CHAIR AND DEPARTMENT OF BIOCHEMISTRY AND MOLECULAR BIOLOGY MEDICAL UNIVERSITY OF LUBLIN

BIOCHEMISTRY PART 6

Nucleic acids' metabolism. Protein synthesis Compendium of lectures

2013/2014

Agnieszka Slenzel- Bembenek, MDrPhD

Cellular nucleic acids exist of two form, deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). They are found in tlie nucleus and cytoplasm. Approximately 90% of the nucleic acid within the cell is RNA, and the remainder is DNA. Both can serve as the genetic material for Prokaryotic and Eukaryotic cells. RNA plays other multiple roles: it serves as the carrier of genetic information - mRNA, it can transfer amino acids to the ribosomes - tRNA, it has the structural function - rRNA and it can have catalytic activity -snRNA. Nucleotides are the monomeric units of tlie nucleic acids - DNA and RNA. Each nucleotide consists of a heterocyclic nitrogenous base, a sugar and phosphate. DNA contains the purine bases adenine (A), and guanine (G) and the pyrimidine bases cytosine (C) and thymine (T). RNA contains A,G and C but it has uracil (U) instead of thymine. In some RNAs eg.; tRNAs are present unusual bases such as pseudouridine, inosine, ribothymidine. In DNA, the sugar is deoxyribose (without the hydroxyl group at the 2' position of sugar ring ); in RNA it is ribose. Nucleotides are linked together by phosphodiester bonds between the 3' hydroxyl on the sugar of one nucleotide through a phosphate molecule to the 5' hydroxyl on the sugar of another nucleotide. The sugar- phosphate linkages form symmetrical "backbone" in which the 5' end of one sugar is always linked through a phosphate molecule to tlie 3' end of the adjacent sugar. To the sugar-phosphate backbone variable bases are attached to. The order of bases determines the coding or structural capacity of the nucleic acid. The symmetry of the sugar-phosphate backbone gives a polarity to nucleic acid polymers. The terminal nucleotide of one end usually has a free 3'-hydroxyl group (3' OH) on its sugar moiety. The other end usually has a free phosphate group (5'P) attached to the 5' position of the sugar. Nucleotides participate and play crucial roles in many biochemical processes: they are precursors of DNA and RNA, some nucleotides are structural components of many coenzymes (eg.: NAD, FAD, CoA),nucleotides serve as donors of phosphoryl groups (eg.: ATP, GTP),nucleotides serve as donors of sugars (eg.: UDP-sugar-glucose), nucleotides serve as metabolic regulators - second messengers in signal transduction pathways (eg.: cAMP, cGMP), nucleotide derivatives are activated intermediates in many biosyntheses of some carbohydrates, lipids and proteins (eg.: UDP-glucose, CDP-diacyloglycerol, S- adenosylmethionine), ATP is a universal biologic transducer of flee energy (cleavage of acid anhydride bonds)and synthetic nucleotide analogs are used in chemiotherapy ( toxic effect for nucleic acids synthesis- inhibition of essential enzymes).

Metabolism of purine and pyrimidine nucleotides

Purines and pyrimidines are required for synthesizing nucleotides and nucleic acids. These molecules can be synthesized either from scratch, "de novo" or "salvaged" from existing bases. Purines and pyrimidines are not essential in our diet. We can synthesize purines and pyrimidines from amphibolic intermediates. The biosynthesis of purines and pyrimidines is stringently regulated and coordinated by feedback mechanisms. This regulation enables that purine and pyrimidine nucleotides are synthesized in vivo in quantities and at time appropriate to varying cellular requirements ( increased during cellular growth, division or tissue regeneration). Degradation of dietary nucleic acids occurs in small intestine where pancreatic enzymes are secreted (nucleases -ribonuclcases and deoxyribonucleases, phosphodiesterases, nucleotidases). The purine bases are produced by pathways that use amino acids as precursors. Most of the synthesis "de novo" occurs in the liver. It is energetically very expensive process. The nitrogenous bases and nucleotides are then transported to other tissues by red blood cells.

5'-phosphoribosU-l-pyrophosphate (PRPP) is an intermediate of major significance in nucleotide metabolism. PRPP is formed from ribose 5- phosphate and adenosine triphosphate (ATP).Three processes contribute to purine nucleotides biosynthesis:

1. synthesis from amphibolic intermediates (de novo synthesis) 2. phosphoribosylation of purines 3. phosphorylation of purine nucleosides

The "de novo" pathway of purine synthesis is complex - it consists of 11 steps and requires 6 molecules of ATP for every purine synthesized. The precursors of the purine ring are: PRPP, glutamine, glycine, carbon dioxide (CO2), aspartate, two one-carbon fragments from one-carbon folate pool. The regulation of purine nucleotide biosynthesis occurs at four points in the pathway. The enzymes: PRPP synthetase, amidophosphoribosyl transferase, IMP dehydrogenase, adenylosuccinate synthetase are the main regulation points through the pathway. De novo synthesis of purine nucleotides is regulated by feedback inhibition: PRPP synthetase and PRPP amidotransferase are inhibited by IMP,AMP and GMP, the synthesis of adenylosuccinate from IMP is inhibited by AMP, the synthesis of XMP from IMP is inhibited by GMP

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There aie two "salvage pathways" or "salvage reactions" that require far less energy than cte novo synthesis. These reactions can convert purines and their nucleosides (ribo- and deoxyribonucleosides) to mononucleotides. There are two specific enzymes that catalyze the transfer of the ribose phosphate from PRPP to free purine bases, which are formed by the degradation of nucleotides: - hypoxauthine -guanine phosphoribosyltransferase (HGPRT ) -it catalyzes the formation of nucleotides from either hypoxanthine or guanine, is inhibited by IMP and GMP - adenine phosphoribosyl transferase (APRT) - it catalyzes the formation of AMP from adenine, is inhibited by AMP

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The pyrimidine ring, unlike the purine ring is not built on a molecule of PRPP. At first, the pyrimidine ring is formed and then it reacts with PRPP to form the nucleotide. Pyrimidine bases are first synthesized as a free base and then converted to a nucleotide. The precursors of the pyrimidine ring are: glutamine, aspartate,C02. Aspartate and carbamoyl phosphate (glutamine + C02) form all components of the pyrimidine ring. Pyrimidine nucleotide synthesis is regulated at the start point- at the first reaction in which carbamoyl -phosphate is synthesized by the cytoplasmic enzyme carbamoyl phosphate

synthetase n (CPSII).

Degradation of purine and pyrimidine nucleotides.

Products of RNA and DNA degradation- nucleotides - are sequentially degraded by specific enzymes inside cells. The final product of purine degradation is uric acid excreted in the urine.

The final products of pyrimidine degradation are highly soluble structures such as: ß-alanine (from cytosine and uracil) and ß-aminoisobutyrate (from thymine) which can serve as precursors of acerylCoA and succinylCoA, respectively.

Diseases associated with defects of nucleotide metabolism.

Mutations in a number of the enzymes included in purine nucleotide pathways (synthesis and degradation) lead to serious diseases.

Gout is a disease caused by accumulation of excess uric acid in body fluids, (normal range-1.5 - 7.0 mg/dl = 0.1-0.4 mmol/l).Uric acid and its urate salts have a low solubility in water. Their excessive accumulation results in the precipitation of needle-shaped sodium urate crystals. These crystals are frequently deposited in the soft tissues, particularly in joints and kidneys - they can damage affected joints ( inflammation, erosion) and impair renal function. Allopurinol is an important drug in the treatment of gout. It inhibits xanthine oxidase and blocks the production of uric acid.

Lesch-Nyhan syndrome is a hereditary X-linked disease that is due to severe or complete deficiency of HGPRT( hypoxantine -guanine phosphoribosyl transferase) activity. The lack of HGPRT causes overaccumulation of PRPP which stimulates purine biosynthesis by up to 200-fold. Elevated uric acid leads to gouty artliritis and severe neuropathology ( mental retardation, spasticity, aggressive behavior).

Adenosine deaminase deficiency (ADA)-in the cells of people with ADA deficiency, deoxyadenosine and adenosine are abundant because they are not degraded to deoxyinosine and inosine. The high levels of dATP inhibit ribonucleotide reductase, which inhibits DNA synthesis. Therefore white blood cells cannot proliferate. T-cell and B-cell functions are defective.ADA deficiency is associated with severe combined immunodeficiency (SCID).

Synthetic inhibitors of purine synthesis.

Sulfonamides are structural analogs of PABA (p-Aminobenzoic acid) that competitively inhibit the synthesis of folic acid in microorganisms. Because purine synthesis requires THF as a coenzyme, the sulfa drugs decrease the synthesis of nucleotides needed for the replication. Sulfonamides inhibit the growth of rapidly dividing microorganisms without interfering with human cell function.

Methotrexate and related compounds are structural analogs of folic acid. They inhibit the reduction of dihydrofolate to tetrahydrofolate catalyzed by dihydrofolate reductase. They decrease (he amount of tetrahydrofolate for purine nucleotide synthesis and slow down DNA replication and RNA synthesis in mammalian cells. Methotrexate and related compounds Are useful in treating rapidly growing cancers, but they are also toxic to all dividing cells in human body ( bone marrow, immune system, skin)

Trimethoprim is structural analog of folic acid. It has antibacterial activity because of its selective inhibition of bacterial dihydrofolate reductase.

5-fluorouracil is an analog of thymine, serves as successful antitumor agent.5-fluorouracil is metabolically converted to 5-fluorouridine 5'monophosphate (F-UMP), which becomes permanently bound to the inactivated thymidylate synthase.

DNA and RNA structure.

DNA and RNA are polynucleotides. The DNA and RNA chains are linear sequences of nucleotides linked by 3' to 5' phosphodiester bonds between sugars. The sugar- phosphate linkages form symmetrical "backbone" in which the 5' end of one sugar is always linked through a phosphate molecule to the 3' end of the adjacent sugar. To the sugar-phosphate backbone variable bases are attached to. They stick out from the backbone. The bases of the nucleotides can interact with oilier bases or with proteins. The symmetry of the sugar-phosphate backbone gives a polarity to nucleic acid polymers. The terminal nucleotide of one end usually has a free 3'-hydroxyl group (3' OH) on its sugar moiety. The odier end usually has a free phosphate group (5'P) attached to the 5' position of the sugar. Genetic information is encoded by the sequence of different nucleotide bases in DNA. DNA is double -stranded, it contains two antiparallel polynucleotide strands. These two strands are joined by hydrogen bonding between their bases to form base-pairs. Adenine pairs with thymine (two hydrogen bonds) and guanine pairs with cytosine (three hydrogen bonds). The two DNA strands run in opposite directions- one runs 5'to 3 ' and the other one runs in 3' to 5'. The two DNA strands form a double helix. In the double-stranded DNA molecules, the genetic information resides in the sequence of nucleotides on one strand- the template strand ( or noncoding strand). This strand is copied during nucleic acid synthesis. The opposite strand is considered the coding strand because it matches the RNA transcript that encodes the protein.

DNA coding strand 5' — A T G C C A G T A G G C C A C T T G T C A — 3'

DNA template strand 3"— T A C G G T C A T C C G G T G A A C A G T — 5'

mRNA 5 - A U G CCA GUA GGC CAC UUG U C A - 3 ' .3

Protein N— M et -P ro -Va I -G I y - H i s - L e u - S e r — C

Fig. 14.3. Relationship between the coding stnlnd of DNA. the DNA template Strand, the mRNA transcript, and the protein produced from the gene. The bases in mRNA are used in sets of three (called codons) to specif)' the order of the amino acids inserted into the growing polypeptide chain during the process of translation (sec Chapter 1.1).

Double- stranded DNA can separate when hydrogen bonds between bases are disrupted (eg.: pH >7, high temperature). The process of separation is called denaturafion, it usually occurs when DNA is heated.The temperature when DNA loss its helical structure is defined as the melting temperature(Tm). DNA with the higher concentrations of G/C pairs has a higher Tm than A/T rich DNA .Unwound, denaturated DNA is needed in the process of replication and transcription. Denaturation is reversible and the process of reassociation is called renaturation or reannealing.

DNA must be packaged into a more tightly compacted form to get into a cell or cell's nucleus. In Prokaryotes, DNA is a circular double -stranded molecule, organized into loops or domains, attached to the cell membrane. Sometimes it is also associated with histone-like proteins (eg.: HU, HLP-1, H-NS) and it resides in the region of tlie cell called the nucleoid. In Prokaryotes are present other circular DNA molecules- the plasmids. They can self-replicate. Eukaryotic cells contain much more DNA than Prokaryotes. In the nucleus, tlie long linear molecule of double-stranded DNA is bound to a complex mixture of proteins ( histones and non-histone proteins) which is called chromatin.

The structure of chromatin is determined and stabilized tlirough tlie interaction of DNA and DNA-binding proteins. There are two classes of DNA-binding proteins: histones and non-histone proteins (transcription factors, hormone receptors, enzymes -polymerases, helicases, topoisomerases and structural proteins). There are five classes of histones: I I 1 , H2A, H2B, H3 and H4.They are small nuclear proteins with high concentration of lysine and arglnine, highly conserved in evolution. They form a structural core (two molecules of each - H2A, H2B, H3 and H4 histone; a histone octamer) around which the double helix of DNA - 146bp long, is wound nearly twice, forming left-handed supercoil. Histone I I 1 is not found in the nucleosome, it is attached to tlie linker DNA chain between nucleosomes which varies from 20 up to 200 bp. long. Histone H 1 helps the packing of nucleosomes into the more compact structures -solenoids. The 30 nm fiber (solenoid) folded into loops is attached to fibrous protein scaffold at the center of a chromosome. During the cell division this complex is organized into tlie next order of packaging - chromosomes.

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Figure 36-3. Shown Is the extent of DNA packaging in metaphase chromosomes (top) to noted duplex DNA (bor torn). Chromosomal DNA is packaged and organized at several levels as shown (see Table 36-2). Each phase of con­densation ot compaction and organization (bottom to top) decreases overall DNA accessibility to an extent that the DNA sequences in metaphase chromosomes are almost totally transcriptionally Inert. In toto, these five levels of DMA compaction result In nearly a 104-fold linear decrease in end-to-end DNA length. Complete condensation and decondensation of the linear DNA in chromosomes occur in the space of houis during the normal replicative cell cycle (see fiyure 36-20).

RNA is generated through tlie transcription of the gene. There are three major types of RNA: messenger RNA (mRNA), ribosomal RNA (rRNA) and transfer RNA (IRNA). mRNAs contain the nucleotide sequence that is converted into the amino acid sequence of a protein in the process of translation. Prokaryotic mRNA is polvcistronic because it carries the information for the production of multiple polypeptides. Eukaryoric mRNA is monocistronic because it carries the information for the production of a single polypeptide. Numerous post-transcriptional modifications (processing events) are needed for mRNA to be functional in cytoplasm: modification of the 5'end (5'cap-7-methylguanylatc), modification of the 3'end (polyadenylated (poly A tail) and splicing (the process by which noncoding sequences are removed to form a functional mRNA) Process of splicing is multistep and it is catalyzed by a large ribonucleoprotein complex called a spliceosome, which contain five snRNAs: UI, U2, U4, U5 and IJ6.

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rRNAs and tRNAs do not encode proteins. They are part of the apparatus for protein synthesis. rRNAs with proteins build the complexes called ribosomes, which bind mRNA and tRNA during translation. Besides structural, rRNA may also have a catalytic function - it can work as an enzyme - a ribozyme.

tRNA binds and activates a specific amino acid for insertion into the polypeptide chain. A unique trinucleotide sequence on each tRNA called an anticodon binds to a complementary codon on the mRNA. tRNA has a "cloverleaf ' structure with arms: the acceptor arm terminates in the nucleotides CCA, the D loop, the PPC loop, the extra arm and the anticodon arm. tRNA contains a number of" unusual" nitrogenous bases- pseudouridine, inosine, methyl guanosine, ribothymidine - all of them are post- transcriptional modifications of tlie four "usual" bases.

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Synthesis of DNA (replication). DNA synthesis occurs by the process of replication. During replication, each of the two parental strands of DNA serves as a template for the synthesis of a complementary strand. Each DNA molecule generated by the replication process contains one intact parental strand and one newly synthesized strand. In Eukaryotes, DNA replication occurs during the S phase of the cell cycle. In both, Prokaryotes and Eukaryotes, the site at which replication is occurring is called the replication fork. During process of replication, the two parental strands separate in the front of the fork. Behind the fork, each newly synthesized strand of DNA base-pairs with its complementary parental template strand. Many proteins (enzymes) are involved in the process of replication- helicases, topoisomerases, single-strand binding proteins (SSB) and the major enzyme -DNA polymerase. DNA polymerase copies each parental template strand in the 3 ' to 5' direction, producing new strands in the 5' to 3' direction. One strand of newly synthesized DNA grows continuously, the other strand is synthesized discontinuously in short fragments known as Okazaki fragments. DNA polymerase cannot initiate the synthesis of new strand- it needs a short stretch of RNA - a primer. To its 3' end DNA polymerase can add the deoxyribonucleotides. In Prokaryotes there is one origin of replication, in Eukaryotes, replication begins at multiple sites along the DNA helix.

The primosome is the complex of the proteins that comprises: topoisomerases, helicase, DnaA protein, SSB proteins, primase and several other proteins. The primosome complex is responsible for:

- the recognition of the origin of replication - unwinding the double helix ahead of the replication forks - maintaining the separation of the parental strands - priming DNA synthesis at the origin

It needs the ATP hydrolysis and moves with the replication forks, making RNA primers for Okazaki fragment synthesis. In Prokaryotes there are three DNA polymerases which besides DNA synthesis are capable of catalyzing other reactions - eg. DNA repair:

a) DNA polymerase I (pol I) - replication of DNA and repair damaged DNA b) DNA polymerase II (pol II) - repair damaged DNA c) DNA polymerase III (pol III) - is a primary DNA polymarase involved in cellular replication It catalyzes leading and lagging strand synthesis.

The replisome is a large macromolecular complex that comprises all the replication enzymes and factors.This complex moves along the DNA during replication. The replicón is a basic unit of replication, what comprises:

- origin of replication - all of the DNA replicated - termination of replication.

In Prokaryotes there is one replicón.

Eukaryotic replication is not as well known as it is in prokaryotes. Process of eukaryotic replication is semiconservative and proceeds bidirectionally from many origins. In Eukaryotes there is a large number of replicons what is needed for replication the large genome. The eukaryotic replication rate is about 10 times slower than the prokaryotic replication rate: - in prokaryotes replication forks progresses at approximately 1000 base pairs per second; - in eukaryotes replication forks progresses at approximately 100 base pairs per second The slow rate of eukaryotic replication is due to interference of nucleosomes and chromosomal proteins. Eukaryotes contain four different nuclear DNA polymerases : a, ß, e and 8 and one mitochondrial DNA polymerase y. - DNA polymerase a - is involved in replication of nuclear DNA, has primase activity - DNA polymerase 8 - acts in conjunction with a during replication - DNA polymerase ß and 8 are involved in repair of nuclear DNA - DNA polymerase y resides and replicates mitochondrial DNA Eukaryotes replicates their DNA only once per cell division cycle during the S (synthesis) phase.

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Process of transcription. Transcription is the copying process, during which a DNA strand serves as a template. During this process several RNAs are synthesized. Following their syntheses, only messenger RNA (mRNA) is translated into sequences of amino acids (polypeptide chains or proteins). Others RNAs - ribosomal rRNAs, transfer tRNAs and small nuclear snRNAs perfomi structural and regulatory functions and they are not translated. There are many regulatory proteins engaged in the process of transcription. Many of the transcription products may undergo various modifications such as terminal additions, base modifications or splicing because of which they can be converted from the inactive primary transcript into a functional molecule. The basic requirements and mechanism of transcription are similar in prokaryotes and eukaryotes, but there are some differences. During transcription, a single strand of DNA acts as a template to direct the formation of complementary RNA. The four ribonucleoside triphosphates: ATP,GTP,CTP and UTP are necessary for RNA synthesis. Cleavage of the high- energy phosphate bond between the a and ß phosphates of these nucleoside triphosphates provides the energy for the addition of nucleotides to the growing RNA chain. RNA synthesis proceeds in the 5'—>3' direction. Prokaryotes have a single RNA polymerase responsible for all cellular RNA synthesis. The structure of the enzyme is complex (a core enzyme-subunits 02, ß and ß' required for the elongation steps of RNA synthesis and subunit 8 required for proper initiation of transcription). Eukaryotes have one mitochondrial and three nuclear RNA polymerases. The nuclear RNA polymerases are distinct enzymes that function to synthesize different RNAs. - RNA polymerase I ( nucleolus) -* rRNA - RNA polymerase II (nucleoplasm) —> mRNA and snRNA - RNA polymerase III ( nucleoplasm) —• tRNA and 5S rRNA Transcriptional initiation does not require a primer.

There are some sequences on DNA (called promoters) which are responsible for directing RNA polymerase to initiate transcription at a particular point. Promoter sequences differ between prokaryotes and eukaryotes.

Promoter is a region of DNA to which RNA polymerase binds just before initiating the transcription of DNA into RNA. The promoters for most Prokaryotic genes have three sequence elements: initiation site or startpoint ( position 1), Pribnow box - sequence TATAAT ( -10 sequence ) and the - 35 sequence ( the sequence TTGACA). The Eukaryotic promoters used by RNA polymerase I and II are similar to the prokaryotic promoters - they are upstream of the startpoint. The promoter used by RNA polymerase III is unique because it is usual downstream of the startpoint.

The promoters of all the Eukaryotic RNA polymerases start the initiation of the transcription at a particular site called startpoint. They have several sequence elements (common to many of promoters): TATA box sequence ( 25-35 bps upstream of the startpoint ), CAAT box and GC box sequences found 40 to 200 bps upstream of the startpoint and other sequence elements called enhancers or silencers, which affect the rate of initiation of transcription ( increase or decrease, respectively). They are short sequences which are functional either downstream or upstream or even at great distances from the initiation site. Their activity is independent of their orientation relative to gene.

During the initiation of the transcription there are also needed different initiation factors. In Prokaryotes - only a single factor, sigma (8) is needed to initiate the transcription. In Eukaryotes, there are many factors associated with the initiation of the transcription. Each of eukaryotic RNA polymerases (I, II and III) needs specific transcription factors to initiate transcription from their respective promoters. These transcriptional factors are proteins which can interact with each other and wiüi RNA polymerase. They recognize and bind to the promoter sequences. In the presence of ribonucleoside triphosphates together with RNA polymerase they initiate the transcription. The process of transcription can be divided into three phases: initiation, elongation and termination. Elongation of transcription: at the site of RNA synthesis the DNA is unwound-one strand of the DNA is a template. RNA synthesis is in a 5'—>3' direction. The addition of a single nucleotide to the 3' end of the growing RNA chain. Cleavage of the high-energy bond between a and ß phosphates of an incoming nucleoside triphosphate provides the energy for formation of a phosphodiester bond with the 3 ' hydroxyl (OH) of tlie preceding nucleotide.

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A transcription unit extends from the promoter to the termination region. Primary transcript is the product of the process of transcription by RNA polymerase. Primary transcript is a linear copy of a transcriptional unit. The primary transcripts of both Prokaryotic and Eukaryotic tRNAs and rRNAs are post-transcriptionally modified by cleavage of the original transcripts by ribonucleases. Ribosomal rRNAs (in Prokaryotes and Eukaryotes) are synthesized from long precursor molecules called pre-ribosomal RNAs, which later are cleaved by ribonucleases to small required ribosomal RNA species. Transfer RNAs (in Prokaryotes and Eukaryotes) are also made from long precursor molecules which must be modified:

- an intron must be removed - 5' and 3' end sequences must be trimmed - CCA sequence must be added to 3 ' end - modification of bases

Prokaryotic mRNA is generally identical to its primary transcript. Eukaryotic mRNA is extensively modified posttranscriptionally. The primary transcript (the RNA molecule synthesized by RNA polymerase II contains the sequences that are not found in cytosolic mRNA). This precursor RNA molecule is known as a heterogeneous nuclear RNA (hnRNA). Just after transcription hnRNA is extensively modified: 5' cap - 7-methyl-guanosine attached to 5'end, addition of a poly-A tail to the 3' end and removal of introns in association with snRNAs.

Transcription is highly regulated process. The regulation of prokaryotic genes is described on the basis of operon theory. Operon it is a group of bacterial genes with a common promoter. In bacteria, the structural genes that code for the enzymes of the metabolic pathway are often found grouped together on die chromosome together with the regulatory genes that determined their transcription. They are controlled as a unit and produce mRNA as a single long piece. In Eukaryotes, the regulation of the transcription is more complicated than transcription in prokaryotes. There are many regulatory genes, DNA sequences (enhancers and silencers) and proteins (transcriptional factors) involved in the process of the transcription. All these factors and mechanisms work together and determine which genes are to be transcribed in the cell. In eukaryotes each cell is specialized and express only these functions which are required for it to perform its role in maintaining organism. The term gene expression usually refers to the entire process in which the information encoded in a particular gene is decoded into a particular protein. At first the genetic information found in DNA is precisely copied (replicated). Then, througli the process of transcription ( the first stage in the expression of genetic information) an appropriate RNA synthesis occurs. The second and complete gene expression is the process of translation (protein synthesis) in which the nucleotide sequence of the mRNA is translated into protein. The regulation of gene expression determines which proteins are synthesized at any time. An appropriate amounts of these synthesized proteins allow cells to undergo development and differentiation, according to environmental and cell conditions and requirements.

19

Translation of mRNA -protein synthesis Translation is the process by which ribosomes convert the information carried by messenger RNA (mRNA) to the synthesis of new proteins. Protein synthesis usually is called translation because the "language" of the nucleotide sequence on the mRNA is translated into the "language" of an amino acid sequence. This translation requires a genetic code as a translator.

The genetic code can express the information contained in nucleic acid sequence as a specific sequence of amino acids in the polypeptide chain or protein. Each "word" in genetic code is composed of three nucleotide bases and is called a codon. Codons are usually presented in the messenger RNA language of adenine (A), guanine (G), cytosine (C) and uracil (U). They are always written from the 5'end to the 3'end.The four nucleotide bases are used to produce the three base codons- there are 64 (4s) combinations of bases (codons). Three of these codons (UAA,UAG,UGA) are called the "stop" codons or "termination" codons. They are used as signals to stop the synthesis of a protein and do not specify an amino acid. The leaves 61 codons specify 20 amino acids. Almost all the amino acids have more than one codon- usual two, four or even six codons ( leucine, serine, arginine).The exception is: methionine (Met) and tryptophan (Trp) which has only one codon -AUG and UGG, respectively.

Any changes (mutations) that cause the addition or deletion of single nucleotides will cause the "frame shift mutation". It results the different amino acid sequence in a protein ( missense mutation) or even premature termination of protein synthesis (nonsense mutation). Sometimes may be no detectable effect in polypeptide chain because of the degeneracy of genetic code. In that time we have a "silent mutation".

A large number of components are required for the protein synthesis (translation). Translation needs: amino acids, mRNA, tRNA, ribosomes, sources of energy -ATP, GTP, enzymes and protein factors required for initiation, elongation and termination. Ribosomes serve as the machinery on which the mRNA nucleotide sequence is translated into the sequence of amino acids of the protein. The message is carried by mRNA and the linkage between the message and the protein (translation) is made by adaptor molecules- aminoacylated tRNAs (also called charged tRNAs). Ribosomes consist of a small and a large subunit. They have three specific sites which bind the tRNAs: an aminoacyl (A site); the peptidyl (P site) and the exit (empty) E site.Together they cover three neighboring codons.

The peptide bond forms between the amino group of the amino acid in the A site and the carboxyl terminus of the nascent peptide attached to the tRNA in the P site. This process is catalyzed by peptidyl transferase.

Peptidyl transferase is not typical enzyme. It is supposed to be an enzymatic activity of the 23S rRNA, so it is sometimes called as a rybozyme.

Activation of amino acids - formation of aminoacyl-tRNAs. Each amino acid is attached to the acceptor stem of the tRNA by an enzyme called aminoacyl-tRNA synthetase.. The attachment of an amino acid to its appropriate tRNA (the aminoacylation reaction) requires adenosine triphosphate (ATP), which is cleaved to AMP and PPi. That is the two step reaction: Amino acid + ATP «-» aminoacyl-AMP + PPi Aminoacyl-AMP + tRNA «-> aminoacyl-tRNA + AMP PPi is then hydrolyzed to form two free phosphates. A total of two high-energy phosphate bonds of ATP are used in the formation of a single aminoacyl-tRNA.

Codon- anticodon recognition. Each tRNA molecule contains a three- base nucleotide sequence- the anticodon- that recognizes a specific codon on the mRNA. This codon specifies the insertion into the growing peptide chain an appropriate amino acid carried by an appropriate tRNA. The anticodons are complementary to their codons in the antiparallel fashion.

The process of protein synthesis involves the interaction of enzymes, tRNAs , mRNA, ribosomes and many necessary proteins which are known as the factors. The translation is divided into three steps: initiation, elongation and termination. Initiation of protein synthesis takes place when the ribosome (both large and small subunits) has assembled on the mRNA exactly in the manner that the P site is on the AUG codon (start codon). In this case P site is also occupied by the first tRNA molecule [initiator tRNA] - in Prokaryotes- N-formyl methionine tRNA (fmet-tRNA) - in Eukaryotes- methionyl-tRNA (met-tRNA)

Initiation step needs also initiation factors (IFs) ( in Prokaryotes - 3 , in Eukaryotes- at least 12) which promote the association of the initiation complex created from ribosome, mRNA, charged met-tRNA or fmet-tRNA what needs additionally the energy from the hydrolysis of guanosine triphosphate (GTP). The movement of this complex down the mRNA is driven by the hydrolysis of ATP.

Elongation step begins with tlie binding of the charged tRNA in the A site in the ribosome (according to covered codon). In Eukaryotes, the charged tRNA molecule is brought to the ribosome by the action of the elongation factor (EF1), associated with GTP molecule as a source of energy. When the correct charged tRNA molecule has been delivered to the A site the peptidyl transferase catalyzes the formation of a peptide bond between the amino acid in the A site and the amino acid in the P site. When peptidyl bond is formed, the ribosome moves down one codon on mRNA- we call this movement the translocation. It needs another elongation factor (EF2) and GTP as a source of energy.

Termination of protein synthesis (in both Prokaryotes and Eukaryotes) occurs when the A site of the ribosome is over the one of termination codon: UAA,UAG or UGA. These codons are recognized by special proteins- releasing factors. Releasing factors transfer a water molecule to the end of the polypeptide chain (protein), attached to the tRNA molecule in the P site of the ribosome. Addition of water releases the protein from the tRNA. Releasing factors use the GTP as a source of energy. When, the protein chain is released, the ribosomal subunits, tRNA and mRNA dissociate from each other.

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A mRNA molecule can be translated by more than one ribosome at the same time. A polyribosome or polysome - is an mRNA molecule with several bound ribosomes. There are two classes of polysomes found in cells;

- free in the cytoplasm - attached to the endoplasmic reticulum

mRNAs encoding proteins destined for the cytoplasm are translated on free polysomes in the cytoplasm. mRNAs encoding membrane and secretory proteins are translated on polysomes attached to the endoplasmic reticulum. Many proteins must be altered before they are biologically active and these alterations are known as post-translational modifications.

Post-translational modifications include: 1. removal of part of the translated sequence

• zymogens (eg.: trypsinogen) are inactive precursors of secreted enzymes, they become activated through cleavage when they are in their proper sites of action

2. covalent alterations (addition of one or more chemical groups may activate or inactivate enzymatic

and structural proteins) - phosphorylation (addition of phosphates) - glycosylation (addition of sugars) - hydroxylation( proline and lysine residues of collagen) - acetylation (addition of acethyl group) - methylation ( addition of methyl group) - carboxylation (addition of carboxy! group) - formation of disulfide bridges (cysteine)

The antibiotics that preferentially inhibit protein synthesis in bacteria.

• STREPTOMYCIN - binds to 30S subunit, inhibits the initiation of p.s. • GENTAMYCIN - binds to 30S subunit, thus interfering with the assembly of the

ribosomal complex, inhibits the initiation of p.s. • TETRACYCLINES - interact with 30S subunit, inhibit the addition of the

aminoacyl-tRNA to the mRNA-ribosome complex, inhibit the elongation of p.s. • PUROMYCIN - is an analog of aminoacyl-tRNA ( structural resemblance),

inhibits the elongation- it acts as a chain terminator in p. s. in Eukaryotes and Prokaryotes

• CHLORAMPHENICOL - inhibits Prokaryotic peptidvltransferase, inhibits the elongation of p.s.

• ERYTHROMYCIN AND CLINDAMYCIN-bind to 50S subunit, inhibit translocation, inhibit the elongation of p.s.

• DIPHTHERIA TOXIN ( Corynebaclerium diphtheriae )- the toxin causes diphtheria, a lethal disease of the respiratory tract; the A fragment of the toxin inactivates the Eukaryotic eEF-2, inhibits the translocation

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