BIOL 240: Exam 3
What will be consequence of the following chemical changes: (a) Depurination; (b) deamination. Why is the deamination of 5-metheyl cytosine more mutagenic than the deamination of cytosine? Pages 409 - 410; slide# 15
(a) depurination- Breakage of the 1′ carbon bond releases a purine base. Loss of a guanine from one strand of DNA creates an AP site. DNA replication adds adenine opposite the AP site. Since adenine is on the opposite site, when replication occurs, adenine will be paired with thymine when it originally should have been G-C, thus the consequence of depurination is a transition mutation. (b) Deamination can result in a missense mutation. Deamination of 5-metheyl cytosine is more mutagenic than the deamination of cytosine because it can result in a transition mutation while the deamination of cytosine results in uracil, which will likely be fixed during mismatch repair
Study Figure 8.22, and read page # 298. Know the following items found in unspliced pre-mRNA: (a) location of 5' splice site. What near invariant sequence does it have? (b) 3' splice site. What near invariant sequence does it have? (c) Branch site. Where is this located relative to the 3'-splice site? What bases are predominantly found in branch site? Name the base that is highly conserved (invariant) in branch site?
(a): the 5' splice site locates at the 5' end of an intron. The near invariant sequence it has is GU at the 5'-most end. (b): the 3' splice site locates at the 3' end of an intron. The near invariant sequence it has is AG at the 5'-most end. (c): the branch site locates 20-40 nucleotides upstream of the 3' splice site. An invariant A, which is called the branch point adenine, is important in mediating intron splicing.
Explain how a transition mutation may arise when adenine changes from normal to rare tautomer. (slide 6)
1) Tautomeric shift occurs in one base pair. Ex. amino A -> imino A. 2) Semiconservative replication occurs where C=A. 3) Semiconservative replication occurs where C=G and originally would have been A=T, thus a transition mutation arises when adenine changes
At what site of the 30S ribosome does the charged initiator tRNA sit? Name the IF that is associated with the initiator tRNA. Write the sequence of the codon that the initiator tRNA base pair with.
Charged initiator tRNA sits in P site. IF2 is associated with the initiator tRNA. AUG is the sequence of the codon that the initiator tRNA base pairs with.
Write the characteristics of genetic codes described slide# 8. Define codon degeneracy and synonymous codons. Why codes are made of three letters?
Codon degeneracy- an amino acid may have more than one codon. Synonymous codons- Codons that code for the same amino acid. Ex. CUU and CUC both code for leucine. Codons are made of three letters because that provides 64 combinations of 3-letter codes, which is enough to cover 24 amino acids (2-letter codes would not be enough and 4-letter codes would be excessive)
What are the three post-transcriptional processing pre-mRNAs go through to become mature mRNA?
1. 5' capping, 2. 3' polyadenylation, 3. Intron splicing
What are the normal tautomers of adenine, guanine, cytosine and thymine? Name their rare tautomers. Explain the chemical change that takes place for each base when it becomes a rare tautomer. When does adenine - cytosine and guanine - thymine base pairing takes place? (slide 5)
1. Adenine and Cytosine - • normal - amino (NH2) • rare - imino (NH) 2. Guanine and thymine - • normal - keto (C=O) • rare - enol (C-OH). 1. Imino C pairs with amino A. 2. Imino A pairs with amino C. 3. Enol T pairs with keto G. 4. Enol G pairs with keto T.
Describe in detail the process of base excision repair.
1. DNA N-glycosylase recognizes a base-pair mismatch 2. DNA N-glycosylase then removes the incorrect uracil creating an AP (apyrimidinic) site. 3. AP endonuclease generates a single-stranded nick on 5' side of the AP site. 4. Then DNA pol removes and replaces several nucleotides of the nicked strand via nick translation.
Explain splicing by two transesterification reactions. What is a lariat? Does it have intron or exon sequence? What would be consequence if the invariant bases of the 5' and 3' splice sites are deleted or changed?
1. First reaction - 2'-OH of branch point adenine bonds with the phosphate between 5'-splice site and upstream exon. 2. Second reaction - the 3'-OH of the upstream exon bonds with the phosphate between 3'-splice site and downstream exon. lariat- a ring of intron segments that has been spliced out of a messenger ribonucleic acid molecule by enzymes. Some introns form a long tail attached to the ring, giving the structure the appearance of a microscopic cowboy lariat. Lariats have intron sequences If the invariant bases of the 5' and 3' splice sites were deleted or changed, splicing would not occur, thus mature mRNA would not occur and cellular function would cease
Write the three steps of translational elongation. Know what happens at each step. What is the function of peptidyl transferase?
1. First step: loading of correct aminoacyl-tRNA i.e., charged tRNA, to the A-site of the ribosome. 2. Second step:A peptide bond is formed between the charged tRNA at the A site and the peptide chain attached to the peptidyl-tRNA at the P site of the ribosome. The growing polypeptide is transferred from the P-site tRNA to the A-site tRNA. This is catalyzed by peptidyl transferase. 3. Third step: The A-site tRNA attached to growing polypeptide and its associated codon on the mRNA are translocated to the P-site. Two factors known as elongation factors control translocation. GTP hydrolysis provides the energy.
How many RNA polymerases are found in eukaryotes? What type of RNA do they transcribe?
1. RNA polymerase I - synthesizes ribosomal RNAs (rRNAs). 2. RNA polymerase II - synthesizes messenger RNAs (mRNAs). 3. RNA polymerase III - synthesizes transfer RNAs (tRNAs).
Name the seven types of RNA with abbreviations and functions of each. (SSMMRTT) Out of the seven types of RNA, which are found in both prokaryotic and eukaryotic cells?
1. Small nuclear RNA (snRNA)- function: to remove introns from precursor mRNA. 2. Small interfering RNA (siRNA)- function: to regulate mRNA stability and translation. 3. Messenger RNA (mRNA)- funtion: used to encode the sequence of amino acids in a polypeptide. 4. MicroRNA (miRNA)- function: regulatory RNAs that function by base pairing with certain mRNAs, altering their stability and efficiency of translation. 5. Ribosomal RNA (rRNA)- function: helps form the large and small ribosomal subunits that unite for translation of mRNA. 6. Transfer RNA (tRNA)- function: carries amino acids to ribosomes and binds there to mRNA via complementary base pairing to add amino acids to the elongating polypeptide. 7. Telomerase RNA- acts as a template to maintain and elongate telomere length of eukaryotic chromosomes. mRNA, tRNA, and rRNA are found in both prokaryotic and eukaryotic cells.
If the new DNA strand slips at the time of replication and four nucleotides loop out, will it cause addition or deletion of bases on the new DNA strand? Slide 11; page 405.
Addition; slippage during synthesis of daughter strand results in a hairpin loop to form.
When does the 50S ribosome join with the 30S initiation complex? Where does the energy come from for this process?
After the IF3 is released from the small subunit, the 30S and 50S subunits will bind. Energy is provided by a molecule of GTP linked to IF2 that is hydrolyzed.
Draw a tRNA. Show the stems and loops, mark the 3'- and 5'-ends. What base sequence is found at the 3'-end? What is anticodon loop?
All t-RNAs end with 5'-CCA-3' base sequence at the 3'-end. All t-RNAs begin with guanine at the 5'-end. Anticodon base pairs with codons on the mRNA.
What is the chemical composition of an RNA nucleotide? Name the carbons of the sugar molecule that are attached to the nitrogenous bases and phosphates.
An RNA nucleotide is composed of a ribose sugar with a nitrogenous base attached at its 1' carbon and a phosphate attached at the 5' carbon
What is the function of aminoacyl tRNA synthetase? How many different types of this enzyme is present in a cell?
An aminoacyl-tRNA synthetase is an enzyme that attaches the appropriate amino acid onto its tRNA, thus charging the tRNA. 20 different types
Name the transcription factor that binds with the TATA box in eukaryotes.
At the TATA box, TFIID, a multisubunit protein containing the TATA-binding protein (TBP) and subunits of a protein called TBP-associated factor (TAF), binds the TATA box sequence
Write the differences between bacterial and eukaryotic ribosomes. What are A, P and E sites? Where are they located? What are these sites meant for?
Bacterial ribosomes are composed of a 50S large subunit and a 30S small subunit that join together to form a 70S ribosome while eukaryotic ribosomes are composed of a 60S large subunit and a 40S small subunit that join together to form a 80S ribosome. The P-site (for peptidyl) is the second binding site for tRNA in the ribosome. The other two sites are the A-site (aminoacyl), which is the first binding site in the ribosome, and the E-site (exit), is the third and final binding site in the ribosome. The sites are located on the ribosome. These sites act as binding sites for tRNA as the mRNA is being translated to an amino acid sequence.
What is cryptic splicing mutation? What is the result of this type of mutation?
Cryptic splicing mutation- a mutation that causes a region that would normally not be spliced to undergo splicing. More of the mRNA sequence will be spliced than necessary
What can happen if mispairing takes place at the time of replication? Slide # 13
DNA base mispairing generates an incorporated error that if not repaired will generate a replicated error (a mutation) by DNA replication. A mutation can arise from mispairing occurring at the time of replication.
In the initiation process, what is name of the amino acid that is charged to the initiator tRNA? What molecules does the charged initiator tRNA associate with?
Methionine, abbreviated Met, is the amino acid that is charged to the initiator tRNA. Charged tRNA interacts with IF2 and P site on small subunit
What would be the sequence of the amino acids in the polypeptide encoded by the following mRNA: 5'-AUGCCCGAUCGUUAA-3'?
N'-Met-Pro-Asp-Arg-C'
Name the nitrogenous bases and pentose sugar found in RNA. How does an RNA molecule differ from a DNA molecule?
RNA contains adenine, uracil, guanine, and cytosine. While DNA contains deoxyribose sugar, RNA contains a ribose sugar. RNA molecule contains another -OH group on its 2' carbon of the sugar molecule while DNA molecule contains only one -OH group on its 3' carbon of the sugar molecule
Where does RNA polymerase holoenzyme bind to initiate transcription? Explain in detail the process of bacterial transcription. Does the sigma factor stay with RNA polymerase until transcription is over?
RNA pol holoenzyme binds to the promoter consensus sequences to initiate transcription. Process of transcription: 1. The RNA polymerase core enzyme and sigma subunit bind to the -10 and -35 promoter consensus sequences. 2. DNA unwinds near the transcription start site to form the open promoter complex. 3. RNA polymerase holoenzyme initiates transcription and begins RNA synthesis. The sigma subunit dissociates shortly after and the core enzyme continues transcription. 4. The core enzyme synthesizes mRNA until it encounters the termination sequence. As RNA synthesis progresses, the DNA duplex unwinds to allow the template strand to direct RNA assembly. The duplex closes following synthesis. 5. Transcription terminates at the termination sequence, and the core enzyme and RNA transcript are released. Sigma factor does not stay with RNA polymerase during full process of transcription
Name the enzyme that synthesizes RNA molecule. Does this enzyme use both DNA strand to synthesize an RNA molecule? During transcription what type of bonds are created between two rNTPs? Are the rNTPs placed randomly? What are pyrophosphates? Where do they come from?
RNA polymerase; RNA pol uses ONE DNA strand to synthesize an RNA molecule. Phosphodiester bonds link two rNTPs during transcription. The rNTPs are not placed randomly; they are placed according to the template of the DNA strand (i.e., if the DNA strand reads 5'-AGT-3', then the corresponding RNA strand would read 5'-ACU-3'). In chemistry, a pyrophosphate are phosphorus oxyanions that contain a P-O-P linkage. Pyrophosphates are formed by the extra phosphates left behind when an rNTP molecule is linked to an RNA strand.
It was discovered that 61 out of 64 codons are sense codons and 3 are non-sense codons. It is expected that there would be 61 different anticodons for 61 sense codons or 61-different tRNAs each carrying 1 of the 61 anticodons. But in reality, there are less than 61 different tRNAs. In fact, the number of tRNAs in different organisms ranges between 30 - 50. E. coli has 47 different tRNAs. How is it that less than 61 different tRNAs can cover 61 different codons?
Relaxation of complementary base pairing at the third position of a codon can reduce the number of different tRNAs required during translation.
What is the function of release factors? Which codons are recognized by RF1 and RF2 of E. coli? What is function of RF3?
Release factors trigger the release of the polypeptide chain. Stop codons are recognized by RF1 and RF2. The three stop codons are UAG, UAA, and UGA. The function of RF3 is to recycle RF1 and eRF1.
Explain the process of how RNA nucleotides are linked to make an RNA molecule. Draw structure of an rNTP.
Ribonucleoside triphosphates (rNTP) are used as substrates for RNA synthesis. The rNTP is recruited to the forming RNA strand by RNA polymerase. The rNTP is joined to the ribonucleotide of the existing RNA strand at the 3' end of the ribonucleotide's sugar molecule. This reaction results in a phosphodiester bond linking the 3' end of the first sugar to the 5' end of the next, and a phosphate group is discarded.
How do silencer sequences impact transcription?
Silencer sequences are DNA elements that act to repress transcription of their target genes
How do silencer sequences repress transcription of their target genes?
Silencers bind proteins that induce bends in DNA. These bends reduce transcription of the target gene by shielding the DNA from transcription activation by RNA pol II
Name the factors that are found in the preinitiation complex. What are general transcription factors?
The assembled TFIID bound to the TATA box forms the initial committed complex. Next, TFIIA, TFIIB, TFIIF, and RNA pol II join the complex, followed by TFIIE and TFIIH to form the preinitiation complex (PIC). TFs of the PIC are commonly referred to as general transcription factors
How do codons and anticodons base pair. What are isoaccepting tRNAs?
The codons and anticodons base pair antiparallel to another. For example, if the mRNA codon is 5'-GAC-3', then the anticodon would be 3'-CUG-5'. Isoaccepting tRNAs- Different tRNA species that bind to alternate codons for the same amino acid residue
What is the function of promoter? Where is the promoter sequence located? Is promoter sequence transcribed?
The function of the promoter is to initiate transcription when RNA polymerase and accessory proteins bind to it. Promoters are located in the 5' region adjacent to the transcriptional start site. Promoter sequence is not transcribed.
Explain alternative splicing. Give an example showing how a gene can produce more than one polypeptide via alternative splicing.
The same transcript can produce more than one type of mature mRNA by alternating which segments will undergo splicing. Ex: the human calcitonin gene is expressed differently in thyroid and neuronal cells.
What is the function of sigma factor? What is the difference between the RNA polymerase core and holoenzyme with respect to transcriptional efficiency?
The sigma factor is essential for initiating transcription in bacteria by binding to the -35 and -10 regions of bacterial promoter consensus sequences. Holoenzyme transcribes more efficiently than RNA polymerase core alone.
Define the template strand.
The strand used to make mRNA. It serves as the template for transcription and runs in the 3' to 5' direction.
Know how DNA, mRNA and polypeptides are aligned.
Things to note in the transition from DNA to mRNA and mRNA to peptide: 1. Promoters and terminators that are present in DNA transcript are not transcribed to mRNA. 2. 5'- and 3'-UTR sequences are not translated to polypeptide sequence
What are thymine dimers? Explain the effect of UV radiation on DNA.
Thymine dimers are a pair of abnormally chemically bonded adjacent thymine bases in DNA, resulting from damage by ultra-violet irradiation. UV irradiation forms photoproducts from adjacent pyrimidines, distorting the double helix and potentially blocking replication.
What is the purpose of transcription?
To synthesize RNA from DNA. The RNA will act as the intermediate molecule between DNA and proteins.
Explain in detail the four steps of capping. What is the final goal of capping? How does capping help the mRNAs?
To the 5'-end of the mRNA a modified guanine is added. This is called capping. It happens in three steps. 1. First, RNA triphosphatase removes the upsilon phosphate of the 5'-terminal base of mRNA. 2. Next, guanylyl transferase catalyzes linkage between the β phosphate of terminal base of mRNA and the alpha phosphate of a GTP molecule. 3. A pyrophosphate is released and the guanine is attached to the 5'-end of the mRNA via unusual 5' to 5' linkage. 4. In the third step, methyltransferase methylates the guanine at the 7th position. CAP is needed for ribosome binding. Capping protects mRNA; facilitates mRNA transport and increases translational efficiency.
Define the following mutations: missense, nonsense, silent, frameshift, null and neutral mutations. Compare transition and transversion mutations. (slide 1-4)
missense- changes one amino acid. nonsense- creates stop codon and terminates translation. silent- no amino acid sequence change. frameshift- wrong sequence of amino acids. null mutation- mutation in a gene that leads to its not being transcribed into RNA and/or translated into a functional protein product. neutral mutations- Neutral mutations are changes in DNA sequence that are neither beneficial nor detrimental to the ability of an organism to survive and reproduce. Transition mutation - when a purine replaces a purine or pyrimidine replaces a pyrimidine. Transversion mutation - when a purine is replaced by a pyrimidine and vice versa.
What is a polyribosome? Notice that in bacteria mRNA is translated as soon as soon as mRNA sticks out its 5'-end (Figure 9-11b). This shows that in bacteria transcription and translation are coupled. Do you think this is also true for eukaryotes?
polyribosome- a structure composed of two or more ribosomes associated with an mRNA engaged in translation. This is not true for eukaryotes. Translation takes place outside the nucleus
How are ribonucleotides chemically linked to form a RNA strand?
ribonucleotides are chemically linked by phosphodiester bonds that link the 3' end of one sugar to the 5' end of the next, thus forming the RNA strand
What is rifampicin? How does it inhibit RNA synthesis? How many classes of RNA ploymerases are found in bacteria?
rifampicin is an antibiotic that can treat several bacterial infections, including tuberculosis and leprosy. It inhibits RNA synthesis by inhibiting RNA polymerase. Bacteria contain only a single type of RNA polymerase, whereas eukaryotes have three types
Describe in detail the process of nucleotide excision repair.
1. UVR AB complex binds opposite a thymine dimer. 2. UVR B denatures the DNA around the lesion. 3. UVR A departs; UVR C binds and catalyzes 3' and 5' cuts. 4. DNA helicase UVR D helps release the damaged single strand; DNA pol and DNA ligase fill and seal the single-stranded gap. The lesion has been removed and the DNA duplex has been restored
How many tRNAs can read 4 codons for Leucine (see codon dictionary in Slide# 8)
2 codons can read all 4 codons for Leucine (using info given in table 9.6)
What is the function of sigma subunit 28, 32, 54, and 70?
28- flagellar synthesis and chemotaxis. 32- heat shock genes. 54- Nitrogen metabolism. 70- housekeeping genes
Know how a mutation is created by the following chemicals: 5-bromodeoxuridine; Nitrous acid; Ethyl methane sulfonate (EMS), proflavin and benzo(a)pyrene. Pp 410-12; Figures 11-11; 11-12; 11-13.
5-bromodeoxuridine: Bu is incorporated in the DNA sequence. A base mispair occurs in the first replication cycle and a transition mutation occurs in the second cycle of replication. Nitrous acid: nitrous acid causes deamination, which converts adenine to hypoxanthine, which then mispairs with cytosine resulting in a transition mutation. Ethyl methane-sulfonate: ethylmethane-sulfonate causes alkylation of guanine, which produces O^6-ethylguanine, which mispairs with thymine resulting in a transition mutation. Proflavin andbenzo(a)pyrene intercalate into the double helix and distort its shape, generating strand nicking that can produce frameshift mutations
Define the termination region.
A region of DNA where transcription of a particular gene ends; located downstream
How do enhancers impact the level of transcription on specific genes?
Enhancers increase the level of transcription of specific genes
Do the enhancers have fixed position? Where are they located relative the gene that they influence? How do enhancers influence initiation complex? What are activator and coactivator proteins? How do they function?
Enhancers may be variable distances from the genes they affect and may be upstream or downstream of the gene. Enhancer sequences bind activator proteins and associated coactivators to form a "protein bridge" that links the transcription complex at the promoter to the activator-coactivator complex at the enhancer. This bridge bends the DNA so that the proteins at both locations are brought close enough together for them to interact. Such interaction dramatically increases RNA pol II efficiency in transcription initiation.
Draw structure of a eukaryotic gene (slide # 34). Compare exons and introns. Do you expect to find intron sequences in a mature mRNA?
Exons become part of mature mRNA and encode protein segments while introns are intervening segments that are removed from pre-mRNA
Explain the molecular basis of Fragile X syndrome. Page 406; Table 11-2
Fragile X syndrome is one of several human trinucleotide repeat disorders. The repeat sequence is CGG, and this sequence normally has a repeat range of 6-50 times; however, in an individual affected by this disease, the repeat range will be 200-2000, resulting in mental retardation.
What causes frameshift mutation? Why is it called frameshift? Explain with a made-up example (Figure 11-3; Slide# 8)
Frameshift mutations result from insertions or deletions of a base pair. It's called frameshift because the reading frame actually shifts. For example, THE CAT SAW THE HOG experiences a deletion, and the frame shifts to THE ATS AWT HEH OG, thus the entire code downstream from the deletion is completely altered.
Name the seven components that come together to initiate translation. What does IF stand for?
IF stands for initiation factor. Factors that come together to initiate translation: small ribosomal subunit, an mRNA molecule, a specific charged initiator tRNA, GTP, Mg2+, and three initiation factors
Where does IF1 bind? What is its role in the process of initiation?
IF1 binds to the A site, thus blocking the A site from being bound to a tRNA
Where does IF3 bind in the initiation complex? What is its role?
IF3 binds to the small subunit. Its role is to temporarily prevent the binding of the small and large subunit.
Explain the mechanisms of intrinsic and rho-dependent transcription termination in E. coli. What are inverted repeats? How is stem-loop structure formed? What is the significance of stretches of adenines on the template strand immediately after the stem-loop structure?
Intrinsic transcription termination (This is also called Rho-independent transcription termination): 1. Intrinsic termination sequences contain inverted repeats separated by a spacer sequence and followed by a polyadenine sequence. 2. Transcription of the template strand forms mRNA. 3. Inverted repeat sequences in the transcript fold into a complementary stem ending in a single-stranded loop. 4. H bonds between U-A base pairs break, releasing the transcript and terminating transcription. Inverted repeats are single stranded sequences of nucleotides followed downstream by its reverse complement. For example 5'-TTACGnnnCGTAA-3' where n represents any set of nucleotides in between. Stem-loop structure is formed by inverted repeat sequences in the transcript that cause the complementary strand to fold into a complementary stem ending in a single-stranded loop. The hairpin followed by a series of U's in the mRNA causes the RNA polymerase to slow down and destabilize, which is why the stretches of adenines in the template strand are important. Rho-dependent transcription termination: 1. Transcription across the rho utilization site produces the rut sequence in mRNA that is the recognition site for Rho protein. 2. Rho protein binds to the rut sequence and moves toward the 3' end of mRNA. 3. RNA polymerase pauses at the termination sequence as a stem-loop forms. 4. Rho protein catches up to the paused RNA polymerase and releases mRNA and RNA polymerase from DNA to terminate transcription.
How is tRNA(fmet) chemically modified?
It's charged from adding a formyl group to the amino group
Explain the peptidyl transferase reaction that attaches the growing polypetide to the incoming amino acid brought in by charged tRNA (slide #17). What bonds are created by this reaction? Where does the energy come from for this reaction? What is the difference between peptidyl and aminoacyl tRNA? What do they hold at their 3'-ends? At which sites of the ribosome these tRNAs are found?
Peptidyl transferase reaction takes place on the ribosomes and is catalyzed by peptidyl transferase. This reaction links the C'-terminal end of the growing polypeptide (attached to the peptidyl tRNA) to the amino group of the incoming amino acid attached to its tRNA (amino acyl tRNA). The peptidyl and aminoacyl tRNAs are brought close to each other by the ribosome. The amino group of the incoming amino acid attacks the carbonyl group of the C'-terminal amino acid of the growing polypeptide and forms a peptide bond. The reaction is driven by breaking the high energy acyl bond of the aminoacyl tRNA. The acyl bond is formed when ATP is hydrolyzed at the time of tRNA charging. Peptidyl tRNA has the growing polypeptide chain attached while aminoacyl tRNA only contains a single attached amino acid group. At their 3' ends, tRNAs hold an amino acid. These tRNAs are found at the A and P sites
Study the following DNA repair mechanisms: Photoreactive repair; Base excision repair; nucleotide excision repair and mismatch repair. Pages 415 - 418; slides #23 - 26.
Photoreactive repair- repair of UV-induced photoproducts catalyzed by photolyase, which is activated by visible light. Base excision repair- removal of an incorrect or damaged DNA base and repair by synthesis of a new strand segment. Nucleotide excision repair- removal of a strand segment containing DNA damage and replacement by new DNA synthesis. Mismatch repair- removal of a DNA base-pair mismatch by excision of a segment of the newly synthesized strand followed by resynthesis of the excised segment.
Explain the mechanism of polyadenylation. Discuss the function of each enzyme involved in this process. Write the sequence of poly (A) signal. Where is this sequence located in the RNA relative to the polyadenylation site? How does polyadenylation help the mRNA (3 ways)?
Polyadenylation is triggered when the poly-A signal is transcribed into RNA at the end of the gene. 1. Poly-A signal triggers recruitment of CPSF (cleavage and polyadenylation specificity factor) and CstF (cleavage stimulating factor). 2. These proteins and other proteins cleave the RNA few bases downstream from the poly-A signal (AAUAAA). 3. Poly-A polymerase (PAP) adds about 200 adenine residues at the 3'-end of the cleaved mRNA. The enzyme uses ATP as substrate and it does not use a template. Sequence of poly (A) signal: AAUAAA. This sequence is located 10-15 base pairs upstream of the polyadenylation site 3 ways that polyadenylation help the mRNA: 1. Facilitating transport of mature mRNA across the nuclear membrane to the cytoplasm. 2. Protecting the mRNA from degradation. 3. Enhancing translation by enabling the ribosomal recognition of mRNA.
Explain the two-step process of tRNA charging. You need to write the names of all reactants and products produced at each step. Name the enzyme that catalyzes these reactions. What does the high energy bond link?
Process of tRNA charging: 1) amino acid binds with ATP to form adenylated amino acid and a pyrophosphate as a byproduct. 2) Carboxyl group of adenylated amino acid binds with 3' end of tRNA molecule to form charged tRNA and AMP as a byproduct. The reaction is catalyzed by amino acyl t-RNA synthetase. High energy bond links the 3'-end of the tRNA to the Carboxyl or COOH (C'-terminal) group of the amino acid
Explain how Ames test is done. Study pages 413-414, and Figure 11.15.
Purpose of test: to test whether chemical or compound is mutagen. Chemicals that result in many revertant colonies are considered carcinogenic chemicals because they increase the number of mutations occurring per replication cycle.
What is spliceosome made of? Name the snRNAs found in spliceosome. Name the snRNAs that bind at 5'- and 3' splice sites.
Spliceosome - has 150 proteins and 5 RNAs. Five RNAs are: U1, U2, U4, U5 and U6. They are small nuclear RNA or snRNA. snRNP U1 binds at 5' splice site. U2 binds at 3' splice site
What types of mutations may arise from base pair substitution? (Figure 11-2.)
Synonymous mutations, missense mutations, and nonsense mutations
What is the difference between coding and template strand? Which of these two strands is complementary to RNA?
Template strand is the strand that is used to make the mRNA sequence while the coding strand is the strand that is identical to the mRNA strand. The template strand is complementary to the mRNA
Know about EPA sites in ribosome. Name the molecules that occupy each site. Which of these three sites is closest to the 5'-end of the mRNA?
The A site is called the aminoacyl site where the charged tRNA enters. The P site is called the peptidyl site. Here the tRNA is linked to the growing polypeptide chain. The E site is the exit site. It is occupied by the tRNA which is about to leave the ribosome. E site is closest to the 5'-end of the mRNA
Write the composition of E. coli RNA polymerase core enzyme. What is the difference between RNA polymerase core enzyme and RNA polymerase holoenzyme?
The E. coli RNA polymerase core enzyme has a 390 kD molecular weight. It is made up of alpha-I, alpha-II, beta, beta', and omega units. The RNA polymerase holoenzyme contains an RNA polymerase core enzyme + a sigma subunit
What is Goldberg-Hogness box; where is it located? Name the other sequences (boxes) that may be found in eukaryotic promoters.
The Goldberg-Hogness box is also known as the TATA box and is located at the -25 position. The other sequences are the CAAT box located at the -80 position and the less common GC-rich box located at the -90 position.
What are Pribnow box and -35 consensus sequence? Where are they located with respect to the transcribed region of the gene? What is +1? What are 5'- and 3'-UTR?
The Pribnow box and -35 consensus sequences are promoter sequences. They are essential promoter regulatory elements. They are located upstream of the transcribed region of the gene. The +1 is the site on DNA from which the first RNA nucleotide is transcribed. The three prime untranslated region (3'-UTR) is the section of messenger RNA (mRNA) that immediately follows the translation termination codon. The 3'-UTR often contains regulatory regions that post-transcriptionally influence gene expression. The 5′ untranslated region (5′ UTR) is the region of an mRNA that is directly upstream from the initiation codon. This region is important for the regulation of translation of a transcript by differing mechanisms in viruses, prokaryotes and eukaryotes.
Where is Shine-Dalgarno sequence located? What is its relationship with the 16S rRNA? What would be the problem if Shine-Dalgarno sequence is missing?
The Shine-Dalgarno sequence is located upstream of the start codon. The Shine-Dalgarno sequence base-pairs with the 16S rRNA in the small subunit to position the start codon (AUG) at the P site. Without Shine-Dalgarno sequence, translation couldn't occur.
Define splicing. What is the end result of splicing?
The process by which introns are removed from hnRNA to produce mature messenger RNA that contains only exons
what is the difference between coding and template strands found in a gene? Why is the non-template strand called coding strand? What are 5'- and 3'-UTRs? What are their functions?
The template strand is the strand that serves as the template to make an opposite strand of mRNA. The coding strand is not used for making the mRNA sequence; however, it is referred to as the coding sequence because this sequence of DNA is most closely related to the mRNA. The 5′ untranslated region (5′ UTR) is the region of an mRNA that is directly upstream from the initiation codon. This region is important for the regulation of translation. The 3'-UTR often contains regulatory regions that post-transcriptionally influence gene expression.
Study Fig 8.19 critically. What is the role of Torpedo RNase in transcription termination? Explain its function. What would be the consequence if Torpedo RNase fails to act?
The torpedo RNase attacks the uncapped 5ˊ end of the residual mRNAand digests it, leading RNA polymerase II to dissociate from the DNA and the torpedo Rnase. RNA pol II would not be able to dissociate from the DNA, which would inevitably cause countless issues to cell function.
Explain the wobble hypothesis.
The wobble hypothesis explains the phenomenon of degeneracy that is seen in the genetic code through tRNA recognition of more than one codon. Each tRNA contains a three-base anti-codon that is complementary to a codon in transcribed mRNA. The first two pairs need to be complementary for binding to occur, but the third pair does not always need to be complementary. This is the wobble, or allowance of degeneracy, in the genetic code.
Write the structure of an amino acid. How many amino acids are found in a cell? Explain peptide bond formation.
There are 20 amino acids found in bacteria and eukaryotes. Amino acids contain a central carbon (the alpha-carbon), an R-group, an amino group, and a carboxyl group. Peptide bond formation: The carboxyl group (COO-) of one amino acid reacts with the amino group (+H3N) of the adjacent amino acid to form a covalent peptide bond that links amino acids in a polypeptide chain.
How exactly do enhancers increase the level of transcription of specific genes?
They bind proteins that interact with the proteins that are bound to gene promoters, and together the promoters and enhancers drive gene expression
What is the product of translation? Name major items required for translation.
Translation results in the biological polymerization of amino acids into polypeptide chains. In simple terms: translation results in a long sequence of amino acids that eventually form polypeptide chains. Major items required for translation: 1. Amino acids, 2. mRNA, 3. Ribosomes, 4. tRNA
Study Figure 9.9 and page 324 to answer the following questions. What is Ef-Tu? What does it do? What is the role of GTP in the elongation process? At which site of the ribosome does the charged tRNA enter? At step-4 which tRNA is holding the growing polypeptide chain? At which site of the ribosome this tRNA is located now? What happens to the tRNA located at P site? Which factor causes translocation (step 5). What does it help translocate and in which direction? What is occupying A site at this point? Which direction does the polypeptide grow?
What is Ef-Tu/what does it do, and what is the role of GTP in the elongation process: Like IF2 during initiation, EF-Tu is a GTPase and is bound by a GTP, the hydrolysis of which provides energy for the process. Charged tRNAs enter at A site. Aminoacyl tRNA is holding the growing polypeptide chain (slide #18 if you'd like to reference). This tRNA then moves to the P site and the tRNA that originally was in the P site has moved to the E site. Elongation factor protein G (EF-G) causes translocation. EF-G helps translocate the ribosome in the 5'-3' direction of the mRNA. Nothing is occupying the A site at this point. It is empty. Polypeptide chains are synthesized from the amino to the carboxy terminus
Explain the difference between closed and open promoter complex.
When the DNA is in the closed promoter state it remains in a closed double-stranded form. When the DNA is in the open promoter state a short region has been melted at the promoter and so it's open. The holoenzyme is tightly bound in this state.
Define promoter.
a region of DNA that initiates transcription of a particular gene. Located near the transcription start point/upstream
Shown below is the template strand of a gene. 3'-TAC CAA CAA CCA AAG GCT ATT-5' a) What would be the sequence of the mRNA transcribed by this template strand? b) If this mRNA is translated, what would be amino acid sequence of the polypeptide? c) If a G is inserted between TAC and CAA at 3'-end, and G of the downstream AAG (in bold) is deleted, what would be the amino acid sequence of the polypeptide? Use codon dictionary given in slide #8 of lecture 11. What type of mutation will it be?
a) 5'-AUG GUU GUU GGU UUC CGA UAA-3'. b) N'-Met-Val-Val-Gly-Phe-Arg-C'. c) New DNA sequence: 3'-TAC GCA ACA ACC AAA GCT ATT-5'. New mRNA sequence: 5'-AUG CGU UGU UGG UUU CGA UAA-3'. New aa sequence: N'-Met-Arg-Cys-Trp-Phe-Arg-C'. Frameshift mutation
Note that in eukaryotes most initiation factors (eIFs) bind with small ribosomes before the mRNA is brought in. Which initiation factors bring in the mRNA? What is scanning? What is Kozak sequence? What is its role in translation?
eIF4 brings in the mRNA. Scanning: The small 40S subunit "scans" the mRNA sequence in search of the start codon to begin translation. The Kozak sequence is a sequence which occurs on eukaryotic mRNA and contains the start codon. Role of Kozak sequence: The Kozak sequence orients the mRNA on the small subunit and places the authentic start codon in position to begin translation
What is a cistron and what are polycistronic mRNAs?
cistron- a gene that encodes a single-polypeptide chain. polycistronic mRNA- a messenger RNA molecule that encodes the amino acid sequence of two or more polypeptide chains in adjacent structural
Define the non-template (or coding) strand.
the sequence of this strand will be the same as the sequence of the new RNA molecule produced (except U will replace T in the mRNA sequence)
What is the transcription start point or +1 nucleotide?
the site on DNA from which the first RNA nucleotide is transcribed.
What does upstream and downstream mean?
upstream= towards the 3' end of the template strand. downstream= towards the 5' end of the template strand.
