Genetics Chapter 15

Arrange the following components of translation in the approximate order in which they would appear or be used in prokaryotic protein synthesis:

30S initiation complex 70S initiation complex release factor 1 elongation factor G initiation factor 3 elongation factor Tu fMet-tRNAfMe The components are in order according to when they are used or play a key role in translation. The potential exception is initiation factor 3. Initiation factor 3 could possibly be listed first because it is necessary to prevent the 30s ribosome from associating with the 50s ribosome. It binds to the 30s subunit prior to the formation of the 30s initiation complex. However, during translation events the release of initiation factor 3, allows the 70s initiation complex to form, a key step in translation.

Assume that the nucleotide at the 5' end of the first tRNA's anticodon (the tRNA on the left) in Figure 15.11 were mutated from G to U. Give all codons with which the new, mutated anticodon could pair.

5'—UCA—3' and 5'—UCG—3'

Referring to the genetic code presented in Figure 15.10, give the amino acids specified by the following bacterial mRNA sequences.

5´-AUGUUUAAAUUUAAAUUUUGA-3´ Solution: Amino fMet-Phe-Lys-Phe-Lys-Phe Carboxyl

Explain how some antibiotics work by affecting the process of protein synthesis.

A number of antibiotics bind the ribosome and inhibit protein synthesis at different steps in translation. Some antibiotics, such as streptomycin, bind to the small subunit and inhibit translation initiation. Other antibiotics, such as chloramphenicol bind to the large subunit and block elongation of the peptide by preventing peptide bond formation. Antibiotics such as tetracycline and neomycin bind the ribosome near the A site yet have different effects. Tetracyclines block entry of charged tRNAs to the A site, while neomycin induces translational errors. Finally, some antibiotics such as erythromycin, block the translocation of the ribosome along the mRNA.

Sense codon

A sense codon is a group of three nucleotides that code for an amino acid. In a standard genetic code there are 61 sense codons that code for the 20 amino acids commonly found in proteins.

Initiation codon

An initiation codon establishes the appropriate reading frame and specifies the first amino acid of the protein chain. Typically, the initiation codon is AUG; however, GUG and UUG can also serve as initiation codons although rarely.

Compare and contrast the process of protein synthesis in bacterial and eukaryotic cells, giving similarities and differences in the process of translation in these two types of cells.

Bacterial and eukaryotic cells share several similarities as well as have several differences in protein synthesis. Initially, bacteria and eukaryotes share the universal genetic code. However, the initiation codon, AUG, in eukaryotic cells codes for methionine, whereas in bacteria the AUG codon codes for N-formylmethionine. In eukaryotes, transcription takes place within the nucleus, whereas most translation takes place in the cytoplasm (although some translation does take place within the nucleus). Therefore, transcription and translation in eukaryotes are kept temporally and spatially separate. However, in bacterial cells transcription and translation are coupled and occur nearly simultaneously. Stability of mRNA in eukaryotic cells and bacterial cells is also different. Bacterial mRNA is typically short-lived, lasting only a few minutes. Eukaryotic mRNA may last hours or even days. Charging of the tRNAs with amino acids is essentially the same in both bacteria and eukaryotes. The ribosomes of bacteria and eukaryotes are different as well. Bacteria and eukaryotes have large and small ribosomal subunits, but they differ in size and composition. The bacterial large ribosomal consists of two ribosomal RNAs, while the eukaryotic large ribosomal subunit consists of three. During translation initiation, the bacterial small ribosomal subunit recognizes the Shine-Dalgarno consensus sequence in the 5' UTR of the mRNA and to regions of the 16S rRNA. In most eukaryotic mRNAs, the small subunit binds the 5' cap of the mRNA and scans downstream until it encounters the first AUG codon. Finally, elongation and termination in bacterial and eukaryotic cells are functionally similar, although different elongation and termination factors are used.

How does the process of initiation differ in bacterial and eukaryotic cells

Bacterial initiation of translation requires that sequences in the 16S rRNA of the small ribosomal subunit bind to the mRNA at the ribosome binding site or the Shine- Dalgarno sequence. The Shine-Dalgarno sequence is essential in placing the ribosome over the start codon (typically AUG). In eukaryotes, there is no Shine-Dalgarno sequence. The small ribosomal subunit recognizes the 5' cap of the eukaryotic mRNA with the assistance of initiation factors. Next, the ribosomal small subunit migrates along the mRNA scanning for the AUG start codon. In eukaryotes, the start codon is located with a consensus sequence called the Kozak sequence (5'-ACCAUGG-3'). Transcription in eukaryotes also requires more initiation factors.

A series of tRNAs have the following anticodons. Consider the wobble rules listed in Table 15.2 and give all possible codons with which each tRNA can pair.

From the wobble rules outlined in Table 15.2, we can see that when A occurs at the 5' of the anticodon it can pair only with U in the 3' end of the codon. When C is present at the 5' of the anticodon, it can only pair with G at the 3' of the codon. However, both U and G, when present at the 5' end of the anticodon, can pair with two different nucleotides at the 3' end of the codon (U with A or G; and G with U or C). The rare base iosine (I) is also found at the 5' of the anticodon of tRNA on occasion. Iosine can pair with A, U, or C at the 3' end of the codon.

Overlapping code

If an overlapping code were present, then a single nucleotide would be expected to be included in more than one codon. The result for a sequence of nucleotides within a single gene would be to encode more than one type of polypeptide. However, because the genetic code is nonoverlapping, codons within the same gene do not overlap. Where overlap occurs is in overlapping genes in some viruses, where the same segment of the genome encodes multiple—two or even three (in case of HIV Env gene)—peptides. In such cases, the codons of different peptides are translated in different frames in nonoverlapping fashion.

Nonoverlapping code

In a nonoverlapping code, a single nucleotide is part of only one codon. This results in the production of a single type of polypeptide from one polynucleotide sequence.

Universal code

In a universal code, each codon specifies, or codes, for the same amino acid in all organisms. The genetic code is nearly universal, but not completely. Most of the exceptions occur in mitochondrial genomes

What role do the initiation factors play in protein synthesis?

Initiation factors are proteins that are required for the initiation of translation. In bacteria, there are three initiation factors (IF-1, IF-2, and IF-3). Each one has a different role. IF-1 promotes the disassociation of the large and small ribosomal subunits. IF-3 binds to the small ribosomal subunit and prevents it from associating with the large ribosomal subunit. IF-2 is responsible for binding GTP and delivering the fMettRNAfMet to the initiator codon on the mRNA. In eukaryotes, there are more initiation factors, but many have similar roles. Some of the eukaryotic initiation factors are necessary for recognition of the 5′ cap on the mRNA. Others possess RNA helicase activity, which is necessary to resolve secondary structures.

Give the amino acid sequence of the protein encoded by the mRNA in Figure 15.21.

Met-Pro-Thr-Thr-Ala-Ser-Val-Pro-Leu-Ar

Nonuniversal codons

Most codons are universal (or nearly universal) in that they specify the same amino acids in almost all organisms. However, there are exceptions where a codon has different meanings in different organisms. Most of the known exceptions are the termination codons, which in some organisms do code for amino acids. Occasionally, a sense codon is substituted for another sense codon.

Mutations that introduce stop codons cause a number of genetic diseases. For example, from 2% to 5% of the people who have cystic fibrosis possess a mutation that causes a premature stop codon in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR). This premature stop codon produces a truncated form of CFTR that is nonfunctional and results in the symptoms of cystic fibrosis. One possible way to treat people with genetic diseases caused by these types of mutations is to trick the ribosome into reading through the stop codon, inserting an amino acid into its place. Although the protein produced may have one altered amino acid, it is more likely to be at least partly functional than is the truncated protein produced when the ribosome stalls at the stop codon. Indeed, geneticists have conducted clinical trials on people with cystic fibrosis with the use of a drug called PTC124, which interferes with the ribosome's ability to correctly read stop codons (C. Ainsworth. 2005. Nature 438:726-728). On the basis of what you know about the mechanism of nonsense-mediated mRNA decay (NMD), would you expect NMD to be a problem with this type of treatment? Why or why not?

No, NMD should not be a problem with this type of treatment. NMD is thought to be dependent on exon-junction proteins that are not removed from the mRNA by the movement of the ribosomes during translation. These proteins are thought to interact with enzymes that degrade the mRNA. If the first ribosome to read the mRNA inserts an amino acid for the stop codon due to the action of PTC124, then it should not stall at the stop codon. The ribosome's continued movement along the entire mRNA molecule without stalling should remove any exon-junction proteins before the RNA degrading enzymes are recruited. The result is that the mRNA will be stabilized, thus allowing more translation events to occur.

Nonsense codon

Nonsense codons or termination codons signal the end of translation. These codons do not code for amino acids.

What are some types of posttranslational modification of proteins?

Several different modifications can occur to a protein following translation. Frequently, the amino-terminal methionine may be removed. Sometimes, in bacteria only the formyl group is cleaved from the N-formylmethionine, leaving a methionine at the amino terminal. More extensive modification occurs in some proteins that are originally synthesized as precursor proteins. These precursor proteins are cleaved and trimmed by protease enzymes to produce a functional protein. Glycoproteins are produced by the attachment of carbohydrates to newly synthesized proteins. Molecular chaperones are needed by many proteins to ensure that the proteins are folded correctly. Secreted proteins that are targeted for the membrane or other cellular locations frequently have 15 to 30 amino acids, called the signal sequence, removed from the amino terminal. Finally, acetylation of amino acids in the amino terminal of some eukaryotic proteins also occurs.

What is the significance of the fact that many synonymous codons differ only in the third nucleotide position?

Synonymous codons code for the same amino acid, or, in other words, they have the same meaning. A nucleotide at the third position of a codon pairs with a nucleotide in the first position of the anticodon. Unlike the other nucleotide positions involved in the codon-anticodon pairing, this pairing is often weak, or "wobbles," and nonstandard pairings can occur. Because the "wobble," or nonstandard base pairing with the anticodons, affects the third nucleotide position, the redundancy of codons ensures that the correct amino acid is inserted in the protein when nonstandard pairing occurs.

How is the reading frame of a nucleotide sequence set?

The initiation codon on the mRNA sets the reading frame.

The following amino acid sequence is found in a tripeptide: Met-Trp-His. Give all possible nucleotide sequences on the mRNA, on the template strand of DNA, and on the nontemplate strand of DNA that could encode this tripeptide.

The potential mRNA nucleotide sequences encoding for the tripeptide Met-Trp-His can be determined by using the codon table found in Figure 15.10. From the table, we can see that the amino acid His has two potential codons, while the amino acids Met and Trp each have only one potential codon. Therefore, there are two different mRNA nucleotide sequences that could encode for the tripeptide. Once the potential mRNA nucleotide sequences have been determined, the template and nontemplate DNA strands can be derived from these potential mRNA sequences. (1) 5'-AUGUGGCAU-3' DNA template: 3'-TACACCGTA-5' DNA nontemplate: 5'-ATGTGGCAT-3 (2) 5'-AUGUGGCAC-3' DNA template: 3'-TACACCGTG-5' DNA nontemplate: 5'-ATGTGGCAC-3'

What events bring about the termination of translation?

The process of termination begins when a ribosome encounters a termination codon. Because the termination codon would be located at the A site, no corresponding tRNA will enter the ribosome. This allows for the release factors (RF-1, RF-2, and RF-3) to bind the ribosome. RF-1 recognizes and interacts with the stop codons UAA and UAG, while RF-2 can interact with UAA and UGA. A RF-3-GTP complex binds to the ribosome. Termination of protein synthesis is complete when the polypeptide chain is cleaved from the tRNA located at the P site. During this process, the GTP is hydrolyzed to GDP.

Reading frame

The reading frame refers to how the nucleotides in a nucleic acid molecule are grouped into codons containing three nucleotides. Each sequence of nucleotides has three possible sets of codons, or reading frames.

Termination codon

The termination codon signals the termination or end of translation and the end of the protein molecule. There are three termination codons—UAA, UAG, and UGA—which can also be referred to as stop codons or nonsense codons. These codons do not code for amino acids.

Give the elongation factors used in bacterial translation and explain the role played by each factor in translation.

Three elongation factors have been identified in bacteria: EF-TU, EF-TS, and EF-G. EF-TU joins with GTP and then to a tRNA charged with an amino acid. The charged tRNA is delivered to the ribosome at the A site. During the process of delivery, the GTP joined to EF-TU is cleaved to form an EF-TU-GDP complex. EF-TS is necessary to regenerate EF-TU-GTP. The elongation factor EF-G binds GTP and is necessary for the translocation or movement of the ribosome along the mRNA during translation.

A nontemplate strand on bacterial DNA has the following base sequence. What amino acid sequence will be encoded by this sequence? 5´-ATGATACTAAGGCCC-3´

To determine the amino acid sequence, we need to know the mRNA sequence and the codons present. The nontemplate strand of the DNA has the same sequence as the mRNA, except that thymine-containing nucleotides are substituted for the uracilcontaining nucleotides. So the mRNA sequence would be as follows: 5'-AUGAUACUAAGGCCC-3'. Assuming that the AUG indicates a start codon, then the amino acid sequence would be starting from the amino end of the peptide and ending with the carboxyl end: fMet-Ile- Leu-Arg-Pro.