| M W F; 2:30 - 3:20 pm |
Molecular Biology |
Douglas W. Smith |
| Centre 101 |
BIMM 100 |
5254 Muir Biology Building |
| Winter, 2000 |
x42620; dsmith@ucsd.edu |
| BIMM100 | Syllabus
| Sections / Off Hrs | Grading
Policy | DNASYSTEM
|
| Lectures | Journal
Articles | Study Qs | Lab
Techniques | Exams |
Name: _____________________
Pages
BIMM 100 2. 50
3. 160
Molecular Biology
4. 85
D. W. Smith
5. 70
Final Examination 6. 65
7. 75
645 Points Possible
8. 65
9. ___75____
Final Exam Score: 645
Total Course Score:
Fall, 1995 Grade:
I hereby give permission to have my graded exam placed in a box
in the hall outside of 3306 Bonner Hall for me to pick up.
I realize this will render my exam available for public
examination and scrutiny.
Name ___________________________________________ Date _________________
15 pts 1. In the Gross et al HtpR paper, runoff transcripts were used as an assay system. (5 pts) a. What is a runoff transcript? A runoff transcript is the product of an in vitro RNA polymerase transcription experiment using a linear DNA template such that transcription elongation proceeds to the end of the DNA template and "runs off" the end. (5 pts) b. What was the purpose of the
assay? The runnoff transcript assay was used to assess ability of fractions obtained from protein purification procedures to serve as a sigma factor for transcription from a heat shock promoter.
(5 pts) c. Two runoff transcripts were
used. What was the difference in templates used to generate the
two runoff transcripts? The difference in DNA templates was the restriction enzyme used to generate the template. Both templates used the same heat shock promoter Hence the difference in size of RNA product was well defined in relation to the restriction sites used as ends of the DNA templates, and hence the transcription start position could be precisely determined, and should be the same for both templates. 35 pts 2. Draw the structure of the
terminal nucleotide (C) of a tRNA molecule and an amino acid,
using R for the R group, in their aminoacyl linkage. Then draw
SCHEMATICALLY the rest of the tRNA molecule showing the secondary
structure features of the tRNA molecule. Label each stem-loop
of the tRNA molecule and explain its function. Assuming the codon
recognized by this tRNA is CUA, draw the anticodon, with its
sequence, on your tRNA drawing. Label 5' and 3' ends of the tRNA
molecule. 10 pts 3. Write the LETTER for the protein
or protein complex that matches each the molecular site in the
protein column.
30 pts 4. Suppose you have the following
completely overlapping, comma (G is
the comma), sequential ...GACCTGACTTGCTATGTTCAGCAATG... (20 pts) a. Write down the codons. ACC, CCT, ACT, CTT, CTA, TAT, TTC, TCA, CAA, AAT
(5 pts) b. How many possible codons are there for this Genetic Code? 3 nucs possible at each of 3 positions (triplet code): 33 = 27
(5 pts) c. Assuming no redundancy, how
many amino acids could a base substitution 1 or 2 if any base is substituted EXCEPT a G If G mutates to A,T,or C: 3 new amino acids are "inserted" If non-G base mutates to a G: 1 or 2 amino acids are "deleted" 30 pts 5. Match one or more of the items
A-L in the two right columns with the
4 gene expression stages in the left column.
90 pts 6. The E. coli gal operon has 3 adjacent, coordinately expressed structural genes galE, galT, and galK, with Promoter (galP) and Operator (galO) just upstream of these genes. Control of gene expression is via a Repressor from a non-adjacent galR gene and via catabolite repression. Galactose is an Inducer of the operon.
(20 pts) a. Draw the structure of the operon and its regulatory elements. (10 pts) b. On your diagram, show the effects of gal Repressor on Gal operon gene expression when Galactose is absent and show the effects when Galactose is present. (5 pts) c. Define coordinate expression. Coordinate expression is the simultaneous or common regulation of a group of genes (structural genes). They are all turned on (induced or derepressed) or turned off (repressed) together. (5 pts) d. How are the effects of an Inducer different from those of a Co-Repressor? Inducer and Co-Repressor molecules are both small molecules that interact with a Repressor of an Operon in prokaryotes. However, an inducer (eg b-galactosides for the lac operon) INACTIVATES a Repressor whereas a Co-Repressor (eg tryptophane for the trp operon) ACTIVATES the Repressor. (5 pts each) e. State the expected phenotypes (Gal operon expression in presence and absence of galactose) of the following types of mutations:
(5 pts each) f. State the expected dominance relations of the following types of merodiploids:
(5 pts) h. Briefly explain Catabolite Repression, using the gal operon as an example. Catabolite Repression is the POSITIVE control system whereby cAMP and CAP (catabolite activator protein) bind to promoters of catabolic operons to ACTIVATE transcription by RNA polymerase. Presence of Glucose reduces cAMP concentrations, turning off operon expression, and vice versa: Glucose Effect. 50 pts 7. Bacteriophage Lambda: N protein functions as an AntiTerminator of transcription, binding with NusB to NutR and NutL sites, permitting RNA polymerase to read-through at the tR1 and tL1 transcription terminator sites. This Positive regulation leads to expression of the CII, CII, O, P, Q genes, and some xis and int expression. (5 pts) b. If Q encoded a new sigma factor, what change in lambda DNA sites would be needed to elicit the normal temporal expression of lambda genes? Simplest answer: a new promoter near tR' specific for CoreRNApol + this Q sigma factor which would then express Late genes (S, R, Head, Tail genes) Alternative: modify the PR' promoter to make it specific for CoreRNApol + this Q sigma factor, and delete the tR' transcription terminator. (25 pts) c. Diagram using arrows the antagonistic behavior of Cro and CI repressors, indicating whether the effect of the arrow is positive or negative. Provide names of the relevant promoters and operators. (5 pts) d. What is the role of cAMP in the lambda life cycle? The major role of cAMP is to control the decision lytic vs lysogenic via its concentration level. cAMP does this at the PRE promoter: cAMP is required for activation of this promoter (together with CII, CIII, CAP proteins) which results in initial CI repressor expressor and turn off of Cro, CIII, O, P, Q expression via the AntiSense RNA activity of the PRE transcript. 80 pts 8. RNA splicing: 1. 3'-OH attack at Donor Site [5' end of intron]; source of 3'-OH: G-OH
in Group I introns, intron A-OH with lariat formation in Spliceosomes, etc
2. 3'-OH attack of the terminal nuc of Exon 1 (left exon) on the last nuc
of intron at intron 3' end, with release of Intron and joining of the 2
exons
3. Debranching, etc of the Intron
(25 pts) b. What are the 5 main classes of RNA splicing? Indicate which use a Ribozyme and which use a Spliceosome. 1. Group 1 introns - Ribozyme - [rRNA genes of lower eukaryotes] 2. Group 2 introns - Ribozyme - [fungal mitochondrial introns] 3. Hammerhead introns in viroids - Ribozyme - [viroids] 4. tRNA processing intron type - - [yeast tRNA genes] 5. Introns using snRNPs - Spliceosome - [nearly all euk mRNA genes] (5 pts) c. Draw an example of alternative splicing. (10 pts) d. Explain what a "snerp" is and how it functions. A Snerp is an snRNP: small nuclear ribonucleoprotein particle composed of protein(s) and a snRNA (small nuclear RNA) species. Snerps function in Spliceosome-mediated Intron splicing events, each playing a specific role in the splicing. When the complete complex of Snerps has been constructed, the complex is called a Spliceosome. (5 pts) e. What is the GT-AG rule? GU is always present at 5' end of Intron; AG always present at 3' end of Intron (5 pts) f. Suppose the Internal Guide
Sequence of a mutant L-19 RNA were: UUUUUU.
(A)5 or AAAAA (10 pts) g. Write down the 2 substrate-product reactions in the overall net reaction with this UUUUUU substrate. (5 pts) h. How did Zaug and Cech show that a deoxyoligonucleotide is a competitive inhibitor of the normal substrate? They did enzyme kinetics, measuring amount of product at varying concentrations of CCCCC (rCrCrCrCrC) substrate as a function of fixed amount of dCdCdCdCdC present. Data are presented as a Lineweaver-Burk plot which shows that the dCdCdCdCdC acts as a Competitive Inhibitor of the rCrCrCrCrC reaction 45 pts 9. Control of Eukaryotic gene
expression: 1. DNA region containing a cluster of Transcription Factor binding sites 2. can be located upstream or downstream of a given gene promoter 3. can be found very distant from the promoter 4. can be in either orientation relative to the promoter 5. must be on same DNA molecule (15 pts) b. Name three types of general biological processes where Enhancers are used for control of gene expression. 1. tissue specific gene expression 2. temporal control of gene expression in development 3. gene expression in response to external signals) 4. yeast catabolism, eg GAL4 Other answers were also possible ... (10 pts) c. What are Response Elements? How are they similar and how are they different from the TATA box? Response Elements are short DNA binding sites for in Enhancers and as Upstream Promoter Elements for Transcription Factors that bind in response to changes in the cell environment (temperature change in heat shock, presence of small molecule, eg heavy metals, steroids, etc) Similarities to TATA: short DNA binding site, bind TFs, enhance transcription,... Differences from TATA: often part of an Enhancer element, not positional element 30 pts 10. In the Metallothionein paper, Deletion mutagenesis with generation of set of specific 5' deletion derivatives or mutants and generation of set of specific 3' deletion derivatives (10 pts) b. what are 2 of the 3 methods used to identify the binding site for the glucocorticoid receptor? 1. filter binding assay
2. DNase I footprinting assay
3. base modification protection experiments [DiMethylSulfate (DMS) specific
for G's]
(10 pts) c. what two external signals activate expression of this gene, and what was the major result from this paper concerning activation by these two types of external signals? External signals: steroid binding to Glucocorticoid Receptor; presence of heavy metal, eg Cadmium Major result: presence of either signal sufficient to activate MT II gene expression ... the response elements to the 2 external signals act independently of each other. 20 pts 11. For lagging strand DNA synthesis in E. coli, diagram and briefly describe the events in synthesis and maturation of two Okazaki fragments adjacent to the mature daughter DNA strand. Name an enzyme that is used to catalyze each event, and label 5' and 3' ends. 55 pts 12. Retroviruses: (5 pts) b. The retrovirus genome is a linear molecule. During its replication, briefly explain how the "end problem" (replication of all nucleotides at the ends of the linear genome) is solved. The end problem is solved by duplicating the 3' end of the RNA genome at the 5' end of the DNA provirus, and the 5' end of the RNA genome at the 3' end of the provirus, resulting in the LTR structures (U3-R-U5) at both ends of the provirus. Transcription of the provirus to yield progeny RNA genomes then can begin at a promoter in U3, to fully duplicate both R sites and all sequences in between in the resulting RNA genome molecules. (10 pts) c. Briefly describe (one sentence
each) how this solution to the "end
problem" differs 1. eukaryotic chromosomes use the telomere solution
2. Lambda circularizes its genome upon infection, permitting replication
with no end problem
(5 pts) d. When in frame, the pol "gene" is translated via nonsense suppression. Briefly describe nonsense suppression. In Nonsense Suppression, a first point mutation results in presence of a Stop codon in the middle of a gene, resulting in a truncated, inactive protein upon translation. The effects of this mutation are Suppressed by a second mutation, usually in a tRNA gene such that the mutant tRNA 1) can still be charged by a cognate Aminoacyl Synthetase and 2) has an anticodon that will recognize the Stop Codon of the first mutation. The result is that an amino acid is put into the growing polypeptide chain during translation when the Stop codon (first mutation) is encountered and no truncation of the protein occurs. (10 pts) e. What are the functions of Reverse Transcriptase and RNaseH in replication of the retrovirus genome? Reverse Transcriptase is an RNA-dependent DNA polymerase. It replicates the retroviral RNA genome into a double-stranded DNA species which, when integrated into the host genome, is called the Provirus. RNaseH is an RNase that degrades the RNA part of an RNA:DNA duplex nucleic acid molecule. It degrades the retroviral RNA genome after the genome has been replicated into DNA by Reverse Transcriptase. The two enzymatic activities work in synchrony during this replication process. 65 pts 13. Translation: Elongation factor EF-Tu brings charged tRNA molecules to the A site of the ribosome during protein synthesis, using energy from GTP to do this. Upon GTP cleavage, EF-Tu is released as a Tu-GDP complex. The GDP is released from this complex via displacement by EF-Ts, with formation of a Tu-Ts complex. Ts is replaced by GTP when Tu interacts with another charged tRNA molecule. The comparable eukaryotic protein is eEF-T or eEF-1. This "T factor" does what Tu and Ts do together in prokaryotes. (35 pts) b. Draw a figure of a ribosome functioning in translation elongation just before peptide bond formation. Include each of the major molecular structures. Label 5' and 3' ends and indicate direction of translation. (20 pts) c. Translation initiation in eukaryotes differs in what major ways from translation initiation in prokaryotes? In prokaryotes, 30S ribosome binds to the Shine-Delgarno sequence near the Start Codon in an mRNA molecule, and the AUG Start Codon is placed in the Psite of the 30S ribosome. fMet-tRNAf then binds to the P site of the 30S-mRNA complex, with the AUG Start Codon of the mRNA. Finally 50S ribosome is added. In eukaryotes, there is no Shine-Delgarno sequence. There is also no fMet. Further, Met-tRNAi binds to 40S ribosome BEFORE 40S ribosome binds to the mRNA. The Met-RNAi-40S ribosome complex then binds to the Cap region at the 5' end of the mRNA. This complex then translocates down the mRNA to the Start Codon, using ATP for energy and placing the Start Codon in the P site of the 40S ribosome. finally 60S ribosome is added. |
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| BIMM100 | Syllabus
| Sections / Off Hrs | Grading
Policy | DNASYSTEM
|
| Lectures | Journal
Articles | Study Qs | Lab
Techniques | Exams |
If you have problems or comments, send email to Doug Smith