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Chap. 4 Problem 2 The two strands of the double-helical plasmid DNA separate (melt, denature) at 90˚C. During cooling down to 25˚C, the strands come back together. However, because the single-stranded DNA sequencing primer is in great excess, it hybridizes preferentially to its complementary region of the plasmid. This prevents the two complementary strands of the plasmid DNA from reannealing at the region where the primer binds. DNA polymerases used in sequencing bind to the 3’-OH group of the primer and extend it in the sequencing reactions. DNA sequencing is covered in Chap. 5. Chap. 4 Problem 4 Prokaryotic mRNAs are ready to be translated immediately after they are transcribed. Prokaryotic mRNAs often are polycistronic, and contain coding sequences for multiple genes that are under the control of a common promoter. Eukaryotic mRNAs typically encode only a single protein. They further are extensively post-transcriptionally modified by capping, intron splicing, and polyadenylation reactions (Fig. 4.15). Chap. 4 Problem 6 An operon is a collection of tandemly linked genes that are transcribed in a single polycistronic mRNA. In the E. coli trp operon, the five gene products all work together in the biosynthesis pathway for the amino acid tryptophan. It is advantageous to link genes together in an operon, so that only one promoter must be regulated to coordinate expression of the related genes. In eukaryotes, the trp genes are transcribed separately (Fig. 4.13b). Thus, multiple promoters must be coordinately regulated to achieve the goal of balanced expression. Chap. 4 Problem 8 DNA polymerases synthesize DNA strands in a 5’ 3’ direction while moving 3’ 5’ relative to a template strand. While one strand (the leading strand) can be synthesized continuously as the replication fork advances, the other strand (the lagging strand) must be synthesized discontinuously in segments (Okazaki fragments) due to the polarity of DNA polymerase chain elongation. Synthesis of the lagging strand occurs from primers laid down on the template strand as it is exposed by movement of the fork. (See Fig. 4.30) Chap. 4 Problem 15 a. If DNA were unwound by helicases for replication, but stabilizing ssDNA binding proteins such as RPA were mutated and could not bind to the ssDNA, then replication would cease due to reformation of dsDNA. b. If a mRNA molecule formed a hairpin loop on itself at the AUG start site, then the AUG start codon could not be located by the ribosomal initiation complex. Translation of the mRNA would be blocked. c. If a cell were unable to produce functional tRNAiMet, then translation of most mRNAs would be blocked at the initiation step. tRNAiMet is the only tRNA that can be used to initiate translation.