PART I

Central Dogma Thought Questions

• Assume that a given length of dsDNA has given rise to a length of RNA which in turn has given rise to a length of protein. How can that same length of DNA lead to the formation of an entirely different length of protein?
• If you know the sequence of a protein, can you reverse your thinking and know the sequence of the DNA that encoded it?
• We are creatures of habit (or we try to be). If a short sequence of DNA is written down in a hurry by someone, which end, according to the typical convention, is on the left and which is on the right?
• In describing RNA structure, what do we mean by the term "stem and loop"? Can stems and loops form anywhere in the molecule, or are there certain locales that favor them?
• Here's another one of those literary descriptions that some molecular biologists are fond of. Protein molecules have been described as "charm bracelets". What are the charms that dangle from the bracelet?
• This question ran against conventional thinking about the central dogma: Is there such a thing as "reverse transcription"? And if there is such a thing, what kind of nucleic acid is the template sequence and what kind of nucleic acid is the product sequence?

DNA mutation and repair. Thought questions.

• Describe three forms of DNA damage that occurs naturally as the result of physical or chemical factors that affect cells.
• UV light causes thymine dimers. If you are a bacterium, how is blue light used to repair the thymine dimer?
• If you are a human, how is that thymine dimer repaired?
• Breaks in DNA are bad news since the ends of chromosomes are unstable and can lead to chromosome translocations where fragments of different chromosomes are joined to one another. Two cellular mechanisms act to repair broken chromosomes. These are non as non-homologous end joining (NHEJ) and homologous recombination (HR). Which of these two often results in insertions and deletions (known as indel mutations) that can cause a gene to stop working even though the break in the chromosome, a much bigger problem, has been fixed. And which of the two (NHEJ or HR) achieves error free repair?
• What does it take to achieve error-free repair? [Hint: What sort of repair template is needed?]
• In homologous recombination, how does strand invasion eventually resolve? Or to say this a little differently, what is a Holliday junction and how does the junction eventually turn back into two similarly sequenced double stranded DNA molecules?

PART II

Thought questions. DNA replication Part 1
• Re-word and re-express the thoughts behind the famous sentence written by Watson and Crick in their molecular biology research article describing the structure of DNA: " It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material."
• How did the manufacture of DNA by E.coli fed with heavy nitrogen nutrients help to verify that DNA replication is semi-conservative?
• How would you describe the direction of DNA manufacture by DNA polymerase?
• What is the solution to the problem at the replication fork in which only one of the strands can be lengthened by DNA polymerase in the same direction as the advancing replication fork?
• If you are shown a portion of a replication fork showing the template strands and the newly synthesized strands, can you label the ends of each strand, making plain the polarity of every DNA molecule in the structure?
• If you draw one replication bubble, how many replication forks have you drawn?
• The number of replication bubbles that are opened to replicate a human genome is not known with much certainty. Make an estimate from the fact that it takes about 6 hours to replicate the genome and the fact that a eukaryotic replication fork advances at about 50 base pairs per second.
• Why do replicating circular chromosomes (such as a replicating bacterial chromosome) resemble the Greek letter theta?

DNA replication Part 2. Thought questions

• What supplies the chemical energy, the driving force, for incorporating dNTP building blocks into a growing DNA strand?
• Enzymes catalyze chemical reactions, i.e. they can be used over and over again to make the reaction happen more quickly. The suffix "-ase" is the designation of an enzyme, hence DNA polymerase is an enzyme. What chemical reaction does DNA polymerase catalyze?
• To begin incorporating dNTP's, DNA polymerase needs a primer and a template. So what exactly is a primer, what is a template, and to which end of which of these are the dNTPs incorporated?
• DNA polymerase I is composed of what type of folded chain?
• Besides having the ability to catalyze the synthesis of DNA, DNA polymerase I can also actively trim the ends of DNA strands. Describe the direction and purpose of such "exonuclease" activities of DNA pol I.
• Why and how is DNA proofread at the time of its synthesis?
• How do we know that DNA pol I is not the replicative polymerase in E. coli cells?
• How does DNA pol III achieve such a high degree of "processivity"?
• A replisome is a complicated structure involving many molecular machines. What is the purpose of the following machines: topoisomerase, helicase, primase, single-strand binding proteins; and DNA ligase.
• Describe an Okazaki fragment and explain why newly formed Okazaki fragments contain a stretch of RNA whereas less recently make Okazaki fragments contain no RNA.
• If a replication mistake gets past the early proofreading, putting a newly incorporated base in a mismatched arrangement with the parental strand, how does the cellular machinery recognize which strand is the parental strand and which is the new strand? And after that recognition takes place, how is the newly synthesized strand fixed so that the mismatch is corrected?

DNA replication Part 3. Thought questions

• Name one of the major difference between the job of DNA replication in eukaryote as compared to prokaryotes?
• Bacterial cells contain 3 kinds of DNA polymerase. Eukaryotic cells contain many more than that. Name one of the common purposes of DNA polymerases besides the replication of chromosomes.
• Define "nucleosome" and explain why they are needed.
• What happens to nucleosomes in the vicinity of the replisome?
• Eukaryotic chromosomes have "telomeres". What is a telomere and why do bacterial chromosomes lack them entirely?
• The ends of chromosomes present a problem for DNA replication in eukaryotes. Explain how RNA primers are the root of the problem.
• How does telomerase counteract telomere shortening? What is the purpose of the RNA sequence carried by telomerase?
• When an RNA sequence, such as telomerase RNA, is used to direct the manufacture of DNA, the information flow runs in the opposite direction dictated by the Central Dogma (which states that DNA directs the synthesis of RNA which directs the synthesis of protein). In what way does such "reverse transcriptase" activity come into play in the life cycle of retroviruses such as human HIV?

PART III

Translation. Thought questions

• Review the central dogma in molecular terms. You should be able to fill in a table asking whether a given item is composed of DNA, of RNA or of protein. For example, choose the composition (DNA, RNA or protein) of the following items: Transcription factors, promoters, operators, introns, exons, codons, anticodons, polyA tails, ribosome binding sites, amino acids, DNA polymerases, RNA polymerases, ribosomes.
• Define "triplet codon" and explain why doublet codons are insufficient for life on earth.
• Use the table of the genetic code to translate the RNA sequence here: 5'-AAU AGU GAU UAG-3'
• In that example, what is the purpose of a STOP codon?
• Now use the table of the genetic code to translate the following example of a DNA coding sequence, beginning at the A on the 5' end: 5'-ATG GTC AAT TTC ...
• Now study the effect of a frame-shift mutation. Insert or delete a DNA letter anywhere and translate the sequence such as in the following example where a T has been inserted after the first A in the preceding sequence: 5'- ATT GGT CAA T TT C ...
• Bioinformatics tools. Computers are ideal helpers for molecular biologists. One of the tools that helps us translate sequences is called ExPASy. It can be found by going to https://web.expasy.org/translate. Insert any nucleotide sequence (from 5' to 3') into the box and see what you get.
• Mechanistically speaking, what are the adaptor molecules that bridge the information at the RNA level to the information at the protein level?
• Describe the structure of a tRNA molecule. To which end is the amino acid attached in a charged tRNA?
• Choose alanine from the list of 20 amino acids and memorize how to draw it. Look away from the page where it is drawn and see if you can recall how, in alanine, the alpha carbon is joined to the amino group and to the carboxylic acid group. Now look back at the structure and note how the side chain of alanine, a methyl group, can be substituted by any other side chain, making it a straightforward though admittedly arduous task to memorize all 20 amino acid structures as having the same structure as alanine, but with a different side chain. Now choose a letter in your name, or someone else's name, and find the amino acid that uses that letter as its 1-letter abbreviation (sorry, you are out of luck if the letter is B, J, O, U, X, or Z; so try another letter). Memorize the structure of that amino acid and be prepared to name it and draw its structure in the final exam.
• Break down a ribosome into its main parts, describe the three cavities within.
• How does a ribosome recognize which AUG in an mRNA is the start codon?
• In the elongation cycle of protein translation, each charged tRNA enters the ribosome, donates its amino acid, and then leaves to be charged again later for reuse. Now consider a protein that was initiated with a methionine as the first amino acid, followed by 100 more amino acids when a STOP codon signaled the finish of protein translation. In all, 101 amino acids were incorporated. How many peptide bonds were formed by the ribosome?
• What keeps a protein from folding prematurely and perhaps incorrectly?
• Describe some of the varieties of 3D chain formations in the folded structures of proteins. (Careful, the scientific community has now described many thousands of protein structures and the list continues to grow with no end in sight.)
• What approach has technology taken to develop new versions of the jellyfish green fluorescent protein? In other words, how have new colors of the protein been invented?

PART I Central Dogma Thought Questions • Assume that a given...