synthesis. Silent mutation Often a change in the base that is present in the third position of a codon of an mrna to another base can result in a synonym that does not alter the amino acid sequence of the encoded protein. Such changes are called silent mutations. Neutral Mutation It is a kind of missense mutation where the change in a base leads to a change in the incorporation of a different amino acid that is chemically similar to the original one, and does not influence the protein function. Frame shift mutations Insertion or deletion of one or more base pairs in the gene sequence affects the reading frame of mrna. As a consequence the amino acid sequence of the protein from the point of mutation to the C-terminus will be modified. For example 5. ABC ABC ABC ABC ABC ABC 3 sequence in mrna is modified by the insertion of a D for example can give a rise a message to ABC ABC DAB CAB CAB CAB C. Even deletion of a base also alters the coding capacity not only at the site of modification but also the rest of the message from the site of modification. However in the above sequence if insertion or deletion of three bases (like D, E and F) instead of one or two bases occurs then the coding specificity of the message is altered at and between the insertions of these bases but the downstream sequences are not altered as shown below. 5. ABC ABC ABC ABC ABC ABC 3 is changed to 5. ABC DAB CAE BCA FBC ABC ABC 3. Suppressor Mutations A suppressor mutation as the name indicates, suppresses the influence of another mutation. Thus the organism that contains a suppressor mutation is a double mutant and it has two mutations: one the original mutation and the second one being the suppressor mutation that will produce a phenotype which is similar to the wild type. However this is not called reverse mutation. In reverse mutation, whatever change occurred in the original nucleotide is restored. A suppressor mutation occurs away from the original mutation site. The suppressor mutation can occur within the same gene that contains already a mutation (intragenic suppressor) or in another gene (intergenic suppressor). Both kinds of suppressor mutations block the effects of an earlier mutation. Gene density and overlapping genes While the genome size and the approximate number of total genes present in an organism is related to the complexity of an organism, gene density that refers to the average number of genes per million bases (Mb) of genomic DNA is however high in lower organisms. An analysis of gene density in different organisms reveals that highest gene densities are found in viruses where 21