PHBC522 Biochemistry I Winter 2024 Lecture Notes PDF

PHBC522 Biochemistry I Winter 2024 Faculty for Pharmacy and Biotechnology Lecture 6-Gene Expression & Protein Synthesis Prof. Sahar Mohamed  By the end of the lecture, you should be able to Know the following: Know all types and structure of RNA Mention the Regulation of...

PHBC522 Biochemistry I Winter 2024 Faculty for Pharmacy and Biotechnology Lecture 6-Gene Expression & Protein Synthesis Prof. Sahar Mohamed  By the end of the lecture, you should be able to Know the following: Know all types and structure of RNA Mention the Regulation of Gene transcription by chromatin remodeling. Describe the promoter region. Know role of enhancers and inhibitors in gene transcription. Illustrate Post transcriptional modification of mRNA Recognize Post transcriptional regulation of Gene expression by RNA interference. Mention Epigenetic regulation Describe RNA Editing. Identify the genetic code and translate it to amino acid. Discuss Protein synthesis steps.  RNA Structure and Synthesis Genetic Key of an organism is contained in the sequence of deoxyribonucleotides in DNA. This information is transferred from DNA through the ribonucleic acid in RNA. Transcription: It is the copying process which uses one of the two DNA strands as a template to synthesize mRNA. mRNA are then translated into sequence of amino acids & polypeptide chains. There are 3 types of RNA: The 3 types differ in terms of size, function and special structure modification. 1-Ribosomal RNA ( rRNA,. 2-Messenger RNA (mRNA) 3-Transfer RNA (tRNA). NB. Small nuclear (Sn) RNA : In  1-Ribosomal RNA(rRNA) They are large complex of proteins & rRNAs. It comprises 80% of RNA in cell. Ribosomes are present in the cytosol or bound to endoplasmic reticulum. They are factories for protein synthesis. rRNA are expressed as (sved berg values)S or sedimentation coefficients (particle moves to the bottom of the test tube under centrifugal force) S depend on shape and molecular mass. The complete ribosome have 3 binding sites each of which extends over both subunits. A site: binds an incoming aminoacyl-tRNA. P site: binds the t-RNA carrying the chain of amino acid which has already been synthesized. E site: is occupied by the empty t-RNA which is about to exit the ribosome. 2-Messenger RNA (mRNA) mRNA comprises only 5% of the RNA in the cell. The mRNA carries genetic information from the nuclear DNA to the cytosol, where it is used as the template for protein synthesis. If the mRNA carries information from more than one gene it is called polycistronic and this is characteristic to prokaryotes. If the m RNA carries information from one gene it is called monocistronic and it is characteristic to eukaryotes. mRNA has a protein coding region (exons), to be translated and untranslated non-coding regions (introns). mRNA include a long sequence of adenine nucleotides (a poyl A tail ) on the 3’end of the RNA 3-Transfer RNA (tRNA). It comprises 15% of total RNA in cell. The translation of the final mRNA transcript into protein requires specific adapters that can recognize a specific sequence on the m RNA and ‘translate’ this into one specific amino acid in the protein. Since there are 20 proteinogenic amino acids, there must be 20 tRNA adapters. These are the tRNA molecules, each having the characteristic 3-loop structure. At Each kind of tRNA has a sequence of 3 the 3’-end, the amino acid is attached, unpaired nucleotides (anticodon) forming aminoacyl-tRNA. which recognize the complementary triplet of nucleotides (codon) in a messenger RNA (mRNA) molecule following the rules of base pairing. The reading of codons in mRNA (5' -> 3') requires that the anticodons bind in the opposite direction (antiparallel binding). Anticodon (tRNA) 3' CUC 5‘ Codon (mRNA) 5' GAG RNA Transcription(Gene expression) It is the copying process which uses one of the DNA strands as a template to synthesize m RNA. Coding strand Template strand Coding (sense) strand: same as mRNA. It is not transcribed Template (antisense) strand: being transcribed, complementary to mRNA  Q1: If the sequence of the coding strand is 5′-TACGTACGTACGTA-3′, what is the sequence of the transcribed RNA? Sol: The answer is 5′- UACGUACGUACGUA-3′. RNA has the same sequence as the coding strand, except in place of thymine it has uracil.  RNA Transcription(Gene expression) RNA polymerase Recognize the promoter region on DNA & initiate RNA synthesis. There are 3 distinct classes of RNA polymerase in the nucleus of eukaryotic cells (I, II, III). 1- RNA polymerase I : synthesizes the large ribosomal RNAs. 2- RNA polymerase II: synthesizes the messenger RNAs and some small nuclear RNAs(snRNAs). 3-RNA polymerase III: It produces the tRNA and small ribosomal RNA and some snRNAs RNA polymerase reads from 3’to 5’ and synthesize from 5’to 3’.  Pre- transcriptional Regulation 1- Chromatin Remodeling DNA is attached to histones , so it is very difficult to be transcribed, so the genes to be transcribed are found in a relaxed form of chromatin called euchromatin. The interconversion between active and inactive forms of Chromatin is called chromatin remodeling. It is influenced by DNA methylation and histone acetylation. DNA methylation takes place on permanent inactive genes which are not going to be transcribed. This is done through methylation of the cytosine on the DNA and modification of the present chromosome. A DNA methyl transferase can methylate cytosines within the promoter sequence , the gene itself and the upstream Histone activator binding sites. acetylation This modification disrupts binding of transcription factors and activators. This could increase or decrease the level of transcription depending on whether the methylation inactive a positive or negative regulatory element. DNA Histone acetylation : The basic amino acids in histone is acetylated by the enzyme histone acetyl methylat transferase (HATs) to make chromatin more looser ion and DNA becomes more accessible to transcription. Histone deacetylase (HDACs) enzyme can remove the acetyl group from the histone. 2-Epigenetic Gene Regulation The inheritance of gene expression pattern through cell division , in the absence of both mutation and the initiating signal is called epigenetic regulation. The figure below show a DNA sequence in which 2 cytosines are present on each strand, one methylated and the other not. This pattern is maintained through cell division, because during replication, a maintenance methylase recognizes the hemimethylated DNA, and adds methyl group to the unmethylated cytosine. The completely unmethylated sequence is not recognized by this enzyme and so remains unmethylated. Thus both daughter DNA ends up with the same pattern of methylation as parent. Unmethylate d cytosine 3-Core Promoter: They are the immediate neighboring regions around the transcription start site (TSS) that serve as the docking site for the transcriptional machinery and pre-initiation complex(PIC) It has the TATA motif ( referred to as TATA box) 30 bp upstream of an initiator sequence which contains the TSS. 4- Proximal Promotor : They are sequences right upstream of the core promoters and are associated with the initiation of transcription and PIC assembly. They are CAAT box & GC box They are thought to have a similar function like TATA box NB. Both promoters are called cis-acting elements 5-Transcription Factors : They are proteins that recognize a specific DNA motif to bind on a regulatory region and regulate the transcription rate of the gene associated with the regulatory region sequences serve as binding sites They are synthesized in cytosol and diffuse to nucleus , so they are called trans –acting elements. The concentration of the transcription factors and the availability of synergistic or competing transcription factors will also affect the transcription rate. 6- Enhancers They are distal regulatory elements in the human genome. They are similar to proximal promoters and they contain binding sites for the same transcriptional activators and they enhance the gene expression. Their activity is independent of their concentration and their distance to the promoter they interact with. Enhancers can act upon their target genes over several kilobases away. Enhancers achieve this by looping the DNA and coming into contact with their target genes. 7- Silencers : Are similar to enhancers, but their effect is opposite and results in decreasing the level of transcription. They contain binding sites for repressive transcription factors. Repressive transcription factors can either block the binding of an activator, directly compete for the same binding site or induce a repressive chromatin 8- Insulators: They are certain motifs on the gene that modify its structure defined by CTCF-binding factor and limit the effect of other regulatory elements for eg.They can block enhancer –promoter communication, they stop the enhancer from binding to the promoter.  Post transcriptional modification of mRNA 1 5’ capping: Cap is 7 – methyl guanosine attached to 5’ terminal end of the mRNA, forming an unusual 5’ ↔ 5’ triphosphate linkage. Methylation of guanine occurs in cytosol after capping. The cap helps stabilize mRNA & permits initiation of translation. Also, capping protects the RNA from being degraded by enzymes that degrade RNA from the 5′ end. 2 Addition of a poly(A) tail. This is a stretch of 40 – 200 adenine (A) nucleotides attached to the 3’end. It is added after transcription. Poly A tail stabilizes the mRNAs & facilitate their exit from the nucleus. Then it is shortened gradually. 3 Removal of introns present in the pre-mRNA and splicing of the remaining exons. Nucleus RNA splicing (splicisoming) https://digfir-published.macmillanusa.com/pol2e/asset/img_ch10/ Post Transcriptional Regulation of mRNA 1- RNA Editing Editing can change the sequence of mRNA after it has been transcribed. Thus the protein produced upon translation is different from that predicted from the gene sequence. During editing individual bases are either inserted , deleted or changed. Mechanisms of Editing: Site-Specific Deamination of adenine or cytosine: A specific targeted cytosine residue within mRNA is converted into uracil by deamination. It is specific for certain tissues or cell types in a regulated manner. E.g. Apoprotein –B –Gene : This gene has several exons with one of it CAA codon, which is targeted for editing (C U). CAA glutamine UAA stop codon Both 2 forms are involved in lipid metabolism. Each form is expressed in different tissue and has different function.  Post Transcriptional Regulation of mRNA 2-Micro RNA (miRNA): They are small non-coding RNA transcribed by RNA polymerase II and hairpinned into primary miRNA(Pri-miRNA) and processed in nuclei by an enzyme called Drosha. to become precursor miRNA (Pre- miRNA). Then the pre-miRNA is transferred from nucleus to cytoplasm via protein Exportin -5 transport mechanism. Then it is digested by a double stranded RNA specific ribonuclease called Dicer to obtain mature single stranded miRNA consisting of 20-22 single stranded ribonucleotides. The miRNA combine with RNA- induced silencing complex (RISC) & bind to mRNA which result in complete or incomplete inhibition of translation. Post Transcriptional Regulation of mRNA 3- Small Interfering RNA (siRNA): The double stranded RNA is cleaved by an RNA endonuclease enzyme called Dicer, which makes the short siRNA. After that the siRNA leaves the nucleus and enter the cell , it binds to RNA-induced silencing complex (RISC). Then siRNA unwound to be a single strand. The siRNA binds to its target mRNA and cleave the mRNA. The mRNA is recognized abnormal and degraded. Thus translation is inhibited.  The genetic code The base sequence in mRNA must encode the amino acid sequence of the protein. Work from Marshal Nirenberg, Francis Crick, Sydney Brenner, led to the establishment of the genetic code in 1961. Genetic codons are usually presented in the messenger RNA language. Characteristics of Genetic code: - Three nucleotides encode an amino acid; two nucleotides would only allow 42 = 16 different residues to be coded. 1- Redundancy:- Since a three letter code of four bases has 43 = 64 different ‘words’, the code is degenerate (more than one genetic code per amino acid). 2- Non-overlapping and commaless: - The genetic code is ABCDEF  ABC /DEF and has no punctuation (i.e. It is read continuously from start to end). 3- Universal : The specificity of the genetic code has been conserved from very early stage of evolution with only slight differences in the manner in which the code is translated. 4- Specific : A specific codon always codes for the same amino acid - The number of codons for each amino acid correlates with its frequency of occurrence in proteins. Leucine(L) ,Arginine(AR), and Serine have 6 codons each, while Tryptophan (W) and Methionine (M)only have one. - Specific codons for start and endpoint of translation exist. - The first and second base of a codon are often more critical than the third. In several cases (L, V, S, P, T, A, R, G), the first two bases already specify the amino acid. In fact, base-pairing in the third position is often less specific (Wobble hypothesis). The genetic code Point mutations Changing a single nucleotide base on the mRNA chain can lead to any of the three results: 1- Silent mutation : The codon containing the changed base may code for the same amino acid. 2- Missense mutation: The codon containing the changed base may code for different amino acid. 3-Nonsense mutation: The codon containing the changed base may code for a termination codon. 4-Other mutations: which can alter the amount or structure of the protein produced by  Translation of the Genetic Code The pathway of protein synthesis is called translation because the language of the nucleotide sequence on the mRNA is translated to an amino acid sequence. Components required for Translation: 1- Amino acids. 2- Transfer RNA(t-RNA). 3- Aminoacyl-tRNA synthetases. 4- Messenger RNA 5- Ribosomes. 6- Protein Factors( Initiation factors (IF), Elongation factors (EF) and releasing factors (RF) 7- ATP and GTP as sources of energy.  Aminoacyl-tRNA synthetases. It is a family of enzymes. Each member of this family recognizes both a specific amino acid and the t-RNA that corresponds to that amino acid. Role of aminoacyl-tRNA synthetases: 1- Activation of amino gp by ATP which is cleaved to AMP and PPi by pyrophosphatase. PPi drives the reaction forward. 2- Covalent attachment of an amino acid between the carboxyl group of the amino acid and the 3’ of its corresponding tRNA. NB. Charged tRNA: tRNA covalently attached to an activated amino acid.  Sites on the ribosomes The ribosome has 3 binding sites A,P and E for the tRNA molecules. The 3 binding sites extend over both subunits of ribosome. A site binds the new upcoming tRNA according to the codon of mRNA on this site. P site binds the tRNA which carries the chain of amino acids that has already been synthesized. E site is occupied by the empty tRNA which is about to exit the ribosome.  Protein Synthesis 1- Initiation  - Assembly of the components of the translation system. -It requires to select the mRNA for translation by ribosomes.  -The 30 S ribosome binds to the mRNA. -This is done by the aid of initiation factor(IF)which recognizes the initiating codon on mRNA. - The initiating codon always starts with methionine. In prokaryotes, a formyl gp is added to methionine by enzyme transformylase while in eukaryotes the methionine is not formylated- (in both euk & prok the N-terminal methionine is usually removed before the translated protein is released from ribosome). - Initiation factors will be released when the Protein Synthesis Protein Synthesis  Protein Synthesis 2. Elongation An aminoacyl-tRNA (a tRNA covalently attached to the amino acid which is corresponding to the codon on mRNA )arrives at the A site associated with: – an elongation factor (called EF-Tu in bacteria) – GTP (the source of the needed energy) The preceding amino acid (Met at the start of translation) is transferred from the t-RNA on P site to the amino acid formed on A site and it is covalently linked to the amino acid with a peptide bond. The initiator tRNA is released from the P site to move to the E site. The ribosome moves one codon downstream. This shifts the more recently-arrived tRNA, with its attached peptide, to the P site and opens the A site for the arrival of a new aminoacyl-tRNA. This last step is promoted by another protein elongation factor (named EF-G) and the energy of another molecule of GTP.  Protein Synthesis 3. Termination The end of translation occurs when the ribosome reaches one or more STOP codons (UAA, UAG, UGA). (The nucleotides from this point to the poly(A) tail make up the 3'-untranslated region [3'- UTR] of the mRNA.) There are no tRNA molecules with anticodons for STOP codons. However, a protein release factor (RF) recognizes these codons when they arrive at the A site. Binding of this protein releases the polypeptide from the ribosome. The ribosome splits into its subunits, which can later be reassembled for another round of protein synthesis.  References Lippincott’sIllustrated Reviews:Biochemistry by Richard A. Harvey& Pamela C. Champe. Stryer Biochemistry by L. Stryer, Freeman & Company New York …. Harper’s Biochemistry by R.K. Murray, D.K. Granner, P.A. Mayes & V.W. Rodwell. Biochemistry. Appleton & Lange, New York,Connecticut, California. Molecular biology of the Gene; Watson, Baker,Bell,Gann,levine,losick;Pearson Sixth Edition