Protein Synthesis. Presented by Dr. Mohammad Saadeh The requirements for the Pharmaceutical Biochemistry I Philadelphia University Faculty of pharmacy

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1 Protein Synthesis Presented by Dr. Mohammad Saadeh The requirements for the Pharmaceutical Biochemistry I Philadelphia University Faculty of pharmacy

2 STRUCTURE OF RNA RNA, adenine forms a base pair with uracil and guanine with cytosine There are three major types of RNA that participate in the process of protein synthesis: 1. ribosomal RNA (rrna) the complex structures that serve as the sites for protein synthesis. 2. transfer RNA (trna) brings specific amino acids to the ribosome to be assembled as proteins. 3. messenger RNA (mrna) mrna carries genetic information from the nuclear DNA to the cytosol, where it is used as the template for protein synthesis.

3 The Central Dogma of Protein Synthesis Transcription is the synthesis of RNA from DNA. Transcription occurs in the nucleus. mrna carries the information encoded in the gene (DNA) to the ribosome. RNA is copied from gene using base pairing, so is complimentary (but U not T). DNA unwinds in a section. RNA polymerase synthesizes the RNA strand.

4 Transcription of RNA from DNA Strand Involves four stages: Binding, Initiation, Elongation and Termination 1. The binding of RNA polymerase to a DNA promoter sequence - triggers the unwinding of double helix (binding site of RNA polymerase called DNA promoter). 2. RNA polymerase initiates the synthesis of an RNA chain. 3. Elongation: RNA polymerase moves along the DNA template, unwinding DNA (similar to helicase activity) and elongating RNA chain. 4. Termination: Terminators are genetic parts that usually occur at the end of a gene and cause transcription to stopand dependent upon the participation of a protein known as the ρ (rho) factor.

5 Transcription of RNA from DNA Strand Involves four stages: Binding, Initiation, Elongation and Termination

6 Action of antibiotics: Some antibiotics prevent bacterial cell growth by inhibiting RNA synthesis (inhibit transcription). For example, 1. rifampin: inhibits the initiation of transcription by binding to the β subunit of prokaryotic RNA polymerase (Figure 30.10). 2. Dactinomycin (actinomycin D) it is intercalating into the narrow groove of the DNA and interferes with the movement of RNA polymerase along the DNA. Figure Inactivation of prokaryotic RNA polymerase by rifampin.

7 Eukaryotic mrna processing: -methylguanosine cap at 5ˋ Increase mrna stability and position mrna on the ribosome for translation. Poly (A) tail mrna stability and recognised by specific proteins involved in exporting mrna into cytoplasm.

8 prokaryotic mrna processing: The Shine-Dalgarno (SD) sequence is a ribosomal binding site in prokaryotic messenger RNA helps to initiate protein synthesis by aligning the ribosome with the start codon, generally located around 8 bases upstream of the start codon AUG see figure Figure 31.10: Complementary binding between prokaryotic mrna Shine-Dalgarno sequence and 16S rrna.

9 Protein Synthesis: The Genetic Code: Each 3 consecutive bases on RNA is a coded word (the codon) (triplets) that specifies an amino acid. Leads to the open reading frame (ORF) The genetic code consists of 64 codons, (4x4x4 resulting from a combination of 4 possible ribonucleotides), but only 61 code amino acids. One codon, AUG, codes for methionine, and is also the start signal for the initiation of translation. Three codons act as signal terminators of translation called stop codon or non sensecodon(uaa, UAG, UGA) by binding release factors (RF), which cause the ribosomal subunits to disassociate, releasing the amino acid sequence.

10 Protein Synthesis Figure 31.2 Use of the genetic code table to translate the codon AUG.

11 Protein Synthesis III. COMPONENTS REQUIRED FOR TRANSLATION 1. Transfer RNA brings specific amino acids to the ribosome to be assembled as proteins. Amino acid attachment site: Anticodon contains 3 bases that are specific for the attached amino acid base pairs to the complementary triplet code on mrna (the codon) (Figure 31.6). [Note: When a trna has a covalently attached amino acid, it is said to be charged; when it does not, it is said to be uncharged.] Anticodon: Each trna molecule also contains a three-base nucleotide sequence, the anticodon, that pairs with a specific codon on the mrna (see Figure 31.6). Figure 31.6 Complementary, antiparallel binding of the anticodon for methionyl-trna (CAU) to the mrna codon for methionine (AUG), the initiation codon for translation.

12 Protein Synthesis III. COMPONENTS REQUIRED FOR TRANSLATION 2. Aminoacyl-tRNA synthetases a) Aminoacyl-tRNA synthetases is required for attachment of amino acids to the 3' end of a trnas. b) Aminoacyl-tRNA synthetases catalyze the covalent attachment of the carboxyl group of an amino acid to the 3'-end of its corresponding (cognate) trna. (Need energy, the enzyme splits ATP to AMP + PPi). c) The Aminoacyl-tRNA synthetases have a proofreading or editing activity that can remove amino acids from the enzyme or the trna molecule. Figure 31.7 Attachment of a specific amino acid to its corresponding trna by aminoacyl-trna synthetase (E).

13 Protein Synthesis III. COMPONENTS REQUIRED FOR TRANSLATION 3. Messenger RNA carries genetic information from the nuclear DNA to the cytosol D. Functionally competent ribosomes A ribosome consists of rrna with proteins. Ribosomes consist of a large subunit and a small subunit. mrna binds to the small subunit. whose relative sizes are given in terms of their sedimentation coefficients, or S (Svedberg) values.

14 Protein Synthesis D. ribosomal RNA (rrna) the complex structures that serve as the sites for protein synthesis. rrna has three binding sites for trna molecules: the A, P, and E sites, A-site (aminoacyl t RNA site) Holds trna carrying next amino acid. P-site (peptidyl trna site) carrying growing polypeptide chain. E- site (exite site) empty trna leaves ribosome from exit site

15 Protein Synthesis IV. CODON RECOGNITION BY trna A. Antiparallel binding between codon and anticodon antiparallel binding of the trna anticodon to the mrna codon. the mrna codon is read 5' 3' by an anticodon pairing in the flipped (3' 5') orientation (Figure 31.9). B. Wobble hypothesis trnas can recognize A Multiple (more one) codons can code for a single amino acid is described by the wobble hypothesis. Wobble hypothesis: (figure 31.9) 1. Nontraditional base-pairing is possible at third position of codon (3ˋ position). 2. Traditional base pairing in first and second positions of codon.

16 Protein Synthesis V. STEPS IN PROTEIN The process of translation is divided into three separate steps: Initiation, elongation, and 3 termination. (Translation need energy, GTP) A. Initiation: (need GTP) see figure 1. Small subunits separated from large subunit of rrna by IF 1, IF s rrna Small subunits rrna bind with mrna by shine dalgarno sequence. 3. IF2-GTP bring charged trna with methionine. 4. Hydrolysis GTP to GDP and all IF are released. B. Elongation: (need GTP) see figure 1. EF-Tu-GTP and EF-Ts bring charged trna with new amino acid at A site then Hydrolysis GTP to GDP and EF-Tu are released. 2. peptidyltransferase 3. Proofreading by aminoacyl-trna synthetases (bring right amino acid). 4. movement of trna and mrna through the ribosome by hydrolysis of EF-G-GTP to EF-G-GDP + Pi.

17 Protein Synthesis V. STEPS IN PROTEIN The process of translation is divided into three separate steps: Initiation, elongation, and 3 termination. (Translation need energy, GTP) A. Termination: (need GTP) see figure 1. Release factor RF1, RF2 and RF3-GTP bind to stop or termination codon. 2. RF3-GTP hydrolysis to RF3-GDP +Pi then rrna, trna and polypeptide are released.

18 Protein Synthesis

19 Protein Synthesis

20 The inhibitors of prokaryotic translation 1. streptomycin inhibit the Binding of formylmethionyl trna to 30S subunit of ribosomes. 2. Tetracycline prevents synthesis of polypeptide or elongation by Preventing binding of aminoacyl trna to the A site. 3. Puromycin causes inhibition of elongation and premature chain termination in both prokaryotes and eukaryotes. 4. Chloramphenicol inhibits the peptidyltranferase activity of 50S ribosomal subunit. 5. Erythromycin bind irreversibly to a site 50S so inhibiting translocation. The inhibitors of eukaryote translation 1. Diphtheria toxin inhibits EF2 and inhibits translation in eukaryotes by preventing translocation.

21 Q. From double-stranded DNA contains the sequence: (very important) (a) What is the base sequence of the mrna that can be transcribed from this strand? (b) What amino acid sequence could be coded by the mrna base sequence? Answer: 3ˋ 5ˋ N-terminal C-terminal

22 Mutations Mutations: changes to the DNA sequence. Consequences of altering the nucleotide sequence: Changing a single nucleotide base on the mrna chain (a point mutation ) can lead to any one of three results (Figure 31.3): 1. Silent mutation: The codon containing the changed base may code for the same amino acid. For example, if the serine codon UCA is given a different third base, U, to become UCU, it still codes for serine. This is termed a silent mutation. 2. Missense mutation: The codon containing the changed base may code for a different amino acid. For example, if the serine codon UCA is given a different first base,c, to become CCA, it will code for a different amino acid, in this case, proline. The substitution of an incorrect amino acid is called a missense mutation. 3. Nonsense mutation: The codon containing the changed base may become a termination codon. For example, if the serine codon UCA is given a different second base,a, to become UAA, the new codon causes termination of translation at that point, and the production of a shortened (truncated) protein. The creation of a termination codon at an inappropriate place is called a nonsense mutation.

23 Mutations Consequences of altering the nucleotide sequence: Figure 31.3 Possible effects of changing a single nucleotide base in the coding region of an mrna chain. A=adenine; C=cytosine; U=uracil.

24 Mutations Consequences of altering the nucleotide sequence: 4. Frame-shift mutations: one or two nucleotides are either deleted from or added to the coding region of a message sequence. different amino acid sequence, or a truncated product due to the creation of a termination codon (Figure 31.5). Figure 31.5 Frame-shift mutations as a result of addition or deletion of a base can cause an alteration in the reading frame of mrna.

25 Recombinant DNA Technology Presented by Dr. Mohammad Saadeh The requirements for the Pharmaceutical Biochemistry I Philadelphia University Faculty of pharmacy

26 Gene cloning What is gene cloning? A DNA fragment carrying the gene is inserted into the plasmid molecules (vector) to produce a recombinant DNA molecules. The technology of how this is done is known as recombinant DNA technology.

27 Restriction enzymes Restriction enzymes or restriction endonuclease is an enzyme that cuts DNA at a specific sequence of nucleotides. 1. Some restriction enzymes such as EcoRI produces "sticky" ends. 2. Some restriction enzyme such as SmaI cleavage produces "blunt" ends.

28 Recombinant DNA Technology Steps of recombinant DNA technology: A. Isolation and purification of both DNA and vector (plasmid). B. Why gene cloned? 1. Recombinant human insulin. 2. Recombinant hepatitis B vaccine. 3. Recombinant human growth hormone 4. Insect-resistant crops 5. Diagnosis of infection with HIV. and joined by DNA ligase and inactivated lacz gene. 5. Identification bacteria that contain recombinant DNA

29 1. DNA fragment can be inserted for cloning in: a) Plasmid b)chromosomal DNA c) foreign DNA d) all of these

30 2. which of the following enzyme is used to cut DNA in rdna technology: a) restriction enzyme b) nuclease enzyme c) DNA POL I D) DNA ligase

31 3. Identification bacteria that contain recombinant DNA: a)activation chromosomal DNA. b) inhibition lacz gene or antibiotic resistance gene c) inhibit transcription process d) all of these

32 4. The termination site for transcription is recognized by: A) α Subunit o RNA polymerase B) β Subunit of RNA polymerase C) Sigma factor D) p(rho) factor

33 5. Anticodons are presented on: A) mrna B) trna C) rrna D) None of these

34 6. Translocation of the newly formed peptidyl trna at the A site into the empty P site involves A) EF-G, GTP B) EF-Tu, GTP C) EF-Ts, GDP D) Peptidyl transferase, GTP 7. If the codon UCA on mrna changes into UAA as a result of a base substitution in DNA, it will result in A) Silent mutation B) Acceptable mis-sense mutation C) Nonsense mutation D) Frameshift mutation

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