CHAPTER 6 Gene Expression: Translation Peter J. Russell CHAPTER 6 Gene Expression: Translation Chapter 6 do Q6.1, Q6.2 Problems * means particularly relevant 6.1-6.5 6.6-6.8* 6.9,10,12 6.13-6.17* 6.18 6.19,20* 6.21,22 6.23* 6.24 6.25* 6.26 Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

Translation and the genetic code What covered in this chapter I am treating a good deal of this as review and will focus on new aspects in light of our interest in genetics, and I am presenting the material of this chapter in a different order than the book the book has a difficult task to decide what to discuss and in what detail. I can not possibly recapitulate the history of the field, but I find its selection somewhat confusing. It wants to include many of the major historical experiments, because they were very clever and use a logic that is very common in genetics. It also wants you to be aware of some model systems. The conflict comes when the book explains how translation is known to work now, the importance and cleverness of the historical experiments with the contemporary technology is not clear On board, a simple overview of the path from the genetic material, DNA, to the gene product protein, the central dogma and some of the major regulatory mechanisms. Still keeping within the simple formulation of the central dogma, the relationship between genotype and phenotype is through how much of which enzyme (or mutant enzyme) is produced. We discussed transcription and mRNA processing last chapter, now we will discuss how the information contained in the sequence of nucleotides is translated into a sequence of amino acids

Fig. 6.17 Diagram of a polysome, a number of ribosomes each translating the same mRNA sequentially this is an overview of translation an mRNA is translated by ribosomes using tRNAs as adapter molecules, the molecules that connect the information in the nucleotide sequence to the sequence of amino acids. The mRNA is the template being read, the tRNAs charged with aminoacids are substrates, the polypeptide chain is the product, the ribosome is the catalyst, and the expended tRNAs are recycled after being recharged. This is an enormously complex process. There is initiation, elongation, and termination, as in transcription, and there are many opportunities for regulations. A recurring theme is that whatever mechanism that one can imagine and more, is probably exploited somewhere. I will try to point out some of the more important and interesting examples as we proceed Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings. Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

Fig. 6.3 Mechanism for peptide bond formation between the carboxyl group of one amino acid and the amino group of another amino acid I assume most of the discussion in the book about protein chemistry and structure is review, if not learn what needed for genetics and wait for your Biochemistry course This to remind you about the nature of the peptide bond. It is not the chemistry actually used by the cell to synthesize proteins. In the chemistry used by the ribosome, the pink part of amino acid 1 is not a free carboxylic acid, but activated by being esterified to the end of a tRNA, and the reaction is not a condensation reaction, but rather a transpeptidation, like a transesterification. Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

Fig. 6.4 Four levels of protein structure also review, to remind you that polymers of amino acids tend to form certain secondary structures and tertiary structures and even quaternary structures, and the end product, the protein or protein complex may have many different biologically important activities, such as oxygen transport, or as discussed in these last chapters, may be an enzyme or a transcription factor or a ribosomal protein. Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

Fig. 6.6 Reversion of a deletion frameshift mutation by a nearby addition mutation The story of how the genetic code was broken is very interesting, but too long to go into here. I would suggest anyone going to graduate school read it. Soon after the central dogma was produced, there were various hypotheses about how nucleic acid could code for polypeptides. Since there were 20 amino acids, and only four bases, and only 16 possible dinucleotides, interest focused on codes using three or more bases. *how many possible triplets An early hypothesis considered an overlapping code of triplets, but this would constrain the amino acid sequences of proteins and was rejected after some simple experiments What is important in this story, is the logic used to decipher the code, it is a logic common to genetics Crick and collegues used mutagens were used that cause the insertion or deletion of base pairs in DNA, using T4 bacteriophage. Insertions and deletions cause frameshift mutations, that sometimes have a clear phenotype. The book discusses the experiments reverting a phenotype of T4 Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

Fig. 6.7 Hypothetical example showing how three nearby + (addition) mutations restore the reading frame, giving normal or near-normal function while a frameshift of 1 or 2 in either direction always produced an altered phenotype, frameshifts of 3 sometimes did not. It was deduced that the code was a triplet code, that the spacing was three, and that sometimes the addition or deletion of one amino acid did not drastically alter protein function. Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

Fig. 6.8 The genetic code This a table of the genetic code. You should learn at least the start and stop codons, and that M and W have only one codon, and that other amino acids have 2, 3, 4, or 6 codons The experiments used to work this out use logic and you will find the problems at the end of the chapter focus on this logic. Two methods were important, translating synthetic RNA polymers such as poly U, UC copolymer, and random copolymer and analyzing the amino acid content of the polypeptide and determining which trinucleotide RNAs would bind ribosomes bound with specific tRNAs. There are only 20 amino acids encoded by 64 possible codons, 61 of which are used. Having multiple codons for one amino acids is called degeneracy, and you will note that there are patterns to the degeneracy Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

Fig. 6.9 Example of base-pairing wobble What is the origin of the degeneracy. One could imagine that there are 61 different tRNAs, but most cells have only 50 or 60 distinct tRNAs Some tRNAs recognize more than one codon Francis Crick (again) proposed the wobble hypothesis, that the 3’-nt of a codon can read by the 5’-end of the anticodon by more than one pairing, using what we now called a non-watson-crick base pair, or a wobble base pair- Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

Fig. 6.11 Molecular details of the attachment of an amino acid to a tRNA molecule How does a tRNA function? tRNAs are the adapter molecule, the molecule that deciphers the genetic code. The set of tRNAs are the cipher, or the key to the code At one end it recognizes the codon on the mRNA, on the other it bears the activated amino acid, the part of the substrate becomes part of the product polypeptide. How is this code maintained? How is the tRNA charged with the correct amino acid? Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

Fig. 6.10 Charging of a tRNA molecule by aminoacyl-tRNA synthetase to produce an aminoacyl-tRNA (charged tRNA) How is this code maintained? How is the tRNA charged with the correct amino acid? Note that while there are 61 codons used, and between 50 and 60 tRNAs used in a cell, there are only 20 tRNA synthetases, one for each amino acid. This figure shows a schematic of the reaction cycle of a tRNA synthetase, with the anticodon being recognized. This seems an elegant solution, but it is not always used, some synthetases do not use the anticodon to recognize their correct substrate. Be aware that tRNA synthetases proof read their product to reduce errors in protein synthesis Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

Fig. 6.12 Initiation of protein synthesis in prokaryotes What should you know? That the AUG initiating codon is recognized as the start of transription in prokaryotes by its location just downstream from a ribosome-binding site, called the Shine-Dalgarno sequence. The 16S RNA of the 30S subunit binds via base pairing to the Shine-Dalgarno sequence. The next step is binding of the fMet initiator tRNA. This is a tRNA used only for the first codon, charged with formyl methionine. Other AUGs in the mRNA are read by a normal tRNA-Met. All proteins begin translation with a formyl methionine, but it is often removed later. Then the 50S subunit binds, with tRNA-fMet in the P site, and other factors are released and this is called the 70S initiation complex. In eukaryotes the process is similar, but: different accessory factors are used, including eIF-4F, cap-binding protein, poly(A) binding protein usually the first AUG from the 5’-end is used there is not a Shine-Dalgarno sequence, but a Kozak sequence the special initiator tRNA has an unmodified methionine Translation initiation is an important regulatory step in gene expression Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

Fig. 6.14 Sequences involved in the binding of ribosomes to the mRNA in the initiation of protein synthesis in prokaryotes Important style of experiment to support the hypothesis that 16S RNA of the 30S subunit recognizes the Shine-Dalgarno binding site was use of compensatory mutations in the Shine-Dalgarno sequence and the 16S RNA. This kind of experiment is very often used, is elegant and powerful, and you should be able apply it to genetics problems Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings. Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

Fig. 6.16 The formation of a peptide bond between the first two amino acids of a polypeptide chain is catalyzed on the ribosome by peptidyl transferase A couple very important points, the actual chemistry is a nucleophilic displacement reaction, the book describes it misleadingly. The amino group of the amino acid attached to the tRNA in the A site attacks the carbonyl group of the amino acid attached to the tRNA in the P site, the last amino acid of the polypeptide being synthesized. This is the peptide bond formation, it is not a condensation reaction, it does not release water, it released the uncharged tRNA. it is catalyzed by the ribosome and does not require hydrolysis of GTP The other point is that when I was in college, the role of the ribosomal RNAs was thought to structural, scaffolds to hold the ribosomal proteins that did the real business. Since then, it has been demonstrated that that view is backward, the RNA does the chemistry, and the proteins have a structural role. Harry Noller’s group has contributed an enormous amount to the understanding of this, and has shown that the rRNAs can catalyze peptide bond formation in the absence of proteins Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings. Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

Fig. 6.15 Elongation stage of translation in prokaryotes The mechanism by which the ribosome works is extremely complicated, and by no means completely understood. You should know the general mechanism, the role of the A, P, and E sites. Only two tRNAs are bound at a time Note that the catalytic step is only part of the process, translocation is complex. Proof-reading also occurs Aminoglycoside antibiotics such as streptomycin and neomycin function by interfering with the fidelity of translation, by interfering with the RNA structure to make it error-prone, and enough mistakes are made to prevent bacteria from replicating Elongation rate and pausing is a mechanism exploited for regulating gene expression. Frameshifting of the translation reading frame can also occur, especially in retroviruses, including HIV Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

Fig. 6.18 Termination of translation Termination does not use special tRNAs with anticodons complementary to the stop codons, but proteins called termination factors or release factors after termination, the initial methionine is usually removed, and the polypeptide begins its sorting and modification process. Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings. Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

Fig. 6.19 Movement of secretory proteins through the cell membrane system I assume this is all review. Proteins destined for secretion or the cell surface or lysosome have a signal peptide that is recognized as it emerges from the ribosome by a Signal Recognition Particle, which binds the peptide and halts translocation. Proteins destined for other destinations are translated into the cytoplasm and directed to their destination by different mechanisms, some examples, nuclear encoded mitochondrial proteins also have signal sequences Protein destined for the nucleus have nuclear localization signals, which can be anywhere in the sequence, the localization may be regulated, phosphorylation is a mechanism to regulate location and other protein functions. Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings. Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

Fig. 6.20 Model for the translocation of proteins into the endoplasmic reticulum in eukaryotes The SRP brings the ribosome to the endoplasmic reticulum and the peptide inserts into the membrane. The SRP is released and translation resumes. The polypeptide is extended into the cisternal space of the endoplasmic reticulum and the signal peptide removed, and other modifications made, primarily glycosylation, such as the for the ABO blood groups we discussed earlier. Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings. Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.