Chapter 2

View the human receptor for the steroid called glucocorticoid bound to DNA. What must happen before a steroid receptor can bind to DNA?

Steroid receptors form homodimers, meaning two receptors bind to each other. Furthermore, it must move into the nucleus where the DNA is located.

How do steroids affect your cells?

Steroids only affect cells that produce a steroid receptor even though they diffuse into every cell in your body. Once bound to its receptor, the steroid and protein receptor migrate to the nucleus and bind to appropriate promoters to activate genes to be transcribed.

Calculate the percent of bases that are A or T inside the box for each of the six sequences in Figure 2.13(Alignment of DNA sequences containing promoters). Compare these values to the percent A or T for the next seven bases downstream, to the right, of the box. If there were no bias in the DNA, you would expect these two percentages to be near 50%. Are they? Review the number of hydrogen bonds between A:T and G:C from IQ #26 in Section 1.4. What functional significance might the percent AT have on the structure of DNA?

The A or T percentages range from 57% (4 out of 7) for sequences 1 and 4, to 100% for sequence number 2. The next 7 bases range from 57% A or T down to 0% A or T. These two percentage ranges do not seem similar to each other or close to 50% on average. AT base pairs have only 2 H-bonds compared to 3 H-bonds between GC base pairs. The two strands of AT-rich dsDNA sequences are held together less firmly than GC-rich dsDNA.

View the structure of LacI+ bound to DNA in Figure 2.11C(Molecular structure of LacI+ bound to lacO+). Describe the shape of lacO+ when LacI+ is bound. Based on the shape of the lacO+ DNA, hypothesize how LacI+ represses β-galactosidase and permease transcription.

The DNA is forced to make a complete loop by the bound LacI protein. Since lacO+ is a promoter that RNA polymerase binds, the loop appears to block RNA polymerase binding.

Hypothesize about the function of lacO+ and lacO- alleles (sixth and seventh rows) given the data in Table 2.1. Hint: Determine whether the mutant lacO- is able to affect both of the diploid lacβ alleles or only one.

The DNA labeled lacO- is a mutated allele of lacO+ which is the wild-type, or functional, version. lacO appears to function as an on/off switch but it cannot "reach" over to affect the second copy.

What proteins determine when a gene is transcribed and by how much?

The RNA polymerase is always involved. Sometimes transcription factors are required to facilitate RNA polymerase binding. Sometimes repressors, such as LacI protein, block transcription but this blockage can be removed with the binding of an activator such as lactose.

Which sequence initiated transcription the best in Figure 2.14B(Electrophoresis results from mRNA (arrowheads) produced using three variations of promoters with the wild-type (wt) sequence TATA shown from panel A)? Rank the three tested promoters from strongest to weakest.

The best is TATA >> TAGA > TAAA.

Look at the full genetic code in Figure 2.23(Full genetic code used by plants, animals, bacteria and archaea); three codons do not encode any amino acid. What is their function? In most species, the first amino acid in a protein is methionine (M). What codon encodes for this amino acid and has the nickname of the "start codon?" Be sure you are able to translate an mRNA using the genetic code.

There are three stop codons that tell the ribosome to stop adding amino acids. ATG (AUG) encodes for methionine. When translating an mRNA, look for the start codon to determine the reading frame.

Go to the interactive Jsmol site showing LacI+ protein bound to lacO+. How many different proteins are bound to the DNA? What effect do they have on the shape of the DNA structure?

There are two copies of LacI+ proteins bound to the lacO DNA. These two proteins cause the DNA to bend about 30 degrees off the vertical.

Which amino acids are encoded by four codons? Which base of these codon can be altered without changing the encoded amino acids? Are there instances when the third base is changed and it does alter which amino acid is encoded?

Valine, alanine, threonine, leucine, serine, proline, arginine and glycine are encoded by 4 or more codons. For those with exactly four codons, you can change the third base and the amino acid does not change. Yes, if you look at the four codons that start with AG_, you can see that changing the third base can alter the encoded amino acid. There are several other examples.

Other than the very prominent rRNA bands, can you see any other size categories of RNA in the two lanes? Describe the size and relative amounts of the different RNA size categories you see. Remember, the gel only appears white where RNA is present.

You can see a fuzzy band at the bottom of the gel which means it is very small molecular weight. You can also see a general smear of faint white from near the wells at the top down to the fuzzy band at the bottom.

In order to understand how proteins are made, we need to examine a cell's RNA content. Look at the RNA gel in Figure 2.3(Black and white photo of gel electrophoresis for two RNA samples), and find the very bright rRNA bands. How many different sized rRNA molecules are in a ribosome?

You can see two rRNA bands of different sizes.

How can you tell when a promoter has transcription factors bound to it in Figure 2.15(X-ray film shows the results of a promoter-binding experiment)? Do the three transcription factors and RNA polymerase each bind independently to the promoter in Figure 2.15?

You can tell because the radioactive promoter DNA is small and normally migrates to the bottom of the gel but when all the proteins are added, the radioactive DNA migrates much slower, indicating its molecular weight has increased dramatically. The three proteins can only bind when all are present. No combination of one or two proteins is able to bind to the DNA.

If ribosomes assemble amino acids into proteins, speculate about the function of tRNA molecules that bind to one amino acid at a time.

he function of tRNAs is to bring the appropriate amino acid to the ribosome to be added to the growing protein.

UV light measures the presence of RNA regardless of radioactivity. Which type of RNA is the most abundant in Figure 2.6(Centrifugation of RNA isolated from virally infected cells)? What differences do you see compared to Figure 2.3(Black and white photo of gel electrophoresis for two RNA samples)?

rRNA not associated with ribosomes and rRNA that is part of intact ribosomes are the most abundant RNAs in Figure 2.6. In Figure 2.3, the ribosomes were intentionally disrupted, so you can see the two sizes of rRNA. In Figure 2.6, the samples were prepared more gently in order to disrupt as few intact ribosomes as possible. In this separation, the two sizes of rRNA are together in the single, tallest peak.

Name the three types of RNA and the role each plays in protein formation.

rRNA: comprise 60% of the ribosome and are responsible for polymerizing amino acids into proteins (translation). tRNA: brings individual amino acids to active, intact ribosomes. The tRNA base pairs with mRNA to make sure the correct amino acid is being added to the growing protein. mRNA: encodes protein amino acid sequence and is read by the ribosome which assembles the amino acids in the correct order.

Review the biological concept behind central dogma, and describe the role of the molecular players required to produce a phenotype.

Central dogma is the transmission of genetic information to functional proteins where DNA is transcribed to RNA which is translated into protein. Transcription factors, repressors and RNA polymerase govern transcription. The ribosome and its rRNA carry out translation along with tRNA that brings amino acids to base pair with mRNA. Proteins are responsible for nearly every phenotype, either directly or indirectly. It is conserved in all three domains of life.

In Figure 2.21(Deciphering the first codon), why did they use U's instead of T's in their synthetic mRNA? Which temperature was able to translate the poly-U code the fastest? How can you tell? Hypothesize why the amount of phenylalanine polymer produced at 37° C began to decline after 5 minutes.

DNA does not contain the base uracil, only RNA does. This way they could be sure that any translation that happened came from RNA and not DNA. 37° C allowed the fastest translation which you can tell from the initial slope, not the end point. 37° C is a warm temperature which could have led to the breakdown of existing protein.

Explain how DNA information is not directly converted to protein information.

DNA encodes proteins, but DNA is never used directly in translation. RNA is the precursor to protein production.

Review the biological concept behind central dogma, and describe the role of the molecular players required to produce a phenotype.

DNA encodes proteins, but DNA is not used directly in protein production (translation). DNA is transcribed by RNA polymerase into three types of RNA (see above). The production of new proteins is governed by the production of specific mRNAs. The tRNAs and rRNAs are produced consistently so they are always ready to translate any available mRNAs (viral or cellular). Central dogma is the transcription of DNA into RNA which is translate into proteins.

Explain how DNA information is not directly converted to protein information.

DNA is transcribed to RNA which is the template for protein synthesis. DNA indirectly encodes proteins via mRNA.

Figure 2.9 (Proposed mechanism for activation of lactose-responsive genes. Jacob)depicts a proposed model of gene regulation with a pair of inhibitors that lactose inhibits. By analogy, construct a sentence that means you like to sleep, but use the word "not" two times in the sentence. Now describe the mechanism of ß-galactosidase gene induction in Figure 2.8(2.8 Induction of β-galactosidase after exposure to lactose) using the nouns "inhibitor" and "lactose" and the verb "inhibits."

Lactose inhibits the gene inhibitor which turns on gene transcription.

Is the promoter in Figure 2.16 (Mapping a promoter) a digital on/off switch that only has two states: full on and full off? Use the different promoter lengths to determine which portions of this promoter contained functionally-important DNA.

No, this promoter has two powers (full and partial). The functional regions of this promoter are between bases 155 and 113 and between 78 and 29. You can tell this because when the promoters is deleted at 155, the strength is full. The next deletion to 113 knocks the power down to partial strength. The same logic applies to the 78-29 region. Perhaps transcription factors bind to these two regions of the promoter.

Compare and contrast RNA synthesis and DNA synthesis. Both RNA and DNA synthesis uses a DNA template to encode the complementary strand.

RNA is composed of RNA nucleotides that have an OH on the 2' carbon while DNA is composed of DNA nucleotides that have only H on the 2' carbon. The bases are identical except DNA has thymine (T) while RNA has uracil (U), though the both base pair with adenine (A). RNA synthesis is called transcription but DNA synthesis is called replication which was discussed extensively in Chapter 1. Both nucleotide polymers are synthesize in the direction of 5' to 3', with the newest nucleotide added to the 3' end.

Is RNA polymerase bigger than a DNA nucleotide, or is it smaller? Use the data from Figure 2.12 (Partial sequences of a T7 promoter and encoded mRNA) to support your answer. Look at the aligned sequences inside the red box of Figure 2.13(Alignment of DNA sequences containing promoters). Can you visually detect a sequence pattern for promoters? Is there a rule that describes which base will be the first one to be transcribed?

RNA polymerase is much bigger than a nucleotide since one protein covers about 50 nucleotide pairs. There is an A in all six sequences at the second position, and a T at the penultimate (next to last) position. It is hard to see a shared pattern in these six sequences. The +1 nucleotide is the sixth or seventh base after the boxed sequence.

Speculate how steroids that first bind to proteins in the cytoplasm can become located in nuclei later. Use the data from Figure 2.19(Tracing progesterone location over time) to support your answer.

Receptors without steroids are located in the cytoplasm, but once they bind a steroid, they move into the nucleus. The loss from the cytoplasm is mirrored by the arrival in the nucleus.

If steroids can diffuse into every cell but activate genes in only a subset of cells, how do organisms regulate which cells will respond to a particular steroid?

Regulation of genes by steroids is controlled by which cells produce steroid receptors. Any cell lacking a receptor cannot activate genes in response to a steroid.

How does a ribosome "know" which amino acids should be used at any given time?

Ribosomes are responsible for forming a covalent bond between the growing protein and the newest amino acid being added on. The tRNA is key to bringing the right amino acid to base pair with the mRNA. We don't have sufficient information to know how active the ribosome is in screening the tRNAs to make sure they are properly paired with the codons.

Describe the activity of a ribosome.

Ribosomes are the focal point for translation. They are central to the polymerization of amino acids in the right sequence to produce a functional protein.

Describe the size distribution of the radioactive RNA in Figure 2.6(Centrifugation of RNA isolated from virally infected cells). Speculate about the function of the radioactive RNA transcribed since the time of infection by the virus?

A large amount of the radioactive RNA is associated with intact ribosomes so this RNA must encode the viral proteins. Now we call this RNA messenger RNA (mRNA). There is also a large amount of very small radioactive RNA. This could be new mRNA not yet associated with ribosomes or perhaps tRNAs. We cannot distinguish these two possibilities in this experiment.

What effects can a DNA-binding protein have on RNA polymerase activity?

A repressor protein can block RNA polymerase from binding the DNA and thus block transcription. Alternately, a transcription factor can facilitate RNA polymerase binding to a promoter and thus increase the transcription of a gene.

List specific examples of post-translational processing.

After translation, proteins are processed by: 1) folding in 3D shapes; 2) disulfide bonds can form to hold their shapes; 3) proteins translated on the rough ER have their signal sequences trimmed off (the first ~25 amino acids); 4) some protein have other portions removed as was seen for insulin.

How do proteins "know" where to bind DNA in order to activate or repress a gene?

All proteins function based on the physical shape and chemical properties of their constituent amino acids. Proteins bind to the complementary shape of the DNA base pairs.

Which components from the experiment in Figure 2.20B(In vitro translation with each column representing a different experiment and + indicating presence of an ingredient) are necessary for translation tooccur? Which parts, if any, were not required for translation?

All the components except DNA are required for translation. DNA has no role if the RNAs are already produced.

Calculate the ratio of ß-galactosidase to total protein using three points from the diagonal line in Figure 2.8(Induction of β-galactosidase after exposure to lactose). Use these data to argue that the transcription of the ß-galactosidase gene is induced by lactose.

At 15g, the amount of ß-galactosidase was 0.2 μg for a ratio of 0.013 (ignoring units since this is the same for all three ratios). At 22g, the amount of ß-galactosidase was 0.75 for a ratio of 0.034 (ignoring units since this is the same for all three ratios). At 45g, the amount of ß-galactosidase was 2.25 for a ratio of 0.05 (ignoring units since this is the same for all three ratios). From these three ratios, you can tell that the proportion of total protein that was ß- galactosidase increased almost four fold (0.05 ÷ 0.013 ≈ 3.85).

Are promoters sequence-specific, or can all strings of As and Ts work equally well? How did the TAGA promoter contradict the hypothesis that AT-rich promoters are the best?

Because TATA is much better promoter than TAAA, you can prove that AT-rich promoters are not all the same. Therefore the promoter is sequence specific and not simply areas with AT-rich DNA.

Use Figure 2.10(Genetic map of DNA involved in lactose metabolism) to explain why the β-galactosidase enzyme was produced before permease.

Because the lacβ gene comes before the lacP gene (from left to right), β-galactosidase is transcribed first and thus translated first.

Review the biological concept behind central dogma, and describe the role of the molecular players required to produce a phenotype.

Central dogma is the transmission of genetic information to functional proteins where DNA is transcribed to RNA which is translated into protein. Transcription factors, repressors and RNA polymerase govern transcription. The ribosome and its rRNA carry out translation along with tRNA that brings amino acids to base pair with mRNA. Proteins are responsible for nearly every phenotype, either directly or indirectly. Central dogma is conserved in all three domains of life.

In Table 2.1(Synthesis of β-galactosidase in diploid and haploid E. coli), how much lacβ gene is transcribed in cells with a lacID allele (fourth and fifth rows) when lactose is present? What can you conclude about the function of the LacI protein? Is the LacI protein able to diffuse within a cell, or is it constrained to only the adjoining lacβ allele?

Essentially no lacβ was transcribed. In biology, it is very rare to get zero signal for a detection method, so 1 or 2 can be considered no biologically significant amount of mRNA produced.

Why are some genes bigger than the mRNA they encode? Do all genes share this trait? Explain your answer.

Genes in most eukaryotes contain introns that are spliced out. Some human genes lack introns and prokaryotes do not have introns as a general rule.

What function is accomplished by the TATA box and other promoter sequences?

The function is to get RNA polymerase to bind at the appropriate time and frequency so that the right amount of RNA is transcribed.

Which sequence is longer, the mRNA or the gene? How can you tell? By how many bases do these two coding segments differ? Use only the first aligned sequences below the graph to determine if any mRNA bases are not present in the gene. Predict how many amino acids would be in the human insulin protein after translation, given the length of the mRNA.

The gene is much longer (8416 bp) than the mRNA (469) for a difference of 7,947 base pairs (bp). All of the bases in the mRNA are in the gene, but there are bases in the gene that do not appear in the mRNA. You should calculate 156 amino acids encoded by the mRNA if you divide 469 by 3, but this is not the biologically correct answer.

Illustrate one example of how a gene's activity is regulated, in part, by environmental factor, such as the availability of sugar.

The lac operon encodes two proteins, β-galactosidase and permease. Normally, the LacI repressor block RNA polymerase from binding to the promoter, called lacO. When lactose is present, the sugar binds to LacI, causing a shape change and LacI no longer can bind to lacO. This release of lacO allows the RNA polymerase to bind and initiate transcription.

Use the data in Figure 2.18(Amount of steroid bound by its receptor) to support the conclusion that the steroids bind to protein receptors.

The only time less steroid bound to its receptor was when the receptor was incubated with a protease that destroys proteins.

Given that rRNA is not radioactive, when was the rRNA transcribed for this experiment? How can intact ribosomes associate with radioactive RNA if the rRNA is not radioactive?

The rRNA must have been produced (transcribed) prior to the infection by virus. Intact ribosomes, with non-radioactive rRNA, co-separate with the radioactive RNA that was produced after infection. The radioactive RNA must encode the viral proteins that are produced shortly after infection.

Use the data in Figure 2.5A(RNA molecules interact with radioactive leucine) to determine which form of RNA binds to individual amino acids prior to protein production. Did the amino acid leucine directly interact with rRNA (ribosomes) where proteins are produced? Support your answer with data.

The radioactive leucine has co-separated with the small molecular weight tRNA and not the two larger molecular weight rRNA. Therefore, individual amino acids interact directly with tRNA prior to protein formation (translation).