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3/6/2016 • Protein synthesis Food Biotechnology-3 1 2 Flow of genetic information is divided into three steps Genes and genetic code Gen: DNA fragment carrying the the genetic information is called as gene. Genes 1 replication are compose of DNA. • Genetic code is determined by the orders of A,G,T, and C bases. • Information on DNA is transferred to RNA, and transformad to proteins by the help of RNAs Moleküler biyolojinin santral dogması • DNA, RNA and protein are called as informational macromolecules, since they all contain genetic information. 3 • Order of amino acids in a polypeptide chain is dtermined by the order of bases in mRNA. 2) Transcription (copying): Genetic information contained in DNA is copied in the form of RNA. This replication process is called as transcription. RNA molecule that copies the DNA code is messeger RNA (mRNA). • Some genes contain codes necessary for Transfer RNA (tRNA) and ribosomal RNA (rRNA) These also take part in protein synthesis, however, they do not contain the codes forr protein synthesis. 3) Translation: process of protein synthesis using the information copied on mRNA 4 As translation process is started by mRNA, genetic code is written in the form of mRNA, not DNA. • There is a linear relation between the order of aminoacid of a polypeptide and base order of a gene. With 4 different nucleotides, a code of 3 nucleotide could code maximum 64 codons (43). • Each 3 bases in mRNA encodes an aminoacid. The nucleotide triplets in mRNA that encode amino acids are called as codons. It has been demonstrated thaat only, 61 codons encode aminoacids. The rest 3 are stop codons and do not encode a.a. 20 aminoacids are encoded by codons, so one amino acid can be encoded by several codons. • Each codon encodes a specific amino acid • Genetic code is translated to proteins. Translation system consists of ribosomes (protein and rRNA), tRNA and some enzymes Thus, if amino acid sequence of a protein molecule is known, it is impossible to predict the codons. 5 On the other hand, If DNA sequence of a gene is known, amino acid sequence of the protein can be known. 6 1 3/6/2016 Protein synthesis PROTEİN SYNTHESIS • Proteins synthesis is a very complex precess, however it is a very rapid process. E.g. İn E. coli ribosomes, a polypeptide chain having 100 amino acids is synthesized in 5 seconds. • In a living cell, many metabolic reactions are carried out by proteins • Proteins are one of the most important elements of the living organisms. E.g: Blood plasma prroteins Hormons Anticores Enzymes Chloroplast Mithocondria Cell wall proteins • In a young cell, there are 5.000-50.000 ribosomes, at which hundreds and thousands of proteins are synthesized simultanously. • Protein sysnthesis is controlled by some other molecules (tiem and amount). So in the protein synthesis many components take part, not only the translation system components. 7 Steps of protein synthesis 8 1) Transcription : synthesis of RNA 1)Transcription: The process in which a particular fragment of DNA is copied into RNA is calle as transcription. 3 key differences between DNA and RNA are: • RNA contains ribose instead of deoxyribose • RNA contains uracil instead of thymin • RNA is single strand 3 types of RNA is produced with transcription. • mRNA is the reoxy copy of gene on DNA. It carries the information from DNA to ribosomes. • Enzymes that attack on DNA do not effect RNA • Enzymes that attack on RNA do not effect DNA • Both T and U can bind with A, so change of base do not effect base pairing. • tRNA and rRNA are involved in the second step of protein synthesis (translation). 2) Translation: protein synthesis is realized by using RNAs. 9 10 • Trancription of genetic information from DNA to RNA is realized by enzyme RNA polymerase. • RNA polymerase catalysis the formation of phosphodiester bonds • The direction of the synthesis is from 5’ to 3’, same as in DNA synthesis. And the templete is antiparallel to the newly synthesized chain. • The enzyme requires DNA as templete. • Substrates of the enzyme are ATP, GTP, UTP and CTP . • RNA polymerase adds ribonucletide triphosphates to the 3’-OH group of the previously added nucleotide . Energy (pyrophosphate) generated by the hydrolysis of NTPs is used for the rowth of the chain. • Unlike DNA polymerase, RNA polymerase does not require primers. 11 12 2 3/6/2016 RNA polymerases • Templete of RNA polymerase is double strand DNA molecule. • Only one strand is trascribed for a gene. However, genes can be any of the strands. So transcription is realized on both of the strands. • Bacterial RNA polymerase has the simplest structure and is known in detail. • RNA polymerase of E.coli has 4 different subunits (polypeptide chain) which are β, β’,α, σ (sigma). • Subunits combine to make RNA polymerase haloenzyme which is the active form. • However, σ factor is not bind tightly, and it can easily remove the haloenzyme structure. The resulting structure after the removal of σ factor is called as RNA polymerase core enzyme (α,β,β’). • These principles are valid for RNA polymerase in all organisms. However, there differences in eucaryotes and procarotes: Procaryotes have one RNA polymerase Eucaryotes have 3 RNA polmerases (l, ll, lll) all of which involve in the trbscription of different genes. Role of sigma factor is the recognition of the correct site where RNA synthesis starts 13 14 Promoters • Poromoter is 35 bases upstream of the start of trancription sites. This is called as -35 position. promoter sequence is not transcribed. • There are two specific sequence (consensus sequences) on the promoter region which provide the recognition and binding of the enzyme to the promoter for the initiation of the transcription. These are: 5’-TTGACA-3’ is in the -35 position 5’-TATAAT-3’ is in the -10 position (TATA box or Pribnow box) • For the initiation of RNA synthesis, firstly, RNA polymerase must recognize the appropriate synthesis sites. These sites are known as promoter. 15 16 Termination of Transcription • -35 consensus sequences determined in E. Coli • All the sequences are recognized by sigma factor For the safety of protein synthesis, termination of the transcription is important as well as the initiation process. There are several ways for the termination of transcription: Hair pin formation: There are specific base sequences on DNA that help termination. • In procaryotes, there are sequence composed of around 40 bases. First bases of this sequence is complementer with last ones, but not the center. So these bases on RNA make pairs (form double strand) which has the shape of an hair pin. • 17 Generally there are Uridins after such sequences. So, this helps RNA to leave from DNA. 18 3 3/6/2016 Life of Messenger, Ribosomal and Transfer RNA • Most genes (mRNA) encodes proteins. And, some of them encodes rRNA and tRNA which are also involved in protein synthesis. Rho-ependent termination • There are special proteins for this type of termination. • In E.coli a proteins called as Rho makes the termination. • Life-spans of mRNAs in procaryotes are very short (a few minutes). At the end of this time, they are digested with ribonucleases. • tRNA and rRNA are stable due to the their folded structures. • mRNAs are not folded. 19 20 2) Translation Ribosomes • Ribosomes are the sites where triplets in mRNA are translated to amino acids in a specified order. Thus ribosomes are considered as translational apparatus. • Translation is the process by which proteins are synthesized using the genetic information transcribed on mRNA. • Translation process can be divided into 5 steps all of which require various molecules and factors. a) activation of amino acids b) initiation of polypeptide synthesis c) Elongation of polypeptide chain d) termination of polypeptide synthesis e) Mpdification on the synthesized chain • Ribosomes are not selective for any kind of translational processes. They translate any kind of genetic information that arrives to the ribosomes. • tRNA molecules are the adaptors that bring the correct amino acids with the proper codons in mRNA. 21 • Ribosomes are one of the the most complicated organels in the cell. 22 • Eucaryotes have 80S, procaryotes have 70S ribosomes. • Ribosomes are mainly composed of RNA and proteins. PROCARYOTES (70 S ribosome) EUCARYOTES (80S ribosome) • There are variety of proteins which make up 60% of ribosomes. Large Subunit (50S) Small Subunit (30S) Large Subunit (60S) Small Subunit (40S) 23S rRNA (2904 nucleotide) + 31 protein 16S rRNA (1541 nucleotide) + 21 protein 28S rRNA (4718 nucleotide) + 49 protein 18S rRNA (184 nucleotide) + 33 protein • Procaryotes have 52, eucaryotes have more than 82 different proteins. • Eventhough, the sizes and number of ribosomes in eucaryotes and procayotes are different, their main structures are similar. 5S rRNA (120 nucleotide) 5.8S rRNA (120 nucleotide) • Both types have one large and one small subunits. 23 S: Svedberg unit (10-13 sn 24 4 3/6/2016 Transfer RNA • Each tRNA carries different amino acids to the ribosomes. • Sedimentation coefficents are 4 S. • They are the smallest nucleic acids in the cells (73-93 nucleotide). • There is at least one tRNA to carry one amino acid. • Some amino acids can be carried with more than one tRNA (2 or 3). • 50-70% of the bases make internal pairings where molecule becomes double stranded. • Procaryotes have 60 and eucartotes have around 100-110 different tRNA. 25 • All tRNAs contain 3 unpaired nucleotides on the acceptor stems. • tRNAs contain some spesific nucleotides which are formed by chemical modification of normal nucleotides (pseudouridine, inosine, dehydrouridine, ribothymidine, methyl guanosine, methyl inosine) • These unpaired nucleotides are CCA in all tRNAs. • Amino acid binds to the 3’ OH ribose of A with covalent ester bond (COOH group of amino acid bind with 3’ OH group of ribose). tRNA has cloverleaf structure. It consists of 4 parts: • • • • 26 Anticodon arm Acceptor stem TψC arm D arm 27 28 a) Activation of amino acids • Anticodon arm contains triplet bases which are complementer to the codons in mRNA. These triplets are called as anticodons. • Enzyme amynoacyl transferase (amino acyl trna synthetase) provides matching the amino acids with proper tRNAs. • Anticodon arm is the most variable part of tRNAs. • Other parts of tRNA interacts both with ribosomes (mRNA and proteins) and translation proteins to activate the enzyme synthetase. 29 30 5 3/6/2016 • Recognition sites on tRNAs for some amino acids • Fistly, enzyme activates the amino acids by reacting them with ATP. And amino acyl-AMP is formed. Amnino asit + ATP aminoaçil-AMP + P-P • In the second step, activated amino acid is transferred to the tRNA. aaminoaçil-AMP + tRNA Aminoaçil TRNA + AMP 31 b) Initiation of Translation 32 • tRNA formyl transferase catalyses the addition of formyl group to methionine. • The translation always starts with methionine (AUG). It is formyl methionine in prpcaryotes (fmet). • The start codon is AUG which encodes methionine. • This modificatin is realized after the transfer of methionine to tRNA. • Fmet is encoded by the same codon with the methionine (AUG). Fmet is used at the beginning. • AUG is the only codon that encodes the methionine. • In procaryotes, initiation of protein synthesis is done on free 30S ribosome subunit. • During the initiation, 30S ribosome subunit, fmet tRNA, and initiation proteins IF1, IF2 and IF3 form initiation complex. 33 • Formyl group provides the attachment of methionine to the specific peptidyl site (P site) on 50 S ribosome subunit. 34 Shine Dalgarno sequence • In addition, it prevent the first a.a (methionine) to be added as an amino acid of synthesized polypeptide chain. • Formyl group is removed after the tRNA attaches to the initiation codon. Thus, the first a.a becomes methionine. • After the protein synthesis is completed, methionine is usualy removed from the protein by methionine aminopeptidase. • It is the sequence which is located around 5-8 bases upstream of the start codon. It is rich in purine bases. • 5’-AGGAGGU-3’ • The sequence is not translated. • It is the ribosimal binding site that assure the initiation of the translation from the correct codons. 35 36 6 3/6/2016 c) Elongation • Complementer of this sequence, 3’-UCCUCCA-5’, is in the 16S rRNA and mRNA and 16S rRNA bind with each other by these sequences. • So, intiation codon (AUG) finds its correct site. Once, ribosome is attached to the Shine –Dalgarno site, initiation codon can be found. • mRNA binds to 30 S subunit of ribosome. • tRNAs interact with two sites at 50S subunit • A-site: acceptor site • P-site: peptide site (where fmettRNA binds) • A-site: tTRNA carries the amino acid attaches (which is the second codon on mRNA) 37 38 • Formation of peptide bond between two amino acids is catalyzed by the enzyme peptidyl transferase (which is the component of 23 S region). • Enzyme breaks the ester bonds of the first amino acid on trna, and binds it with the amino group of the second a.a to form a peptide bond. • Factors named as EF-Tu, EF-Ts and EF-G are required for elongation of the chain. • EF-Tu provides the transfer and attachment of the amino acyl tRNA to A-site. • A GTP molecule is required for this. 39 40 Translocation • Unloaded tRNA moves to the exit site of the ribosome snd leaves. • Process of movement of trna from A-site to P-site is called as translocation. • For translocation, EF-G factor and a GTP are required. • During translocation ribosomes move (not mRNA). • At each movement process, ribosome moves 3 nucleotides by which it bind with a new codon at A-site. 41 42 7 3/6/2016 • Tek bir mRNA molekülü, birbirini takip eden çok sayıda ribozom tarafından polizom denilen bir kompleks oluşturarak translasyona uğrayabilir. • Polizomlar translasyonun hızını ve verimliliğini arttırır. • Her bir ribozomun aktivitesi, komşusu olan diğer ribozomdan bağımsızdır. • Böylece bir polizom kompleksindeki her bir ribozom birbirinden bağımsız tam polipeptitleri yapar. 43 44 c) Sentezin sonlanması (terminasyon) • Protein sentezinin terminasyonu, ribozom bir anlamsız (stop) kodona geldiği zaman gerçekleşir. • Stop kodonlar : UAA, UAG, UGA • Hiçbir tRNA stop kodonlarına bağlanmaz. • Salınma faktörü adında özel bir protein (RF faktörü) zincir sonlama işaretini tanır ve polipeptiti uçtaki tRNA’dan keserek oluşan ürünü salar. • Daha sonra ribozom alt ünitelere ayrılır. 45 8