Chapter 12 Exam 2 (Minimal promoter TF2D TATA, splicing mechanism, sherps involed in finding 5' donor 3' acceptor sites, RNAi, regulates genes expression, experiments to determine genetic code)

Transcription Doc Explain the work of Nirenberg

1. Synthesized RNA homopolymers, each with only one type of ribonucleotide. (AAAAAA,CCCCCCC, UUUUUU, or GGGGGG) Labeled 1 of the 20 amino acids added to the in vitro system and conducted a series of experiments, each with different radioactively labeled amino acid. 2. Then turned to RNA heteropolymers Two or more different ribonucleoside diphosphates were added in combination to form the synthetic mRNA. (Could predict the composition but not specific sequence of triplets) By examining the percentages of any given amino acid incorporated into the protein synthesized, we can propose probable base compositions for each amino acid. Specific sequences are still unknown with these two methods. The use of RNA homopolymers and mixed copolymers in a cell-free system allowed the determination of the composition, but not the SEQUENCE, of triplet codons designating specific amino acids. 3. Developed the triplet binding assay. Observed that ribosomes, when presented in vitro with an RNA sequence as short as three ribonucleotides, will bind to it and form a complex similar to that found in vivo. The triplet acts like a codon in mRNA, attracting the complementary sequence within tRNA. The triplet sequence in tRNA that is complementary to a codon of mRNA is an anticodon. 4.The amino acid to be tested was made radioactive, and a charged tRNA was produced. Because codon compositions were known, researchers could narrow the range of amino acids that should be tested for each specific triplet. The radioactively charged tRNA, the RNA triplet, and ribosomes were incubated together and then passed through a nitrocellulose filter, which retains the larger ribosomes but not the other smaller components, such as unbound charged tRNA. If radioactivity is not retained on the filter, an incorrect amino acid has been tested. But if radioactivity remains on the filter, it is retained because the charged tRNA has bound to the triplet associated with the ribosome. When this occurs, a specific codon assignment can be made. This made up 50 of the 64 triplets. Led to belief that the genetic code is degenerate;that is one amino acid can be specified by more than one triplet and second, the code is unambiguous; a single triplet specifies only one amino acid.

PS12 Below is a partial DNA sequence used to answer the following questions. 5' - TATGCAGCACATT - 3' 3' - ATACGTCGTGTAA - 5' If transcription occurred from right to left, what RNA sequence would be produced? 5' - UAUGCAGCACAUU - 3' 3' - AUACGUCGUGUAA - 5' 3' - ATACGTCGTGTAA - 5' 5' - TATGCAGCACATT - 3' 5' - AUGCAGCACAUU - 3'

3' - AUACGUCGUGUAA - 5'

PS12 Below is a partial DNA sequence used to answer the following questions. 5' - TATGCAGCACATT - 3' 3' - ATACGTCGTGTAA - 5' If transcription occurred from right to left, which strand is the template strand? 3' - TATGCAGCACATT - 5' 5' - TATGCAGCACATT - 3' 5' - ATACGTCGTGTAA - 3' 3' - ATACGTCGTGTAA - 5'

5' - TATGCAGCACATT - 3'

PS12 Below is a partial DNA sequence used to answer the following questions. 5' - TATGCAGCACATT - 3' 3' - ATACGTCGTGTAA - 5' If transcription occurred from left to right, what the RNA sequence would be produced? 5' - TATGCAGCACATT - 3' 5' - UAUGCAGCACAUU - 3' 5' - AUGCAGCACAUU - 3' 3' - ATACGTCGTGTAA - 5' 3' - AUACGUCGUGUAA - 5'

5' - UAUGCAGCACAUU - 3'

PS12 Which of the following is most likely attributable to a base substitution at a 5' splice junction? A longer than usual final transcript A shorter than usual final transcript Failure of the U2 snRNP to bind the transcript An amino acid substitution in the final protein product

A longer than usual final transcript

PS12 Which three statements correctly describe the processing that takes place before a mature mRNA exits the nucleus? A poly-A tail (50-250 adenine nucleotides) is added to the 3' end of the pre-mRNA. A cap consisting of a modified guanine nucleotide is added to the 5' end of the pre-mRNA. A translation stop codon is added at the 3' end of the pre-mRNA. Noncoding sequences called introns are spliced out by molecular complexes called spliceosomes. Coding sequences called exons are spliced out by ribosomes.

A poly-A tail (50-250 adenine nucleotides) is added to the 3' end of the pre-mRNA. A cap consisting of a modified guanine nucleotide is added to the 5' end of the pre-mRNA. Noncoding sequences called introns are spliced out by molecular complexes called spliceosomes.

CQ8 Why does E. coli have several different sigma factors? A) They allow RNA polymerase to recognize and bind to a different subset of promoters. B) They allow the different subunits of the RNA polymerase holoenzyme to bind to each other. C) They allow different RNA polymerases to bind to the promoters. D) This locus recently duplicated in evolutionary time. E) One is needed to transcribe mRNA. A second is needed to transcribe tRNA. And a third is needed to transcribe rRNA.

A) They allow RNA polymerase to recognize and bind to a different subset of promoters.

CQ8 In 1964, Nirenberg and Leder used the triplet-binding assay to determine specific codon assignments. A complex of which of the following components was trapped in the nitrocellulose filter? A) charged tRNA, RNA triplet, and ribosome B) sense and antisense strands of DNA C) ribosomes and DNA D) uncharged tRNAs and ribosomes E) free tRNAs

A) charged tRNA, RNA triplet, and ribosome

The poly(A) tail of mRNAs A) is added to the 3′ end of mRNAs. B) is found on most mature eukaryotic mRNAs. C) helps prevent degradation of eukaryotic mRNAs. D) All of these answers are correct.

A) is added to the 3′ end of mRNAs. D) All of these answers are correct.

CQ8 Shown here are the amino acid sequences of the wild-type and two mutant forms of a short protein. Use this information to answer the following questions: Wild type: met-trp-tyr-arg-gly-ser-pro-thr Mutant 1: met-trp-his-arg-gly-ser-pro-thr Mutant 2: met-cys-ile-val-val-val-gln-his Using the information from each mutant protein, determine the ribonucleotide sequence of the wild-type protein. For each mutant, a single nucleotide change occurs. Place spaces between each codon. For ambiguous bases, bracket the possible nucleotides with parentheses. Do not include the stop codon in your answer.

AUG UGG UAU CGU GGU AGU CAA ACA

Transcription Doc Be able to explain RNA splicing.

After an endonucleolytic "cut" is made at each end of an intron, the intron is removed, and the terminal ends ofthe adjacent exons are ligated by an enzye. Figure 12-12

Textbook How did we determine the compositions of codons encoding specific amino acids?

An initial understanding about the composition (unordered) of codons came from homopolymer and heteropolymer RNAs introduced into an in vitro translation system. Assays of relative amino acid composition of resulting polypeptides indicated the composition of the bases in codons, but not their actual sequence.

Textbook How were the experimentally derived triplet codon assignments verified in studies using bacteriophage MS2?

Because the complete sequence of the RNA phage MS2 was known, scientists matched that sequence with protein products of MS2, which supported the code as derived from previous studies.

CQ8 tRNAs have A) consist of two subunits. contain unusual nucleotides coded B) by genes with nonstandard bases. can be recognized by several C) aminoacyl-tRNA synthetases. D) have double-stranded regions. E) can carry more than one type of amino acid per molecule.

D) have double-stranded regions.

Transcription Doc Describe the activity of RNA polymerase

Directs the synthesis of RNA on a DNA template. It has the same general substrate requirements as does DNA polymerase, the major difference being that the substrate nucleotides contain the RIBOSE instead of the DEOXYRIBOSE form of the sugar.

CQ8 Peptidyl transferase A) is a polypeptide subunit of the small ribosomal subunit. B) catalyzes the reaction that joins a tRNA to its amino acid. C) is active during initiation of translation. D) is not found in eukaryotes. E) is a function of an rRNA in the large ribosomal subunit.

E) is a function of an rRNA in the large ribosomal subunit.

PS12 Which of the following is a characteristic of RNA splicing in Eukaryotes? Exon/intron boundaries are typically characterized by a 5' GU splice junction and a 3' AG splice junction. It involves removal of introns from a gene sequence followed by transcription and subsequent splicing of exons. It involves recognition of sequence-specific intron/exon boundary sites by cytoplasmic proteins. After splicing occurs, the U1, U2, U5, U6 snRNP complex removes remaining exons for degradation.

Exon/intron boundaries are typically characterized by a 5' GU splice junction and a 3' AG splice junction.

Transcription Doc Be able to explain the spliceosome mechanism.

Figure 12-13 Splicing reactions are mediated by a splicosome.

What is a frameshift mutation?

Insertion of a single nucleotide shifts all subsequent reading frames.

Transcription Doc How does the sigma factor effect recruitment of RNA polymerase in prokaryotes?

Plays a regulatory function in the initiation of RNA transcription. Allows the elongation of RNA.

Transcription Doc How is RNA polymerase II recruited to the TATA box in eukaryotes? How does this differ from prokaryotes?

Prokaryotes,RNA polymerase binds directly to the -10 promoter region. Not the case in eukaryotes. A wide range of core-promoter and proximal-promoter elements are also found within eukaryotic gene-regulatory regions, and each can have an effect on the efficiency of transcription initiation from the start site.

Textbook/Transcription Doc In a written paragraph, describe the abbreviated chemical reactions that summarize RNA polymerase-directed transcription.

Ribonucleoside triphosphates and a DNA template in the presence of RNA polymerase and a divalent cation (Mg2+) produce a ribonucleoside monophosphate polymer, DNA, and pyrophosphate (diphosphate). Equimolar amounts of precursor ribonucleoside triphosphates and product ribonucleoside monophosphates and pyrophosphates (disphosphates) are formed. In E. coli, transcription and translation can occur simultateously. Ribosomes attach to the 5' end of the nascent mRNA and progress to the 3' end during translation. Although transcription/translation can be "visualized" in E. coli, the predominant components "visualized" are the strings of ribosomes (polysomes).

Transcription Doc Explain how the genetic code was determined. Explain the work of Nirenberg. What was his success based off of? How could he force reactions in the opposite direction to synthesize RNA?

Success of Nirenberg was dependent on in vitro (cell-free) protein-synthesizing system and polynucleotide phosphorylase (enabled production of mRNAs. Through the use of high concentrations of ribnucleoside diphosphates, the reaction can be forced in the opposite direction to synthesize RNA. *Since polynucleotide phosphorylase does not require a DNA template, Each addition of a ribonucleotide is random, based on the relative concentration of the four ribonucleoside diphosphates added to the reaction mixtures. Together, the cell-free system and the availability of synthetic mRNAs provided a means of deciphering the ribonucleotide composition of various triplets encoding specific amino acids. *

Textbook How were the specific sequences of triplet codes determined experimentally?

The specific sequences of the triplet codes were determined by the triplet binding assay and repeating copolymers of known sequence. When added to a cell-free translation system, a direct analysis of codon assignments was possible.

Transcription Doc The genetic code is nearly universal. What exceptions are there?

Triplets have a normal code word but they are altered Found primarily in Human and yeast mitochondira, paramecium,tetrahymena, and stylonychia.

PS12 Suppose that a portion of double-stranded DNA in the middle of a large gene is being transcribed by an RNA polymerase. As the polymerase moves through the sequence of six bases shown in the diagram below, what is the corresponding sequence of bases in the RNA that is produced? 3'-CCGAGT-5' 5'-GGCTCA-3' Enter the sequence of bases as capital letters with no spaces and no punctuation. Begin with the first base added to the growing RNA strand, and end with the last base added.

UGAGCC

Ch. 12 Quiz Is it possible that a single-base substitution in GGA (gly) could result in a stop codon? no yes

Yes

Textbook Wild type: met-trp-tyr-arg-gly-ser-pro-thr Mutant 1: met-trp Mutant 2: met-trp-his-arg-gly-ser-pro-thr Mutant 3: met-cys-ile-val-val-val-gln-his a) predict the type of mutation that created each altered protein. b) Determine the specific ribonucleotide change that leg to the synthesis of each mutant protein. c) The wild-type RNA consists of nine triplets. What is the role of the ninth triplet? d) For the first eight wild-type triplets, which, if any, can you determine specifically from an analysis of the mutant proteins? In each case, explain why or why not. e) Another mutation (mutant 4) is isolated. Its amino acid sequence is unchanged, but mutant cells produce abnormally low amounts of the wild-type proteins. As specifically as you can, predict where this mutation exists in the gene.

a) #1: nonsense mutation #2: missense mutation #3: frameshift mutation b) #1: mutation in third position to A or G #2: change U to C in third triplet #3: removal of a G in the UGG triplet (trp) c) termination d) all of the amino acids can be assigned specific triplets including the third base of each triplet. Compare the sequences for the wild type and mutant #2. After removal of a G in the UUG triplet of tryptophan, the frameshift mutation shifts the first base of the following triplet to the third (often ambiguous) base of the previous triplet. The only tricky solution is with serine, which has six triplet possibilities, but it can still be resolved. AUG UGG UAU CGU GGU AGU CCA ACA e) The mutation may be in a promoter or enhancer, although many posttranscriptional alterations are possible.

Textbook A short RNA molecule was isolated that demonstrated a hyperchromic shift indicating secondary structure. Its sequence was determined to be 5'-AGGCGCCGACUCUACU-3' a) What DNA sequence would give rise to this RNA molecule through transcription? b) If the molecule were a tRNA fragment containing a CGA anticodon, what would the corresponding codon be?

a) 3'-TCCGCGGCTGAGATGA-5' (Use complementary bases, substituting T for U) b) 3'-GCU-5'

Ch. 12 Quiz What is the correlation (if any) between the number of codons for each amino acid and the frequency of these amino acids in proteins? a) There is a positive correlation (more codons = higher frequency). b) There is a negative correlation (more codons = lower frequency) c) There is no correlation.

a) There is a positive correlation (more codons = higher frequency).

Ch. 12 Quiz Have to flip card to see table Which of the following amino acids could result from a single-base substitution in GGA (gly)? Select all that apply. alanine (ala) arginine (arg) aspartic acid (asp) cysteine (cys) glutamic acid (glu) leucine (leu) proline (pro) serine (ser) valine (val)

alanine (ala) arginine (arg) aspartic acid (asp) cysteine (cys) glutamic acid (glu) valine (val)

Ch. 12 Quiz If the codon specifying glycine is GGA, how many single-base substitutions will result in an amino acid substitution at position 210? Include any changes from an amino acid codon to a different amino acid codon and any changes from an amino acid codon to a stop codon. Include any changes from an amino acid codon to a different amino acid codon and any changes from an amino acid codon to a stop codon. a) 20 b) 14 c) 12 d) 6 e) 4 f) 2

b) 4 Four substitutions: GGU, GGG, GGA, GGC

Ch. 12 Quiz Which subunit of RNA polymerase establishes its binding to a promoter in prokaryotes? See Section 12.9 ( page 226) . a) α b) σ c) β d) β'

b) σ

PS12 Below is a partial DNA sequence used to answer the following questions. 5' - TATGCAGCACATT - 3' 3' - ATACGTCGTGTAA - 5' Part F If transcription occurred from right to left, what is the second amino acid in the growing polypeptide chain? cys val ala gln

cys

Ch. 12 Quiz When scientists were attempting to determine the structure of the genetic code, Crick and coworkers found that when three base additions or three base deletions occurred in a single gene, the wild-type phenotype was sometimes restored. These data supported the hypothesis that ________. a) the code is overlapping b) AUG is the initiating triplet c) the code contains internal punctuation d) there are three amino acids per base e) the code is triplet

e) the code is triplet

PS12 Below is a partial DNA sequence used to answer the following questions. 5' - TATGCAGCACATT - 3' 3' - ATACGTCGTGTAA - 5' If transcription occurred from left to right, what is the second amino acid in the growing polypeptide chain? cys gln ala val

gln

PS12 vBased on your analysis, Mutant1 is a ... transition mutation transversion mutant nonsense mutation insertion mutant deletion mutant none of the above

insertion mutant

PS12 A snRNP is best described as _______. small RNAs associated with protein complexes in the nucleus short mRNA transcripts combined with nuclear protein complexes the loop structure that forms when two exons are brought into close proximity a specific sequence of 7 bases in the mRNA transcript

small RNAs associated with protein complexes in the nucleus

PS12 Which of the following initially determines which DNA strand is the template strand, and therefore in which direction RNA polymerase II moves along the DNA? which of the two strands of DNA carries the RNA primer the position of the gene's promoter on the chromosome the specific sequence of bases along the DNA strands the location of specific proteins (transcription factors) that bind to the DNA the location along the chromosome where the double-stranded DNA unwinds

the specific sequence of bases along the DNA strands

PS12 Based on your analysis, Mutant2 is a .. insertion mutant transition mutation nonsense mutation transversion mutation deletion mutant none of the above

transition mutation

PS12 A partial peptide sequence for the wild type and three mutant alleles of a gene, PET1, are shown below. Each mutant was caused by a single point mutation. Wild Type: met ile arg met asp lys trp... Mutant 1: met ile gln asn gly ... Mutant 2: met ile arg met gly lys trp... Mutant 3: met ile ser met asp lys trp... The ... represent a stop codon. Using the amino acid sequence of the wild-type and the three mutants, deduce the exact DNA sequence of the coding strand of the wild-type allele. On a test I would ask you to write this sequence, but I cannot easily do this using MG. Question: After there any amiguous or degenerate bases in this DNA sequence. These would be nucletides that have multiple possibilities. Yes or no

yes