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INTRODUCTION • The translation of the mRNA codons into amino acid sequences leads to the synthesis of proteins • A variety of cellular components play important roles in translation – These include proteins, RNAs and small molecules • In this chapter we will discuss the current state of knowledge regarding the molecular features of mRNA translation Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display THE GENETIC BASIS FOR PROTEIN SYNTHESIS • Proteins are the active participants in cell structure and function • Genes that encode polypeptides are termed structural genes – These are transcribed into messenger RNA (mRNA) • The main function of the genetic material is to encode the production of cellular proteins – In the correct cell, at the proper time, and in suitable amounts Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display The Genetic Code Translation involves an interpretation of one language into another In genetics, the nucleotide language of mRNA is translated into the amino acid language of proteins This relies on the genetic code The genetic information is coded within mRNA in groups of three nucleotides known as codons Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Three codons do not encode an amino acid. These are read as STOP signals for translation Triplet codons correspond to a specific amino acid Multiple codons may encode the same amino acid. These are known as synonymous codons Special codons: AUG (which specifies methionine) = start codon UAA, UAG and UGA = termination, or stop, codons The code is degenerate More than one codon can specify the same amino acid For example: GGU, GGC, GGA and GGG all code for lysine In most instances, the third base is the degenerate base This defines the reading frame for all following codons AUG specifies additional methionines within the coding sequence It is sometime referred to as the wobble base The code is nearly universal Only a few rare exceptions have been noted Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display An overview of gene expression Note that the start codon sets the reading frame for all remaining codons A Polypeptide Chain Has Directionality Polypeptide synthesis has a directionality that parallels the 5’ to 3’ orientation of mRNA During each cycle of elongation, a peptide bond is formed between the carboxyl group of the last amino acid in the polypeptide chain and the amino group in the amino acid being added The first amino acid has an exposed amino group Said to be N-terminal or amino terminal end The last amino acid has an exposed carboxyl group Said to be C-terminal or carboxy terminal end Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display R1 O H3N+ C C H R2 O N C H H R1 O H3N+ C C H R3 O C C H H + N C C H H O– R2 O N R4 O H3N+ R3 O C N C H H C C C H O– R4 O N C C + H2O H H O– Last peptide bond formed in the growing chain of amino acids (a) Attachment of an amino acid to a peptide chain OH CH3 S CH2 OH CH2 CH2 H3C H + Amino- H3N terminal end C C H O Methionine N H C C H O Serine SH CH3 CH CH2 H N C C H O Valine N CH2 H C C H O Tyrosine N C C H O O– Carboxylterminal end Cysteine Peptide bonds 5′ AUG AGC GU U UAC UGC Sequence in mRNA (b) Directionality in a polypeptide and mRNA Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 3′ • There are 20 amino acids that may be found in polypeptides – Each contains a different side chain, or R group – Each R group has its own particular chemical properties Nonpolar amino acids are hydrophobic They are often buried within the interior of a folded protein Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Nonpolar and charged amino acids are hydrophilic They are more likely to be on the surface of the protein Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Levels of Structures in Proteins There are four levels of structures in proteins 1. 2. 3. 4. Primary Secondary Tertiary Quaternary A protein’s primary structure is its amino acid sequence Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display • Within the cell, the protein will not be found in this linear state – Rather, it will adapt a compact 3-D structure – Indeed, this folding can begin during translation 129 amino acids long • The progression from the primary to the 3-D structure is dictated by the amino acid sequence within the polypeptide Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Levels of Structures in Proteins The primary structure of a protein folds to form regular, repeating shapes known as secondary structures There are two types of secondary structures a helix b sheet Certain amino acids are good candidates for each structure These are stabilized by the formation of hydrogen bonds Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display The short regions of secondary structure in a protein fold into a three-dimensional tertiary structure This is the final conformation of proteins that are composed of a single polypeptide Proteins made up of two or more polypeptides have a quaternary structure This is formed when the various polypeptides associate together to make a functional protein Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Functions of Proteins To a great extent, the characteristics of a cell depend on the types of proteins it makes Proteins can perform a variety of functions A key category of proteins are enzymes Accelerate chemical reactions within a cell Can be divided into two main categories Anabolic enzymes Synthesize molecules and macromolecules Catabolic enzymes Break down large molecules into small ones Important in generating cellular energy Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display STRUCTURE AND FUNCTION OF tRNA • In the 1950s, Francis Crick and Mahon Hoagland proposed the adaptor hypothesis – tRNAs play a direct role in the recognition of codons in the mRNA • In particular, the hypothesis proposed that tRNA has two functions – 1. Recognizing a 3-base codon in mRNA – 2. Carrying an amino acid that is specific for that codon Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Recognition Between tRNA and mRNA During mRNA-tRNA recognition, the anticodon in tRNA binds to a complementary codon in mRNA tRNAs are named according to the amino acid they bear The anticodon is anti-parallel to the codon Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display tRNAs Share Common Structural Features The secondary structure of tRNAs exhibits a cloverleaf pattern It contains Three stem-loop structures; Variable region An acceptor stem and 3’ single strand region The actual three-dimensional or tertiary structure involves additional folding In addition to the normal A, U, G and C nucleotides, tRNAs commonly contain modified nucleotides More than 80 of these can occur Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Found in all tRNAs Not found in all tRNAs Other variable sites are shown in blue as well • • • • • • • Structure of tRNA The modified bases are: I = inosine mI = methylinosine T = ribothymidine UH2 = dihydrouridine m2G = dimethylguanosine y = pseudouridine Charging of tRNAs The enzymes that attach amino acids to tRNAs are known as aminoacyl-tRNA synthetases There are 20 types One for each amino acid Aminoacyl-tRNA synthetases catalyze a two-step reaction involving three different molecules Amino acid, tRNA and ATP Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Charging of tRNAs The aminoacyl-tRNA synthetases are responsible for the “second genetic code” The selection of the correct amino acid must be highly accurate or the polypeptides may be nonfunctional Error rate is less than one in every 100,000 Sequences throughout the tRNA including but not limited to the anticodon are used as recognition sites Many modified bases are used as markers Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display The amino acid is attached to the 3’ end by an ester bond tRNAs and the Wobble Rule The genetic code is degenerate With the exception of serine, arginine and leucine, this degeneracy always occurs at the codon’s third position To explain this pattern of degeneracy, Francis Crick proposed the wobble hypothesis In the codon-anticodon recognition process, the first two positions pair strictly according to the A – U /G – C rule However, the third position can actually “wobble” or move a bit Thus tolerating certain types of mismatches Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display RIBOSOME STRUCTURE AND ASSEMBLY • Translation occurs on the surface of a large macromolecular complex termed the ribosome • Bacterial cells have one type of ribosome – Found in their cytoplasm • Eukaryotic cells have two types of ribosomes – One type is found in the cytoplasm – The other is found in organelles • Mitochondria ; Chloroplasts Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display RIBOSOME STRUCTURE AND ASSEMBLY • Unless otherwise noted the term eukaryotic ribosome refers to the ribosomes in the cytosol • A ribosome is composed of structures called the large and small subunits – Each subunit is formed from the assembly of • Proteins • rRNA Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Functional Sites of Ribosomes During bacterial translation, the mRNA lies on the surface of the 30S subunit As a polypeptide is being synthesized, it exits through a hole within the 50S subunit Ribosomes contain three discrete sites Peptidyl site (P site) Aminoacyl site (A site) Exit site (E site) Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display STAGES OF TRANSLATION • Translation can be viewed as occurring in three stages – Initiation – Elongation – Termination Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Initiator tRNA Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display The Translation Initiation Stage The mRNA, initiator tRNA, and ribosomal subunits associate to form an initiation complex This process requires three Initiation Factors The initiator tRNA recognizes the start codon in mRNA In bacteria, this tRNA is designated tRNAfmet It carries a methionine that has been covalently modified to N-formylmethionine The start codon is AUG, but in some cases GUG or UUG In all three cases, the first amino acid is N-formylmethionine Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display The binding of mRNA to the 30S subunit is facilitated by a ribosomal-binding site or Shine-Dalgarno sequence This is complementary to a sequence in the 16S rRNA Component of the 30S subunit Hydrogen bonding Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display IF1 and IF3 bind to the 30S subunit. IF3 30S subunit The mRNA binds to the 30S subunit. The Shine-Dalgarno sequence is complementary to a portion of the 16S rRNA. Portion of 16S rRNA IF3 5′ IF1 IF1 Start Shinecodon Dalgarno sequence (actually 9 nucleotides long) 3′ IF2, which uses GTP, promotes the binding of the initiator tRNA to the start codon in the P site. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display tRNAfMet Initiator tRNA GTP IF2 IF1 IF3 3′ 5′ IF1 and IF3 are released. IF2 hydrolyzes its GTP and is released. The 50S subunit associates. 70S initiation complex tRNAfMet E 5′ P A 70S initiation complex 3′ Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display This marks the end of the first stage The Translation Initiation Stage In eukaryotes, the assembly of the initiation complex is similar to that in bacteria However, additional factors are required Note that eukaryotic Initiation Factors are denoted eIF The initiator tRNA is designated tRNAmet It carries a methionine rather than a formylmethionine Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display The start codon for eukaryotic translation is AUG Scanning ribosome may pass over the first AUG But in most cases, the start codon for eukaryotic translation is usually the first AUG after the 5’ Cap! Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Translational initiation in eukaryotes can be summarized as such: A number of initiation factors bind to the 5’ cap in mRNA These are joined by a complex consisting of the 40S subunit, tRNAmet, and other initiation factors The entire assembly moves along the mRNA scanning for the right start codon Once it finds this AUG, the 40S subunit binds to it The 60S subunit joins This forms the 80S initiation complex Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display The Translation Elongation Stage During this stage, the amino acids are added to the polypeptide chain, one at a time This process, though complex, can occur at a remarkable rate In bacteria 15-18 amino acids per second In eukaryotes 6 amino acids per second Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display The 23S rRNA (a component of the large subunit) is the actual peptidyl transferase Thus, the ribosome is a ribozyme! Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display tRNAs at the P and A sites move into the E and P sites, respectively Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display The Translation Elongation Stage 16S rRNA (a part of the 30S ribosomal subunit) plays a key role in codon-anticodon recognition It can detect an incorrect tRNA bound at the A site It will prevent elongation until the mispaired tRNA is released This phenomenon is termed the decoding function of the ribosome It is important in maintaining the high fidelity in mRNA translation Error rate: 1 mistake per 10,000 amino acids added Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display The Translation Termination Stage The final stage occurs when a stop codon is reached in the mRNA In most species there are three stop or nonsense codons UAG UAA UGA These codons are not recognized by tRNAs, but by proteins called release factors Indeed, the 3-D structure of release factors mimics that of tRNAs Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display The Translation Termination Stage Bacteria have three release factors RF1, which recognizes UAA and UAG RF2, which recognizes UAA and UGA RF3, which does not recognize any of the three codons It binds GTP and helps facilitate the termination process Eukaryotes only have one release factor eRF, which recognizes all three stop codons Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Bacterial Translation Can Begin Before Transcription Is Completed Bacteria lack a nucleus As soon an mRNA strand is long enough, a ribosome will attach to its 5’ end Therefore, both transcription and translation occur in the cytoplasm So translation begins before transcription ends This phenomenon is termed coupling A polyribosome or polysome is an mRNA transcript that has many bound ribosomes in the act of translation Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Coupling between transcription and translation in bacteria Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display tRNAs that can recognize the same codon are termed isoacceptor tRNAs inosine 5-methyl-2-thiouridine 5-methyl-2’-O-methyluridine 2’-O-methyluridine 5-methyluridine 5-hydroxyuridine lysidine Wobble position and base pairing rules Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Recognized very poorly by the tRNA