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Translation of RNA The Genetic Code and Protein Metabolism The Process of Protein Synthesis The Three Stages of Protein Synthesis Post-translational Processing of Protein Regulation of Protein Synthesis 1 Characteristics of protein synthesis Occurs on a ribosome - a complex of ribosomal RNA and proteins. Made of two subunits • large - three rRNA and about 49 proteins • small - one rRNA and about 33 proteins • Together they provide a platform for synthesis You can think of a ribosome like a tape player, where the tape is mRNA. 2 Ribosomes Two ribosomal subunits join to form a polysome. large subunit small subunit synthesis platform 3 Where protein synthesis begins It has been verified that protein synthesis begins at the amino terminus. R' R growing chain N N H C H COO - R O C H C + H2N C H COO - R' growing chain N N H N H C H COO 4 Role of tRNA Transfer RNA • Serves as an adapter molecule to translate the 4 letter language of mRNA into 20 amino acids. • Binds to a single amino acid at one end. • Serves to bring the proper amino acid to the right spot on the mRNAribosome complex. 5 tRNA HO- C Site of amino acid attachment G A U G U C G G U Three base anticodon site A C G G C A C A C G G U G C G C G U C G G C U U G C A G G C C U U A G U C G C C C G G C G C U G C G U A C G C G C G A U G U G C G C G Point of attachment to mRNA 6 Attachment of amino acids Amino acids are linked to tRNAs by aminoacyl-tRNA synthetases. These enzymes are able to recognize both the correct tRNA and amino acid. amino acid + ATP aminoacyl adenylate + PPi aminoacyl adenylate + tRNA PPi + H2O aminoacyl-tRNA + AMP 2Pi 7 Attachment of an amino acid amino acyl group G A R O CH C HO- U G C G U A C G G C ester bond A C + NH3 U G O C A C G G U G C G C G U C G G C U U G C A G G C C U C C G G U U A G C G C C G C U G C G U A C G C G C G U A G G U G C G C 8 Genetic code, mRNA and protein amino acids Important terms Triplet A set of three nucleotide bases on mRNA for one amino acid. Nonoverlapping A set of three adjacent bases are treated as a complete group - codon. No punctuation. There are no intervening bases between triplets. 9 Genetic code, mRNA and protein amino acids Important terms Degenerate A single amino acid may have more than one triplet code. There is usually a sequential relationship between these codes. Universal The same genetic code is used by all organisms except mitochondria and some algae. 10 Amino acid codons alanine GCA, GCC, GCG GCU, AGA, AGG arginine AGA, AGG, CGA CGC, CGG, CGU asparagine AAC, AAU aspartate GAC, GAU cysteine UGC, UGU glutamate GAA, GAG glutamine CAA, CAG glycine GAA, GCC, GGG GGU histidine CAC, CAU isoleucine AUA, AUC, AUU leucine CUA, CUC, CUG CUU, UUA, UUG lysine AAA, AAG methionine AUG phenylalanine UUC, UUU proline CCA, CCC CCG, CCU serine UCA, UCC UCG, UCU AGC, AGU threonine ACA, ACC ACG, ACU tryptophan UGG tyrosine UCA, UCU valine GUA, GUC GUG, GUU 11 Three stages of protein synthesis Production of a protein requires several players. aminoacyl - tRNA source of amino acids mRNA the information source for construction of the protein sequence Ribosomes platform for synthesis - rRNA and protein. 12 Steps in protein synthesis Step One - Initiation A special protein is required to bring the ribosome parts and mRNA together. It recognizes the initiation (START) codon (AUG). Once formed, the ribosome complex will - hold the mRNA in place. - provide binding sites for the growing protein and incoming amino acids. 13 Steps in protein synthesis Step Two - Chain elongation Amino acid is added sequentially to the peptide chain. An enzyme, peptidyl transferase, is used to move the ribosome down the mRNA strand - translocation. 14 Steps in protein synthesis Step Three - Termination When one of three codons (UAA, UAG or UGA) is encountered, there is no tRNA that matches. Protein synthesis stops. A releasing factor is attracted to the site. This results in the growing protein being released from the ribosome. The ribosome complex then falls apart into the original subcomplexes. 15 Protein synthesis Protein synthesis and energy The energy requirements for synthesis are quite high. • Two anhydride bonds in ATP are cleaved on activation of each amino acid and synthesis of an aminoacyl-tRNA. • One GTP is required for entry of each amino acid into the ribosomal unit. • One GTP is required during each translocation step. 17 Post-translational processing of proteins Protein synthesis establishes the primary structure for a protein. Additional processing is required to convert it to it’s biologically active form. This may include: folding chemical modification attachment of other groups 18 Protein folding Results from interaction of side chains. Proteins called chaperones act as catalysts to guide this process. Possible side chain interactions: • Similar solubilities • Ionic attractions • Attraction between + and - side chains • Covalent bonding 19 Protein folding Disulfide Crosslink Hydrophobic interaction -S-S- -COO- + H3N - Salt bridge Hydrogen bonding 20 Protein folding Side chain interactions Help maintain specific structure. Oxidation of cystine - crosslink formation. O || HO-C-CH-CH2-SH | NH2 oxidation [O] O || HS-CH2-CH-C-OH | NH2 covalent disulfide bond O O || || HO-C-CH-CH2-S - S-CH2-CH-C-OH +H2O | | NH2 NH2 21 Protein folding An example of how a S-S crosslink can affect folding 20 glycines - cysteine 40 glycines crosslink 20 glycines - cysteine - helix structure 22 Biochemical modification Proteolytic cleavage About half of all proteins require the removal of their N-terminus amino acid to become active. In addition, many enzymes are produced in an inactive form - zymogens. They require that one or several specific bonds be cleaved to produce the active form. 23 Biochemical modification Amino acid modification Phosphorylation Serine, threonine and tyrosine residues may be modified by transfer of a phosphoryl group. Hydroxylation Proline and lysine may be converted to hydroxyproline or hydroxylysine. 24 Biochemical modification Attachment of carbohydrates This process is used for the production of glycoproteins. Attachment of prosthetic groups. The addition of small organic, inorganic or organometallic groups - heme, FAD, biotin, ... 25 Protein targeting Most proteins are synthesized by the ribosomes in the cytoplasm. However, they may be required in other cellular regions and organelles Protein targeting deals with the process of sorting out and moving proteins to where they are needed. 26 Protein targeting • In general, proteins that must be transported are produced with an extra sequence of 15-36 amino acids - at the amino terminus. • The sequence marks it for transport. • The sequence is removed by hydrolysis upon arrival. • Proteins sent to the nucleus use an internal sequence that is not cleaved. 27 Protein degradation After a protein has served it’s purpose or becomes damaged, it is marked for destruction. The turnover rate varies by protein. Protein Rat liver RNA polymerase I Rat liver cytochrome c Human hemoglobin half-life 1.5 min 150 min 100 days 28 Protein degradation Ubiquitin pathway Important route for protein labeling and degradation in eukaryotic cells. Ubiquitin • A small protein with 76 amino acid residues. • It is highly conserved. • Yeast and human ubiquitin differ at only 3 of the 76 residues. 29 Ubiquitin 30 Ubiquitin pathway • Ubiquitin is covalently attached via a peptide bond to lysine residues. • Several ubiquitin molecules are often attached to a single protein. • Degradation then occurs via proteolytic action. O ubiquitin C N N Lys protein H C 31 Regulation of protein synthesis • A typical bacterial cell has about 4000 genes in its DNA genome. • The human genome has an estimated 100,000 to 150,000 • Only a small fraction of these genes is used by a cell at any given time, if at all. • The amount of each protein generated must be carefully regulated to account for the needs of the cell. 32 Regulation of gene expression There are many steps that can be regulated. • transcription • post-transcriptional processing • mRNA degradation • protein synthesis • post-translational processing • protein degradation Most gene expression is controlled at the level of transcription initiation. 33 Regulation of gene expression Nucleotides mRNA degradation protein degradation Amino acids posttranslational processing transcription DNA posttranscriptional processing primary transcript mature RNA translation inactive protein Active protein 34 Regulation of gene expression Two fundamental types of gene expression. Expression of constitutive genes. Continuous transcription which produces a constant level of certain proteins. Expression of inducible or repressible genes. Genes that can be activated (induced) or deactivated (repressed). This allows for varied levels of RNA. They are regulated by RNA polymers and molecular signals. 35 Principles of regulating gene expression In many prokaryotic cells, genes for proteins of related function are clustered in units. The components in these units are: • Structural genes - the gene which is to be transcribed & translated. • Promoter region - responsible for RNA polymerase binding to the initiation site. • Binding site for inducers. • Binding site for repressors - called operators. All structural genes are regulated by nucleotide sequences upstream from the start site (+1). 36 Principles of regulating gene expression RNA polymerase • The key participant in transcription. • It initiates transcription by binding to a DNA promoter region. • The sequences in each promoter region determines the affinity for its binding. • For inducible or repressible genes, other levels of control are superimposed regulatory proteins and other signals. 37 Principles of regulating gene expression Eukaryotic gene regulation is a more complicated process. • Complex sets of regulatory elements are present in promoter regions. • Three classes of RNA polymerase using different modes of regulation are present. • The DNA is much more complex in both size and structure. 38 Principles of regulating gene expression RNA polymerase activity is mediated by regulatory proteins of two major types. Activators. Bind to promoter regions and assist the binding of RNA polymerase to the adjacent promoter. Repressors. Bind to specific base sequences in the promoter region and prevent RNA polymerase in gaining access. 39 Principles of regulating gene expression Most regulatory process can be classified into one of four mechanistic types. Positive regulation Transcribes until a molecular signal is sent. Transcribes after a signal is sent. Negative regulation Blocks transcription until a signal is sent. Stops transcription when the signal is sent. 40 Positive regulation activator RNA polymerase molecular signal Binding of molecular signal causes dissociation of activator from DNA Binding of molecular signal causes strong binding of of activator to DNA 41 Negative regulation operator Binding of the molecular recognition signal causes dissociation of the operator from the DNA Binding of the molecular recognition signal causes stronger binding of the operator to DNA 42 Principles of regulating gene expression Regulatory proteins have common, discrete binding domains. • 20-100 amino acid residues. • They bind because the domain is an exact fit for the outer edge of the DNA helix. • Held together by hydrogen bonding that is not disruptive to DNA. • Lysine, arginine, glutamate, asparagine and glutamine form the hydrogen bonds. 43 Classes of regulatory proteins Helix-turn-helix motif About 20 amino acid residues, in two helical regions and a turn. 44 Classes of regulatory proteins Zinc finger motif Only found in eukaryotic regulatory proteins. Cys His Cys Zn His 45 Classes of regulatory proteins Leucine zipper motif Features an -helix region of approximately 30 residues. Leucine occurs as every seventh one. This allows two molecules of the protein to form a zipper like region. 46