Microbial Molecular Genetics BMS424 Translation PDF

Microbial Molecular Genetics BMS424 Topic 4: TRANSLATION IN PROKARYOTES AND EUKARYOTES BY NORASHIRENE MJ Primary source for figures and content: iGenetics: A Molecular Approach, 2nd Edition, 2006, Peter J....

Microbial Molecular Genetics BMS424 Topic 4: TRANSLATION IN PROKARYOTES AND EUKARYOTES BY NORASHIRENE MJ Primary source for figures and content: iGenetics: A Molecular Approach, 2nd Edition, 2006, Peter J. Russell, Pearson, Benjamin Cummings. Molecular Genetics of Bacteria, 2nd edition, 2012, Snyder, L. and Champness, W., ASM Press. LESSON LEARNING OUTCOME At the end of this topic, you will be able to: 1. Understand the stages of translation in prokaryotic cells 2. Understand the characteristics of genetic code  THE GENETIC CODE Genetic code is a set of instructions for transferring genetic data stored in the form of DNA/RNA into proteins. The genetic code is DNA or RNA sequence from which living cells make amino acids. This sequence ultimately dictates the components of proteins, the end products of most genes. Genetic code consists of 64 triplets of nucleotides. These triplets are called CODONS. GENETIC CODE = TRIPLET CODE = CODON  Why should each CODON in the genetic code consist of 3 bases (triplet codon) and not of one base each or 2 bases each?  There are 20 different amino acids which can be used in the synthesis of proteins in the cells. There must be at least one specific codon for each amino acid. Thus, there has to be at least 20 different codons in the genetic code. There are only four bases (AUGC). A minimum of 3 bases per codon is necessary to have (a minimum of) 20 codes. Because at each position of a three-letter codon any of the four different nucleotides (A, U, G, C) may be used, there are 64 different possible three-letter codons (4 x 4 x 4 = 64) in the genetic code. GENERAL STRUCTURE OF AN AMINO ACID There are 20 amino acids used in biological proteins. They are divided into subgroups according to the properties of their R groups (acidic, basic, neutral and polar, or neutral and nonpolar) CHAINS OF AMINO ACIDS = POLYPEPTIDES Polypeptides are chains of amino acids joined by covalent peptide bonds. A peptide bond forms between the carboxyl group of 1 amino acid, and the amino group of another. Polypeptides are unbranched, and have a free amino group at one end (the N terminus) and a carboxyl group at the other (the C terminus). The N-terminal end defines the beginning of the polypeptide. Figure: Mechanism for peptide bond formation between the carboxyl group of one amino acid and the amino group of another amino acid  How are the instructions for assembling amino acids into proteins encoded into DNA? PROCESS OF GENE EXPRESSION RNA is the intermediate between genes and the proteins for which they code Transcription is the synthesis of RNA under the direction of DNA Transcription produces messenger RNA (mRNA) Translation is the synthesis of a polypeptide, which occurs under the direction of mRNA Ribosomes are the sites of translation  TRANSLATION IS HIGHLY CONSERVED IN PROKARYOTES AND EUKARYOTES The major events are very similar The main differences are in the initiation process with related effects on mRNA structure Prokaryotes have polycistronic mRNA, with internal initiation by ribosomes Eukaryotes have monocistronic mRNA, with initiation only at 5’ end cap structures Prokaryotes couple transcription and translation in the same cellular compartment Eukaryotes separate transcription (nuclear) from translation (cytoplasmic)  COMPONENTS OF TRANSLATION 1. mRNA: 1. Eukaryotes: made in the nucleus, transported to the cytoplasm. 2. Prokaryotes: transcription and translation occur concurrently. 2. tRNA: Adaptor molecules that mediate the transfer of information from nucleic acids to protein. Its job is to match an mRNA codon with the amino acid it codes for. 3. Ribosomes: manufacturing units of a cell; located in the cytoplasm. Contain ribosomal RNA and proteins. 4. Enzymes: required for the attachment of amino acids to the correct tRNA molecule, and for peptide bond formation between amino acids. 5. Proteins: soluble factors necessary for proper initiation, elongation and termination of translation.  RIBOSOME STRUCTURE Prokaryotic (70S) and cytoplasmic eukaryotic (80S) ribosomes are each composed of a large subunit and a small subunit of differing sizes between the two groups. Each subunit is composed of rRNA and protein. Organelle ribosomes in eukaryotic cells resemble prokaryotic ribosomes. Responsible for aligning tRNA anticodons with mRNA codons Each ribosome has a binding site for mRNA and two binding sites for molecules of tRNA: P site: Holds tRNA with growing peptide chain A site: hold tRNA containing the next AA to be added to the polypeptide. Ribosome also has an exit slot for tRNA called the E site (binding site for release factor during the translation termination).  tRNA STRUCTURE Transfer RNAs (tRNAs) are structural RNA molecules Serving as adaptors, each tRNA type binds to a specific codon on the mRNA template and adds the corresponding amino acid to the polypeptide chain. The tRNA molecule interacts with three factors: aminoacyl tRNA synthetases, ribosomes, and mRNA. Mature tRNAs take on a three-dimensional structure when complementary bases exposed in the single-stranded RNA molecule hydrogen bond with each other This shape positions the amino-acid binding site, called the CCA amino acid binding end, which is a cytosine-cytosine-adenine sequence at the 3ʹ end of the tRNA, and the anticodon at the other end. The anticodon is a three-nucleotide sequence that bonds with an mRNA codon through complementary base pairing. An amino acid is added to the end of a tRNA molecule through the process of tRNA “charging,” during which each tRNA molecule is linked to its correct amino acid by a group of enzymes called aminoacyl tRNA synthetases. OVERVIEW: THREE STAGES IN TRANSLATION  frame INITIATION OF TRANSLATION 23 00) And-determina : reduce codon Ostart The initiation codon is an AUG Found towards the 5’ end of the mRNA molecule that is being translated. NOT the first 3 nucleotides. will stabilize MoA of It determines the reading frame. ni locatio In prokaryotes, there is a conserved region about 7 nucleotides upstream from the initiating AUG: - this region contains a 6-nucleotide sequence called the Shine-Dalgarno box: AGGAGG. The Shine-Dalgarno sequence is complementary to a region at the 3’ end of the 16 rRNA of the small subunit; base pairing between these complementary sequences stabilizes the binding of the small ribosomal subunit (30S) to the mRNA for proper assembly. onl cit Continenta ↑  INITIATION OF TRANSLATION IN PROKARYOTES IF STEP 1: Prokaryotic translation begins with binding of the 30S ribosomal subunit to a comie & mRNA near the AUG codon. The 30S comes c to the mRNA bound to: ALL THREE Initiation Factors, IF-1, IF-2 and IF-3 Structions IF1: Prevents tRNA from binding to A site IF2: GTPase; binds GTP; interacts with initiator tRNA, IF1, and small 30S subunit. IF3: Prevents 30S-50S subunit association. GTP Mg2+ (final product  INITIATION OF TRANSLATION IN PROKARYOTES STEP 2: Next, the initiator tRNA binds the AUG to which the 30S subunit is bound. ↓AUG universally encodes methionine. start codon Newly made proteins begin with methionine (Met), which is often subsequently removed. eukaryotes metironine o biass pit : In prokaryotes, the first AUG is recognized - 7 by a specialized initiator tRNA (tRNAfMet) carrying a modified methionine: - methionine. (first And only) /lepag formyl methionine) - STEP 3: The large subunit of the ribosome (50S) - now attaches to the small subunit, to sodig temptied newly complete the initiation process. camino exit - - tRNA RNA ↑ elongativean r for to -ready process  INITIATION OF TRANSLATION IN EUKARYOTES - curangotes In eukaryotes, the small ribosomal unit binds first to the methylated cap (7-methyl guanosine) at the 5’ end of the mRNA. It then migrates to the initiation site, usually the first AUG it encounters as it scans the mRNA in the 5’ to 3’ direction. In eukaryotes, the methionine need not be modified. What's3 modification ? i' cap  LESSSON PLAN MID SEMESTER BREAK [21 – 27 Nov 2022] WEEK 7 4.0 Gene Expression: Translation 28/11-2/12 Chemical and molecular structure of Proteins The Nature of genetic codes Ribosome structure and functions WEEK 8 TEST 1 5/12-9/12 Stages of Translation 5.0 Recombination / Gene Transfer WEEK 9 Horizontal Gene Transfer 12/12 – 16/12 Transformation Generalised and specialised transduction Conjugation WEEK 10 Significance of gene transfer 19/12 – 23/12 SPECIAL BREAK [26 Dec 2022 – 1 Jan 2023] CHAPTER 6.0 REGULATIONS OF GENE EXPRESSION (WEEK 11 & 12), WEEK 13 (TEST 2) Microbial Molecular Genetics BMS424 Topic 4: TRANSLATION IN PROKARYOTES AND EUKARYOTES BY NORASHIRENE MJ Primary source for figures and content: iGenetics: A Molecular Approach, 2nd Edition, 2006, Peter J. Russell, Pearson, Benjamin Cummings. Molecular Genetics of Bacteria, 2nd edition, 2012, Snyder, L. and Champness, W., ASM Press.  At the end of this topic, you will be able to: LESSON 1. Understand the stages of translation in LEARNING prokaryotic cells OUTCOME 2. Understand the characteristics of genetic code OVERVIEW: THREE STAGES IN TRANSLATION  REVIEW transcribing RNATRNA FrankNY mRNA > amimo - and ran start for 1. Define Transcription? Translation? eart cod on + whatare genes and how are they different from 2. How many nucleotides make a codon? chromosomes ? 3 3. The anticodon can be found on which object? tRNA 4. The anticodon must match the _______________. cod on 5. Place the following steps in order from start to finish: ↑ Proteins are assembled Z Ribosome reads a codon one at a time ① mRNA arrives at the ribosome 3 tRNA delivers amino acids to the ribosome get  ELONGATION OF TRANSLATION Elongation of the amino acid chain has three steps: tRNAyang amino acid X bawa a. Binding of aminoacyl-tRNA to the ribosome. b. Formation of a peptide bond. between the amino acid end ?- Ke c. Translocation of the ribosome In-terminal and to the next codon. carboxyl terminal)  The ribosome has VA three tRNA binding sites: 1. Aminoacyl- reads that depend what i tRNA poption codun 2. Peptidyl-tRNA - I 3. Exit bila pidch andbond impr dan averenations - amino yentris Aus avain , resit diam das-up Yo gite- ELONGATION OF (procesut a r) TRANSLATION stop 1. At the start of elongation, the mRNA is bound to the complete two subunit ribosome, with the initiating tRNA in the P site, and the A site free for binding to the next tRNA. 2. The ribosome moves along the mRNA in a 5’ to 3’ direction, in a step-wise process, recognizing each subsequent codon. 3. The peptidyl transferase enzyme then catalyses the formation of a peptide bond between the free N terminal of the amino acid at the A site, and the Carboxyl end of the amino acid at the P site, which is actually connected to the tRNA. 4. This disconnects the tRNA fMet from the amino acid, and the tRNA at the A site now carries two amino acids, with a free N terminal and the Carboxyl terminal of the second aa connected to its tRNA.  TERMINATION OF TRANSLATION When the ribosome encounters a STOP CODON, there is no tRNA available to bind to the A site of the ribosome, instead a release factor binds to it. The details are not very clear, but once the release factor binds, the ribosome unit falls apart, releasing the large and small subunits, the tRNA carrying the polypeptide is also released, freeing up the polypeptide product. Table 1. Comparison of Translation in Bacteria versus Eukaryotes Property Bacteria Eukaryotes 70S 80S 30S (small subunit) with 16S rRNA subunit 40S (small subunit) with 18S rRNA subunit Ribosomes 50S (large subunit) with 5S and 23S rRNA 60S (large subunit) with 5S, 5.8S, and 28S subunits rRNA subunits Amino acid carried fMet Met by initiator tRNA Shine-Dalgarno Present Absent sequence in mRNA Simultaneous transcription and Yes No translation  THINK ABOUT IT 1. Describe the structure and composition of the prokaryotic ribosome. 2. In what direction is the mRNA template read? 3. Describe the structure and function of a tRNA. 4. What are the components of the initiation complex for translation in prokaryotes? 5. What are two differences between initiation of prokaryotic and eukaryotic translation? 6. What occurs at each of the three active sites of the ribosome? 7. What causes termination of translation? Practice Problem Always use mRNA codon to find the amino acid DNA mRNA tRNA Amino Acid codon anticodon Practice Problem Always use mRNA codon to find the amino acid DNA mRNA tRNA Amino Acid codon anticodon Practice Problem Always use mRNA codon to find the amino acid DNA mRNA tRNA Amino Acid codon anticodon TRANSLATION PART II: THE CHARACTERISTICS OF GENETIC CODE LESSON LEARNING OUTCOME At the end of this topic, you will be able to: 1. Understand the stages of translation in prokaryotic cells 2. Understand the characteristics of genetic code  CHARACTERISTICS OF THE CODE To what extent is the genetic code universal? What does it mean that the Explain the Wobble code is “comma free” and Hypothesis “non-overlapping”? GENETIC CODE How many codons are there in What are the start and the genetic code that code for stop signals? amino acids? What does it mean that the code is “degenerate”?  CHARACTERISTICS OF THE CODE The following are features to note in the genetic code: 1. It is a TRIPLET CODE. Each three-nucleotide codon in the mRNA specifies 1 amino in the polypeptide. 2. The Genetic Code is written in linear form. The ribonucleotide sequence is derived from the complementary nucleotide bases in DNA. 3. Each “word” within the mRNA consists of 3 ribonucleotide letters  THE TRIPLET CODE. With only THREE exceptions, each CODON specifies ONE amino acid. 4. The code is NON-OVERLAPPING. Each nucleotide is part of only one codon, and is read only once during translation. 5. The code is COMMALESS. No internal punctuation is used in the code. Once translation of mRNA begins, the codons are read one after the other with no breaks between them (until a STOP signal is reached).  CHARACTERISTICS OF THE CODE 5. The code is UNAMBIGUOUS (specific) – each triplet specifies only a single amino acid.  In general, no codon specifies more than one amino acid. The exceptions so far are AUG, UGA and UAG. AUG In the first case, AUG specifies both methionine and N-formyl-methionine (START CODON) which is used to initiate protein synthesis in bacteria. sol· UGA In the second case, UGA specifies the 21st amino-acid, Selenocysteine as well as being a STOP CODON. This codon usage has been found in certain archaea, eubacteria, and animals (humans synthesize 25 different proteins containing selenium). UAD- anregis wo neve UAG In the last case, UAG specifies the 22nd amino acid (the most recent to be added to the list), Pyrrolysine as well as being a STOP CODON. Found in a member of the Archaea. How the translation machinery knows when it encounters UAG whether to insert a tRNA with pyrrolysine or to stop translation is not yet known.  THE RNA CODON Of the 64 codons, 61 specify one of the 20 amino acids. The other 3 codons are chain- terminating codons (STOP CODONS) and UAA do not specify any amino acid. AUG, one of the 61 codons that specify an amino acid, is used in the initiation of protein synthesis. Using this table, you should be able to translate mRNAs  CHARACTERISTICS OF THE CODE 6. The code is DEGENERATE Most amino acids are specified by more than one codon. This is the case for 18 of the 20 amino acids. In fact, only Met and Trp are specified by a single codon: No. of codons amino acids 1 Met, Trp 2 Asn, Asp, Cys, Gln, Glu, His, Lys, Phe, Tyr 3 Ile 4 Ala, Gly, Pro, Thr, Val 6 Arg, Leu, Ser Degeneracy is found only in the third nucleotide of the codon. 7. The code contains one “START” and three “STOP” signals, triplets that initiate and terminate translation, respectively.  1 start codon: AUG (However, note that GUG and UUG are occasionally found as start codons).  3 stop codons: UAA, UAG, and UGA.  CHARACTERISTICS OF THE CODE The code is DEGENERATE Most amino acids are specified by more than one codon.  CHARACTERISTICS OF THE CODE 8. The code is Universal. The genetic code is remarkably the same in all organisms. The same codons are assigned to the same amino acids and to the same START and STOP signals in the vast majority of genes in animals, plants, and microorganisms. However, some exceptions have been found. Most of these involve assigning one or two of the three STOP codons to an amino acid instead. The most common exception is the use of UGA as a codon for Tryptophan in mitochondria. Table above shows how some of the known exceptions to universality in both the genetic code used in the nucleus and in the genetic code used in mitochondria.  REVIEW What are the 8 characteristics of the genetic code? How gene encodes protein? What are the differences of gene expression in prokaryote & eukaryote?