Chapter 16 - Transcription, RNA Processing, and Translation Proteins are synthesized in a two-step process: transcription of genes intomessenger RNAs and translation of messenger mRNAs into proteins. Learning Objectives: You should be able to … • Relate the structure of RNA polymerase to its function in transcription (16-istudy guide) Table 16.2• Describe the steps in initiation, elongation, and termination of transcription andtranslation. • Explain how RNA is processed in eukaryotes (Pre MRNA  Mature mRNA). • Relate the structure of ribosomes and tRNA to their functions in translation. 16-ii An Introduction to Translation A. Ribosomes are the site of protein synthesis.1. Translation is the conversion of a sequence of nucleotides in an mRNA into a sequence of amino acids in a protein. 2. Cells that synthesize lots of protein have lots of ribosomes, and vice versa. 3. Studies showed that the basic mechanism of translation was the same throughout the tree of life. B. Comparing translation in bacteria and eukaryotes1. Electron microscopy of Escherichia coli DNA being transcribed shows that: Ribosomes have attached to mRNA even before transcription is complete, while the mRNA is still linked to DNA  Ribosomes translate mRNA as it is being synthesized by RNA polymerase (transcription and translation occur simultaneously in bacteria).  Multiple ribosomes attach to each mRNA, forming a polysome(polyribosome). 2. In eukaryotes: mRNAs synthesized using DNA template in the nucleus.  mRNAs are then processed before leaving the nucleus.  Then they become associated with ribosomes in the cytoplasm (transcription and translation are separate processes in eukaryotes). (Fig. 16.9) Processing of primary transcript   mature transcript?C. How does an mRNA triplet specify an amino acid?Crick hypothesis: Adapter molecules hold amino acids in place while interacting with an mRNA codon (predicted the existence of transfer RNA molecules). (Fig. 16.10b) -The Structure and Function of Transfer RNA a. A tRNA that becomes covalently linked to an amino acid is called an aminoacyl tRNA. b. The addition of amino acids to tRNAs (charging a tRNA) is catalyzed by aminoacyl tRNA synthetases. c. Each of the 20 amino acids has a different aminoacyl tRNA synthetase andone or more tRNAs. d. Amino acids are transferred from aminoacyl tRNAs to proteins synthesized inribosomes. (Fig. 16.12) What do tRNAs look like?1. Sequencing revealed that tRNAs are 75−85 nucleotides long. 2. Secondary structure was inferred from the nucleotide sequence. a. Hydrogen bonding occurs between complementary bases in differentregions of the tRNA molecule.  The tRNA assumes a cloverleaf shape ofstems and loops (stems are double-stranded; loops are single-stranded regions).(Fig. 16.13) b. All tRNAs have the sequence CCA at their 3' end; it is a binding site for an amino acid. 1c. One of the loops has a triplet of bases that varies in each tRNA; it is the anticodon (i.e., the nucleotides that form base pairs with an mRNA codon). How many tRNAs are there? There are 61 different mRNA codons but only about 40 tRNAs. Why? Crick’s wobble hypothesis: a. Many amino acids are specified by more than one codon.  refer to the Genetic Codeb. Codons for the same amino acid tend to differ only in the third position. Example: Codons 5'-CAA-3' and 5'-CAG-3' code for glutamine. c. A tRNA with the anticodon 3'-GUU-5' can pair with either 5'-CAA-3' or 5'-CAG-3'. d. The third codon position is the wobble position; it often contains an inosine base (a modified base)that can pair with A, U, or C. (third position  more flexible base pairing)Clicker question  DNA sequence in a template strand is 5’TGA3’. What is the anticodon sequence on tRNAthat will bind to this codon? 3’AGU5’ (convert to mRNA, then tRNA)D. The Structure and Function of Ribosomes:I. Characteristics of protein synthesis 1. The sequence of bases in an RNA message is translated into a sequence ofamino acids in a polypeptide. 2. The conversion of each mRNA codon begins when the anticodon of anaminoacyl tRNA binds to the codon. 3. The conversion is complete when a peptide bond forms between the tRNA’samino acid and the growing polypeptide. N terminal  C terminal4. Conversion occurs inside a ribosome, which is composed of two subunits. (Fig. 16.14) a. There is a larger 50S subunit and a smaller 30S subunit (in Prokaryotes). b. Both subunits are composed of multiple RNA molecules (rRNA) and numerous proteins. c. The small subunit holds the mRNA in place during translation. d. The large subunit is where peptide bond formation takes place.  Ribozyme?e. The large ribosomal subunit has three tRNA binding sites. (Fig. 16.14a) (1) During translation, the acceptor or aminoacyl (A) site holds the tRNA with an amino acid that is about to be added to the polypeptide (Exception: Initiation step?). (2) The peptidyl (P) site holds the tRNA that is holding the growing polypeptide chain. (3) The exit (E) site holds the empty tRNA that is about to be released. 5. Three-step sequence of protein synthesis: a. Aminoacyl tRNA diffuses into the A site; its anticodon binds to a mRNA codon b. A peptide bond forms between the amino acid on the aminoacyl tRNA in the A site and the growing polypeptide (held by a tRNA) in the P site. c. The ribosome moves ahead, and all the tRNAs move one position down the mRNA. The tRNA in the E site exits, the tRNA in the P site moves to the E site, and the tRNA in the A site moves to the P site. 6. The polypeptide has grown by one amino acid. 7. The process occurs up to 20 times per second in bacteria and about 2 times per second in eukaryotes. 8. Protein synthesis begins at the amino end (N-terminus) of the protein and proceeds to the carboxy end (C-terminus). II. Initiating translation (Prokaryotic)1. In bacteria, the 30S ribosomal subunit binds to a sequence on mRNA about six nucleotides upstream from an AUG start codon  The sequence to which the 30S subunit binds is the Shine-Dalgarno sequence 22. The initial interaction is mediated by initiation factors. In eukaryotes, initiation factors bind to the 5′ cap on mRNA. 3. An aminoacyl tRNA with N-formylmethionine (f-met) binds to the AUG codon. (Fig. 16.15, step 2) In eukaryotes, the first amino acid is normal methionine. 4. Initiation is complete when the large ribosomal subunit binds and the tRNA-bearing f-met occupies the P site.III. Elongation: extending the