Molecular Biology Central Dogma (Ch. 7-8)

"The bonds that form between the anticodon of a tRNA molecule and the three nucleotides of a codon in mRNA are _____." Complete this sentence with each of the following options and explain whether each of the resulting statements is correct or incorrect. A. covalent bonds formed by GTP hydrolysis B. hydrogen bonds that form when the tRNA is at the A site C. broken by the translocation of the ribosome along the mRNA

A. Incorrect. The bonds are not covalent, and their formation does not require an input of energy. B. Correct. The aminoacyl-tRNA enters the ribosome at the A site and forms hydrogen bonds with the codon in the mRNA. C. Correct. As the ribosome moves along the mRNA, the tRNAs that have donated their amino acid to the growing polypeptide chain are ejected from the ribosome and the mRNA. The ejection takes place two cycles after the tRNA first enters the ribosome (see Figure 7-37).

In the electron micrograph in Figure 7-8, are the RNA polymerase molecules moving from right to left or from left to right? Why are the RNA transcripts so much shorter than the DNA segments (genes) that encode them?

Actually, the RNA polymerases are not moving at all in the micrograph, because they have been fixed and coated with metal to prepare the sample for viewing in the electron microscope. However, before they were fixed, they were moving from left to right, as indicated by the gradual lengthening of the RNA transcripts. The RNA transcripts are not fully extended because they begin to fold up and interact with proteins as they are synthesized; this is why they are shorter than the corresponding DNA segments.

Could the RNA polymerase used for transcription also be used to make the RNA primers required for DNA replication (discussed in Chapter 6)?

At first glance, the catalytic activities of an RNA polymerase used for transcription could replace the primase that operates during DNA replication. Upon further reflection, however, there would be some serious problems. (1) The RNA polymerase used to make primers would need to initiate every few hundred bases, which is much more often than promoters are spaced on the DNA. Initiation would therefore need to occur in a promoterindependent fashion or many more promoters would have to be present in the DNA, both of which would be problematic for the synthesis of mRNA. In addition, RNA polymerase normally begins transcription on doublestranded DNA, whereas the DNA replication primers are synthesized using single-stranded DNA. (2) Similarly, the RNA primers used in DNA replication are much shorter than mRNAs. The RNA polymerase would therefore need to terminate much more frequently than during transcription. Termination would need to occur spontaneously (i.e., without requiring a terminator sequence in the DNA) or else many more terminators would need to be present. Again, both of these scenarios would be problematic for mRNA production. Although it might be possible to overcome this problem if special control proteins became attached to RNA polymerase during replication, the problem has been solved by the evolution of separate enzymes with specialized properties. Some small DNA viruses, however, do utilize the host RNA polymerase to make RNA primers for their replication.

The Lacheinmal protein is a hypothetical protein that causes people to smile more often. It is inactive in many chronically unhappy people. The mRNA isolated from a number of different unhappy individuals in the same family was found to lack an internal stretch of 173 nucleotides that is present in the Lacheinmal mRNA isolated from happy members of the same family. The DNA sequences of the Lacheinmal genes from the happy and unhappy family members were determined and compared. They differed by a single nucleotide substitution, which lay in an intron. What can you say about the molecular basis of unhappiness in this family? (Hints: [1] Can you hypothesize a molecular mechanism by which a single nucleotide substitution in a gene could cause the observed deletion in the mRNA? Note that the deletion is internal to the mRNA. [2] Assuming the 173-base-pair deletion removes coding sequences from the Lacheinmal mRNA, how would the Lacheinmal protein differ between the happy and unhappy people?)

Because the deletion in the Lacheinmal mRNA is internal, it likely arose from incorrect splicing of the pre-mRNA. The simplest interpretation is that the Lacheinmal gene contains a 173-nucleotide-long exon (labeled "E2" in Figure A7−8), and that this exon is lost ("skipped") during the processing of the mutant precursor mRNA (pre-mRNA). This could occur, for example, if the mutation changed the 3ʹ splice site in the preceding intron ("I1") so that it was no longer recognized by the splicing machinery (a change in the CAG sequence shown in Figure 7-20 could do this). The snRNP would search for the next available 3ʹ splice site, which is found at the 3ʹ end of the next intron ("I2"), and the splicing reaction would therefore remove E2 together with I1 and I2, resulting in a shortened mRNA. The mRNA is then translated into a defective protein, resulting in the Lacheinmal deficiency. Because 173 nucleotides do not amount to an integral number of codons, the lack of this exon in the mRNA will shift the reading frame at the splice junction. Therefore, the Lacheinmal protein would be made correctly only through exon E1. As the ribosome begins translating sequences in exon E3, it will be in the wrong reading frame and will therefore will produce a protein sequence that is unrelated to the Lacheinmal sequence normally encoded by exon E3. Most likely, the ribosome will soon encounter a stop codon, which would be expected to occur on average about once in every 21 codons (there are 3 stop codons in the 64 codons of the genetic code).

(T/F) An mRNA may contain the sequence "ATTGACCCCGGTCAA" .

F. RNA contains uracil but not thymine.

(T/F) The large and small subunits of an individual ribosome always stay together and never exchange partners.

F. Ribosomal subunits can exchange partners after each round of translation. After a ribosome is released from an mRNA, its two subunits dissociate and enter a pool of free small and large subunits from which new ribosomes assemble around a new mRNA.

(T/F) Ribosomes are cytoplasmic organelles that are encapsulated by a single membrane.

F. Ribosomes are not individually enclosed in a membrane.

(T/F) An individual ribosome can make only one type of protein.

F. Ribosomes can make any protein that is specified by the particular mRNA that they are translating. After translation, ribosomes are released from the mRNA and can then start translating a different mRNA. It is true, however, that a ribosome can only make one type of protein at a time.

(T/F) The amount of a protein present in a cell depends on its rate of synthesis, its catalytic activity, and its rate of degradation.

F. The level of a protein depends on its rate of synthesis and degradation but not on its catalytic activity.

(T/F) Because the two strands of DNA are complementary, the mRNA of a given gene can be synthesized using either strand as a template.

F. The position of the promoter determines the direction in which transcription proceeds and therefore which of the two DNA strands is used as the template. Transcription of the other strand would produce an mRNA with a completely different (and in most cases meaningless) sequence.

(T/F) All mRNAs fold into particular three-dimensional structures that are required for their translation.

F. mRNAs are translated as linear polymers; there is no requirement that they have any particular folded structure. In fact, such structures that are formed by mRNA can inhibit its translation, because the ribosome has to unfold the mRNA in order to read the message it contains.

One remarkable feature of the genetic code is that amino acids with similar chemical properties often have similar codons. Thus codons with U or C as the second nucleotide tend to specify hydrophobic amino acids. Can you suggest a possible explanation for this phenomenon in terms of the early evolution of the protein-synthesis machinery?

It is likely that in early cells the matching between codons and amino acids was less accurate than it is in present-day cells. The feature of the genetic code described in the question may have allowed early cells to tolerate this inaccuracy by allowing a blurred relationship between sets of roughly similar codons and roughly similar amino acids. One can easily imagine how the matching between codons and amino acids could have become more accurate, step by step, as the translation machinery evolved into that found in modern cells

Which of the following types of mutations would be predicted to harm an organism? Explain your answers. A. Insertion of a single nucleotide near the end of the coding sequence. B. Removal of a single nucleotide near the beginning of the coding sequence. C. Deletion of three consecutive nucleotides in the middle of the coding sequence. D. Deletion of four consecutive nucleotides in the middle of the coding sequence. E. Substitution of one nucleotide for another in the middle of the coding sequence.

Mutations of the type described in (B) and (D) are often the most harmful. In both cases, the reading frame would be changed, and because this frameshift occurs near the beginning or in the middle of the coding sequence, much of the protein will contain a nonsensical and/or truncated sequence of amino acids. In contrast, a reading-frame shift that occurs toward the end of the coding sequence, as described in (A), will result in a largely correct protein that may be functional. Deletion of three consecutive nucleotides, as described in (C), leads to the deletion of an amino acid but does not alter the reading frame. The deleted amino acid may or may not be important for the folding or activity of the protein; in many cases, such mutations are silent—that is, they have no or only minor consequences for the organism. Substitution of one nucleotide for another, as in (E), is often completely harmless. In some cases, it will not change the amino acid sequence of the protein; in other cases, it will change a single amino acid; at worst, it may create a new stop codon, giving rise to a truncated protein.

The charging of a tRNA with an amino acid can be represented by the following equation: amino acid + tRNA + ATP → aminoacyl-tRNA + AMP + PPi where PPi is pyrophosphate (see Figure 3−41). In the aminoacyl-tRNA, the amino acid and tRNA are linked with a high-energy covalent bond; a large portion of the energy derived from the hydrolysis of ATP is thus stored in this bond and is available to drive peptide bond formation during the later stages of protein synthesis. The free-energy change of the charging reaction shown in the equation is close to zero and therefore would not be expected to favor attachment of the amino acid to tRNA. Can you suggest a further step that could drive the reaction to completion?

One effective way of driving a reaction to completion is to remove one of the products, so that the reverse reaction cannot occur. ATP contains two high-energy bonds that link the three phosphate groups. In the reaction shown, PPi is released, consisting of two phosphate groups linked by one of these high-energy bonds. Thus PPi can be hydrolyzed with a considerable gain of free energy, and thereby can be efficiently removed. This happens rapidly in cells, and reactions that produce and further hydrolyze PPi are therefore virtually irreversible (see Figure 3−41).

Consider the expression "central dogma," which refers to the flow of genetic information from DNA to RNA to protein. Is the word "dogma" appropriate in this context?

Perhaps the best answer was given by Francis Crick himself, who coined the term in the mid-1950s: "I called this idea the central dogma for two reasons, I suspect. I had already used the obvious word hypothesis in the sequence hypothesis, which proposes that genetic information is encoded in the sequence of the DNA bases, and in addition I wanted to suggest that this new assumption was more central and more powerful.... As it turned out, the use of the word dogma caused more trouble than it was worth. Many years later Jacques Monod pointed out to me that I did not appear to understand the correct use of the word dogma, which is a belief that cannot be doubted. I did appreciate this in a vague sort of way but since I thought that all religious beliefs were without serious foundation, I used the word in the way I myself thought about it, not as the world does, and simply applied it to a grand hypothesis that, however plausible, had little direct experimental support at the time." (Francis Crick, What Mad Pursuit: A Personal View of Scientific Discovery. Basic Books, 1988.)

List the ordinary, dictionary definitions of the terms replication, transcription, and translation. By their side, list the special meaning each term has when applied to the living cell.

Replication. Dictionary definition: the creation of an exact copy; molecular biology definition: the act of copying a DNA sequence. Transcription. Dictionary definition: the act of writing out a copy, especially from one physical form to another; molecular biology definition: the act of copying the information stored in DNA into RNA. Translation. Dictionary definition: the act of putting words into a different language; molecular biology definition: the act of polymerizing amino acids into a defined linear sequence using the information provided by the linear sequence of nucleotides in mRNA. (Note that "translation" is also used in a quite different sense, both in ordinary language and in scientific contexts, to mean a movement from one place to another.)

Identify which of the following nucleotide sequences would code for the polypeptide sequence arginine-glycine-aspartate: 1: 5ʹ-AGA-GGA-GAU-3ʹ 2: 5ʹ-ACA-CCC-ACU-3ʹ 3: 5ʹ-GGG-AAA-UUU-3ʹ 4: 5ʹ-CGG-GGU-GAC-3ʹ

Sequence 1 and sequence 4 both code for the peptide Arg-Gly-Asp. Because the genetic code is redundant, different nucleotide sequences can encode the same amino acid sequence.

Figure 7−8 shows many molecules of RNA polymerase simultaneously transcribing two adjacent genes on a single DNA molecule. Looking at this figure, label the 5ʹ and 3ʹ ends of the DNA template strand and the sets of RNA molecules being transcribed.

The RNA transcripts that are growing from the DNA template like bristles on a bottlebrush tend to be shorter at the left-hand side of each gene and longer on the right-hand side. Because RNA polymerase synthesizes in the 5ʹ-to-3′ direction it must move along the DNA template strand in the 3ʹ-to-5ʹ direction (see Figure 7−7). The longest RNAs, therefore, should appear at the 5ʹ end of the template strand—when transcription is nearly complete. Hence the 3′ end of the template strand is toward the left of the image (Figure A7−18). The RNA transcripts, meanwhile, are synthesized in the 5ʹ-to-3ʹ direction. Thus, the 5ʹ end of each transcript can be found at the end of each bristle (see Figure A7−18); the 3ʹ end of each transcript can be found within the RNA polymerase molecules that dot the spine of the DNA template molecule.

A mutation in DNA generates a UGA stop codon in the middle of the mRNA coding for a particular protein. A second mutation in the cell's DNA leads to a single nucleotide change in a tRNA that allows the correct translation of this protein; that is, the second mutation "suppresses" the defect caused by the first. The altered tRNA translates the UGA as tryptophan. What nucleotide change has probably occurred in the mutant tRNA molecule? What consequences would the presence of such a mutant tRNA have for the translation of the normal genes in this cell?

The codon for Trp is 5ʹ-UGG-3ʹ. Thus a normal tRNATrp contains the sequence 5ʹ-CCA-3ʹ as its anticodon (see Figure 7-33). If this tRNA contains a mutation so that its anticodon is changed to UCA, it will recognize a UGA codon and lead to the incorporation of a tryptophan instead of causing translation to stop. Many other protein-encoding sequences, however, contain UGA codons as their natural stop sites, and these stops would also be affected by the mutant tRNA. Depending on the competition between the altered tRNA and the normal translation release factors (Figure 7-41), some of these proteins would be made with additional amino acids at their C-terminal end. The additional lengths would depend on the number of codons before the ribosomes encounter a nonUGA stop codon in the mRNA in the reading frame in which the protein is translated.

Discuss the following: "During the evolution of life on Earth, RNA lost its glorious position as the first selfreplicating catalyst. Its role now is as a mere messenger in the information flow from DNA to protein."

The first statement is probably correct: RNA is thought to have been the first self-replicating catalyst and, in modern cells, is no longer self-replicating. We can debate, however, whether this represents a "loss." RNA now serves many roles in the cell: as messengers, as adaptors for protein synthesis, as primers for DNA replication, as regulators of gene expression, and as catalysts for some of the most important reactions, including RNA splicing and protein synthesis.

A sequence of nucleotides in a DNA strand—5ʹ-TTAACGGCTTTTTTC-3ʹ— was used as a template to synthesize an mRNA that was then translated into protein. Predict the C-terminal amino acid and the N-terminal amino acid of the resulting polypeptide. Assume that the mRNA is translated without the need for a start codon.

The mRNA will have a 5ʹ-to-3ʹ polarity, opposite to that of the DNA strand that serves as the template. Thus the mRNA sequence will read 5ʹ-GAAAAAAGCCGUUAA-3ʹ. The N-terminal amino acid coded for by GAA is glutamic acid. UAA specifies a stop codon, so the C-terminal amino acid is coded for by CGU and is an arginine. Note that the usual convention in describing the sequence of a gene is to give the sequence of the DNA strand that is not used as a template for RNA synthesis; this sequence is the same as that of the RNA transcript, with T written in place of U.

In an alien world, the genetic code is written in pairs of nucleotides. How many amino acids could such a code specify? In a different world, a triplet code is used, but the order of nucleotides is not important; it only matters which nucleotides are present. How many amino acids could this code specify? Would you expect to encounter any problems translating these codes?

With four different nucleotides to choose from, a code of two nucleotides could specify 16 different amino acids (= 42), and a triplet code in which the position of the nucleotides is not important could specify 20 different amino acids (= 4 possibilities of 3 of the same bases + 12 possibilities of 2 bases the same and one different + 4 possibilities of 3 different bases). In both cases, these maximal amino acid numbers would need to be reduced by at least 1 because of the need to specify translation stop codons. It is relatively easy to envision how a doublet code could be translated by a mechanism similar to that used in our world by providing tRNAs with only two relevant bases in the anticodon loop. It is more difficult to envision how the nucleotide composition of a stretch of three nucleotides could be translated without regard to their order, because base-pairing can then no longer be used: AUG, for example, will not base-pair with the same anticodon as UGA.