Genetics Chapter 14

Describe two types of alternative processing pathways. How do these pathways lead to the production of multiple proteins from a single gene?

Alternative processing of pre-mRNA can take the form of either alternative splicing of pre-mRNA introns or the alternative cleavage of 3′ cleavage sites in a pre-mRNA molecule containing two or more cleavage sites for polyadenylation. Alternative splicing results in different exons of the pre-mRNA being ligated to form mature mRNA. Each mRNA formed by an alternative splicing process will yield a different protein. In pre-mRNA molecules with multiple 3′ cleavage sites, cleavage at the different sites will generate mRNA molecules that differ in size. Each alternatively cleaved RNA potentially could code for a different protein depending on the location of the alternative 3′ cleavage site. A single pre-mRNA transcript can undergo both alternative processing steps, thus potentially producing multiple proteins.

How would the deletion of poly (A) tail most likely affect a eukaryotic pre-mRNA?

Polyadenylation increases the stability of the mRNA. If eliminated from the pre- mRNA, then the mRNA would be degraded quickly by nucleases in the cytoplasm.

3′ untranslated region

The 3′ untranslated region is a sequence of nucleotides at the 3′ end of the mRNA that is not translated into proteins. However, it does affect the translation of the mRNA molecule as well as the stability of the mRNA.

Are the 5′ untranslated regions (5′ UTR) of eukaryotic mRNAs encoded by sequences in the promoter, exon, or intron of the gene? Explain your answer.

The 5′ UTR is located in the first exon of the gene. It is not part of the promoter because promoters for RNA polymerase II (which transcribes pre-mRNA) are not normally transcribed, and the 5′ UTR is in the mRNA. It is not located in an intron because introns are removed during processing of pre-mRNA, and the 5′ UTR is part of the mRNA.

5' Cap

The 5′ cap functions in the initiation of translation and mRNA stability.

How would the deletion of 5' cap most likely affect a eukaryotic pre-mRNA?

The deletion of the 5′ cap would most likely prevent splicing of the intron that is nearest to the 5′ cap. Ultimately, elimination of the cap will affect the stability of the pre-mRNA as well as its ability to be translated.

How would the deletion of AAUAAA consensus sequence most likely affect a eukaryotic pre-mRNA?

The deletion of the AAUAAA consensus sequence would prevent binding of the cleavage and polydenylation factor (CPSF), thus resulting in no cleavage or polyadenylation of the pre-mRNA. This would affect the stability and translation of the mRNA.

Promoter

The promoter is the DNA sequence that the transcription apparatus recognizes and binds to initiate transcription.

what are the three primary regions of mRNA sequences in bacterial cells?

(1) The 5′ untranslated region, which contains the Shine-Dalgarno sequence (2) The protein-encoding region, which begins with the start codon and ends with the stop codon (3) The 3′ untranslated region

What are some of the modifications in tRNA that take place through processing?

(1) The precursor RNA may be cleaved into smaller molecules. (2) Nucleotides at both the 5′ and 3′ ends of the tRNAs may be removed or trimmed. (3) Standard bases can be altered by base-modifying enzymes.

Poly (A) Tail

A poly(A) tail is added to the 3' end of the pre-mRNA. It affects mRNA stability.

For the ovalbumin gene shown in Figure 14.3, where would the 5′ untranslated region and 3′ untranslated regions be located in the DNA and in the RNA?

Assuming that alternative splicing is not occurring then the 5′ UTR would be located in exon 1 and the 3′ UTR would be located in exon 8 for both the DNA and RNA molecules.

How do the mRNAs of bacterial cells and the pre-mRNAs of eukaryotic cells differ? How do the mature mRNAs of bacterial and eukaryotic cells differ?

Bacterial mRNA is translated immediately upon being transcribed. Eukaryotic pre- mRNA must be processed and exported from the nucleus. Bacterial mRNA and eukaryotic pre-mRNA have similarities in structure. Each has a 5′ untranslated region as well as a 3′ untranslated region. Both also have protein-coding regions. However, the protein-coding region of the pre-mRNA is disrupted by introns. The eukaryotic pre- mRNA must be processed to produce the mature mRNA. Eukaryotic mRNA has a 5′ cap and a poly(A) tail, unlike bacterial mRNAs. Bacterial mRNA also contains the Shine-Dalgarno consensus sequence. Eukaryotic mRNA does not have the equivalent.

Suppose that a mutation occurs in the middle of a large intron of a gene encoding a protein. What will the most likely effect of the mutation be on the amino acid sequence of that protein? Explain your answer.

Because introns are removed prior to translation, an intron mutation would have little effect on a protein's amino acid sequence unless the mutation occurred within the 5′ splice site, the 3′ splice site, or the branch point. If mutations within these sequences altered splicing such that a new exon is recognized by the spliceosome, then the mature mRNA would be altered, thus altering the amino acid sequence of the protein. The result could be a protein with additional amino acid sequence. Or, possibly, the inclusion of the new exon that was previously intron could introduce a stop codon that stops translation prematurely. If a mutation in the new exon or inclusion of the new exon induced a frameshift, the reading frame and the amino acid sequence would be altered from that point onward in the protein.

What do these RNA molecules do in the cell?

Both siRNAs and microRNAs silence gene expression through a process called RNA interference. Both function by shutting off gene expression of a cell's own genes or to shut off expression of genes from the invading foreign genes of viruses or tranposons. The microRNAs typically silence genes that are different from those from which the microRNAs are transcribed. However, the siRNAs usually silence genes from which they are transcribed. The Piwi-interacting RNAs interact with Piwi proteins to silence gene expression of transposons. The silencing of transposon gene expression by the Piwi-interacting RNAs occurs in animal germ cells and can result in transposon mRNA degradation, chromatin rearrangement that inhibits transcription of the transposons gene, and inhibition of translation of transposon encoded proteins.

What is the function of the 5′ cap?

CAP binding proteins recognize the 5′ cap and stimulate binding of the ribosome to the 5′ cap and to the mRNA molecule. The 5′ cap may also increase mRNA stability in the cytoplasm. Finally, the 5′ cap is needed for efficient splicing of the intron that is nearest the 5′ end of the pre-mRNA molecule.

exons

Exons are transcribed regions that are not removed in intron processing. They include the 5′ UTR, coding regions that are translated into amino acid sequences, and the 3′ UTR.

How is the 5′ cap added to eukaryotic pre-mRNA?

Initially, the terminal phosphate of the three 5′ phosphates linked to the end of the mRNA molecule is removed. Subsequently, a guanine nucleotide is attached to the 5′ end of the mRNA using a 5′ to 5′ phosphate linkage. Next, a methyl group is attached to position 7 of the guanine base. Ribose sugars of adjacent nucleotides may also be methylated, but at the 2′-OH.

introns

Introns are noncoding sequences of DNA that intervene within coding regions of a gene.

Explain how rRNA is processed.

Most rRNAs are synthesized as large precursor RNAs that are processed by methylation, cleavage, and trimming to produce the mature mRNA molecules. In E. coli, methylation occurs to specific bases and the 2′-OH of the ribose sugars of the 30S rRNA precursor. The 30S precursor is cleaved and trimmed to produce the 16S rRNA, 23S rRNA, and the 5S rRNA. In eukaryotes, a similar process occurs. However, small nucleolar RNAs help to cleave and modify the precursor rRNAs.

Explain the process of pre-mRNA splicing in nuclear genes.

Removal of an intron from the pre-mRNA requires the assembly of the spliceosome complex on the pre-mRNA, cleavage at both the 5′ and 3′ splice sites of the intron, and two transesterification reactions ultimately leading to the joining of the two exons. Initially, snRNP U1 binds to the 5′ splice site through complementary base pairing of the U1 snRNA. Next, snRNP U2 binds to the branch point within the intron. The U5 and U4-U6 complex joins the spliceosome, resulting in the looping of the intron so that the branch point and 5′ splice site of the intron are now adjacent to each other. U1 and U4 now disassociate from the spliceosome and the spliceosome is activated. The pre- mRNA is then cleaved at the 5′ splice site, producing an exon with a 3′-OH. The 5′ end of the intron folds back and forms 5′-2′ phosphodiester linkage through the first transesterification reaction with the adenine nucleotide at the branch point of the intron. This looped structure is called the lariat. Next, the 3' splice site is cleaved and then immediately ligated to the 3′-OH of the first exon through the second transesterification reaction. Thus, the exons are now joined and the intron has been excised.

Describe the basic structure of ribosomes in bacterial and eukaryotic cells.

Ribosomes in both eukaryotes and bacteria consist of a complex of protein and RNA molecules. A functional ribosome is composed of a large and a small subunit. The bacterial 70S ribosome consists of a 30S small subunit and a 50S large subunit. Within the small subunit are a single 16S RNA molecule and 21 proteins. The 23S and the 5S RNA molecules, along with 31 proteins, are found in the large bacterial subunit. The eukaryotic 80S ribosome is comprised of a 60S large subunit and a 40S small subunit. Three RNA molecules, the 28S RNA, the 5.8S RNA, and the 5S RNA, are located in the large subunit as well as 49 proteins. The eukaryotic small subunit contains only a single 18S RNA molecule and 33 proteins.

What is the 5′ cap?

The 5′ end of eukaryotic mRNA is modified by the addition of the 5′ cap. The cap consists of an extra guanine nucleotide linked 5′ to 5′ to the mRNA molecule. This nucleotide is methylated at position 7 of the base. The ribose sugars of adjacent bases may be methylated at the 2′ -OH.

5′ untranslated region

The 5′ untranslated region lies upstream of the translation start site. The eukaryotic ribosome binds at the 5′ cap of the mRNA molecule and scans to the first methionine codon (AUG). The region 5′ of this start codon is the 5′ UTR.

What role do CRISPR-Cas systems naturally play in bacteria?

The CRISPR-Cas system essentially serves as an adaptive RNA defense system or immune system for bacterial cells and protects cells against foreign DNA genomes from plasmids or infecting bacteriophages. The spacers between the palindromic repeat sequences consist of short nucleotide sequences captured from previous invading DNA molecules and can help to provide adaptive protection by targeting homologous invading foreign DNA molecules for cleavage by the Cas proteins.

What is the function of the Shine-Dalgarno consensus sequence?

The Shine-Dalgarno consensus sequence functions as the ribosome-binding site on the mRNA molecule.

Duchenne muscular dystrophy is caused by a mutation in a gene that comprises 2.5 million base pairs and specifies a protein called dystrophin. However, less than 1% of the gene actually encodes the amino acids in the dystrophin protein. On the basis of what you now know about gene structure and RNA processing in eukaryotic cells, provide a possible explanation for the large size of the dystrophin gene.

The large size of the dystrophin gene is likely due to the presence of many intervening sequences or introns within the coding region of the gene. Excision of the introns through RNA splicing yields the mature mRNA that encodes the dystrophin protein.

In the early 1990s, Carolyn Napoli and her colleagues were working on petunias, attempting to genetically engineer a variety with dark purple petals by introducing numerous copies of a gene that codes for purple petals (C. Napoli, C. Lemieux, and R. Jorgensen. 1990. Plant Cell 2:279-289). Their thinking was that extra copies of the gene would cause more purple pigment to be produced and would result in a petunia with an even darker hue of purple. However, much to their surprise, many of the plants carrying extra copies of the purple gene were completely white or had only patches of color. Molecular analysis revealed that the level of the mRNA produced by the purple gene was reduced 50-fold in the engineered plants compared with levels of mRNA in wild-type plants. Somehow, the introduction of extra copies of the purple gene silenced both the introduced copies and the plant's own purple genes. Provide a possible explanation for how the introduction of numerous copies of the purple gene silenced all copies of the purple gene.

The overexpression of the purple gene mRNA led potentially to the formation of double-stranded regions by these RNA molecules because of areas of homology within the mRNAs being produced. These double-stranded molecules stimulated RNA silencing mechanisms or the RNA-induced silencing complex (RISC), leading to rapid degradation of the mRNA molecules. The result would be a reduction in translation of the protein needed for the production of purple petals and the phenotypic loss of pigmentation.

A geneticist discovers that two different proteins are encoded by the same gene. One protein has 56 amino acids and the other 82 amino acids. Provide a possible explanation for how the same gene could encode both of these proteins.

The pre-mRNA molecules transcribed from the gene are likely processed by alternative processing pathways. Two possible mechanisms that could have produced the two different proteins from the same pre-mRNA are alternative splicing or multiple 3′ cleavage sites in the pre-mRNA. The cleavage of the pre-mRNA molecule at different 3′ cleavage sites would produce alternatively processed mRNA molecules that differ in size. Translation from each of the alternative mRNAs would produce proteins containing different numbers of amino acids.Alternative splicing of the pre-mRNA could produce different mature mRNAs, each containing a different number of exons and thus the mRNAs differ in size. Again, translation from each alternatively spliced mRNA would generate proteins that differ in the number of amino acids contained.

A geneticist induces a mutation in a cell line growing in the laboratory. The mutation occurs in a gene that encodes a protein that participates in the cleavage and polyadenylation of eukaryotic mRNA. What will be the immediate effect of this mutation on RNA molecules in the cultured cells?

The protein is needed as part of the process for cleavage of the 3′ UTR and for polyadenylation. A nonfunctional protein would result in mRNA lacking a poly(A) tail, and the mRNA would be degraded more quickly in the cytoplasm by nucleases.

Suppose that a 20-bp deletion occurs in the middle of exon 2 of the gene depicted in Figure 14.12a. What will be the likely effect of this deletion in the proteins produced by alternative splicing?

The proteins produced by the mRNA with exons 1, 2, and 3 will likely contain more amino acids and be larger than the protein produced by the mRNA with exons 1 and 3.

What is the origin of small interfering RNAs, microRNAs, and Piwi-interacting RNAs?

The siRNAs originate from the cleavage of mRNAs, RNA transposons, and RNA viruses by the enzyme Dicer. Dicer may produce multiple siRNAs from a single double-stranded RNA molecule. The double-stranded RNA molecule may occur due to the formation of hairpins or by duplexes between different RNA molecules. The miRNAs arise from the cleavage of individual RNA molecules that are distinct from other genes. The enzyme Dicer cleaves these RNA molecules that have formed small hairpins. A single miRNA is produced from a single RNA molecule. The Piwi- interacting RNAs are typically 24 to 30 nucleotides in length and originate from a longer single-stranded precursor RNA molecule.

What makes up the spliceosome? What is the function of the spliceosome?

The spliceosome consists of five small ribonucleoproteins (snRNPs). Each snRNP is composed of multiple proteins and a single small nuclear RNA molecule or snRNA. The snRNPs are identified by which snRNA (U1, U2, U3, U4, U5, or U6) each contains. Splicing of pre-mRNA nuclear introns occurs through the action of the spliceosome

How is the poly(A) tail added to pre-mRNA? What is the purpose of the poly(A) tail?

mRNA molecule. Proteins necessary for cleavage and polyadenylation bind to the AAUAAA consensus sequence, which is located upstream of the 3′ cleavage site, and to a site downstream of the cleavage site. Once the complex has formed, the pre-mRNA is cleaved. Other proteins add adenine nucleotides to the 3′ end creating the poly(A) tail. The presence of the poly(A) tail increases the stability of the mRNA molecule through the interaction of proteins at the poly(A) tail. The length of the poly(A) tail influences the time in which the transcript remains intact and available for translation. The poly(A) tail also assists with the binding of the ribosome to the mRNA and nuclear export.