Chem 221 Sample Exam III Questions The questions on this sheet are somewhat indicative of the questions

that may be given on the exam. Exam questions will not necessarily be limited to those given on this handout. 1. Give three differences between A-DNA and Z-DNA? Which form is more likely to be found in vivo. 2. Name all of the components required for the synthesis of the lagging strand in E. coli during DNA replication? 3. Which would be more harmful to a cell, a mutation in DNA or a transcription mistake that leads to an incorrect mRNA? Explain why? A mutation in DNA more harmful, because this mutations is propagated to future generations A transcription mistake only affects one RNA A mutated DNA affects all the RNA transcribed from it. 4. The core RNA polymerase enzyme combines with another protein to form the holoenzyme. What is the name given to this protein and what purpose does this protein serve? Sigma subunit The core RNA Pol without the sigma subunit doesnt bind and transcribe DNA efficiently. Different sigma subunits exists which direct RNA Pol holoenzyme (core+sigma subunit) to bind preferentially to specific promoter sites thus regulation gene expression.

5. When a metabolite- protein complex binds to a DNA site on an operon, transcription is increased. Is this operon under negative control or positive control? Is this an example of induction or repression? Under positive control. Induction. 6. What are Okazaki fragments? Explain how Okazaki fragments are generated. Okazaki fragments are fragments of DNA resulting from the semi-discontinuous replication of DNA in the lagging stand. Since DNA synthesis only occurs in the 53, DNA Pol III require by a primer from RNA. Therefore DNA can be synthesized 53 by Pol III. DNA Pol I removes the RNA primer leaving stretches of DNA called Okazaki fragements. 7. List four differences between DNA replication in eukaryotes and prokaryotes? Eukaryotes Proteins complexed with DNA Small Okazaki fragments (100200bps) Replication begins at hundreds of origins of replications Not all polymers are exonucleases Prokaryotes Very little complexing with protein DNA Okazaki fragments (10002000 bps) Replication starts at only one origin of replication All polymers are exonucleases

8. What is the Pribnow (TATA) box in E.coli? How does the Pribnow (TATA) box function? Pribnow box is the predominant promoter site in E-coli located -10 bps Pribnow box sequences that deviate from the consensus. Pribnow box sequences do not bind RNA Pol as well as Pribnow box sequences that deciate very little from the consensus sequence. The amount of transcription is proportional to the strength with which the RNA Pol binds to the Pribnow box. The more alike the more transcription. 9. What three binding sites on the 70S ribosome of E. coli are used during translation. What are their roles during elongation in protein synthesis? E-EXIT P-PEPTIDYL A- AMINOACYL LOOK AT MODEL OF EPA 1) P site occupied by peptidyl Trna 2) Elongination Factors take part in bringing in aminoacyl tRNA to A- site. 3) Peptide bond formation between aa in A site and peptide in P site. 4) P- site tRNA now uncharged. So no amino acid and no link. 5) Trna moves from P- site to the E-site and then released. 6) The peptidyl Trna moves from A-site to P-site. 7) A-site is not empty 8) The mRNA moves up 3 bases. 9) The cycle repeats. 10. What conditions are necessary for E. coli to transcribe the maximum amount of the lac operon structural genes? Be specific.

 As glucose becomes low cyclic AMP concentration are high. So you need high concentration of cAMP and high lactose concentration. Need both of these conditions for E-coli to transcribe the maximum amount of the lac operon structural genes.

11. What prevents bacterial DNA from being hydrolyzed by their own restriction enzymes. Ecoli methylate their DNA. Methylation. Restriction enzymes do not hydrolyze methylated DNA. 12. What are three major differences between protein synthesis in bacteria compared to eukaryotes? PROKARYOTES PROTEIN SYN. Transcription and translation coupled Mrna often code for multiple proteins (e.g. operons) N-formylated methionine Have a Shine-Dalgarno sequence EUKARYOTES PROTEIN SYN. Not coupled Mrnas code for single proteins. no N-formyl methionine No Shine-Delgarno sequence.

Shine-Delgarno sequence- makes sure translation starts at the proper codon. What makes it difficult for prokaryotes to translate from eukaryotes? 13. Give two major advantages of screening for a gene in a cDNA library over screening for a gene in a

DNA library. 1) The clones in the Cdna library contain copies of sequences that are expressed. No unexpressed copies of the clones are contained in the Cdna library. Hence the Cdna library will be much smaller than the DNA library. Fewer clones must be screened to obtain your desired gene. 2) Eukaryotic system would splice out introns and exons would come together. Second advantage is that once the clone is found, the cDNA is a copy of the processed gene without the introns, thus less work is required to express the gene. 14. How does one construct a cDNA library? 1) Isolate the RNA 2) Column chromatography to purify the cellular RNA. RNA in 0.5M NaCl put on oligo dT column. Solid matrix has oligo dT covalently bound to it. 3) Wash with 0.5 NaCl 4) Elute Mrna with Poly A tails with water 5) Collect Mrna 6) Reverse transcriptase creates complimentary DNA from mRNA 7) Degrade RNA and make the second strand of DNA 8) New DNA incorporated into plasmid 9) Screening for the desired gene 15. What components are necessary to do PCR? PCR-polymerase chair reaction 1) DNA to be amplified 2) Primers that flank the sequence to be amplified 3) Taq DNA Polymerase 4) Dttp, Datp, dgtp, dctp 5) PCR machine 16. What are VNTRs?

VNTR- Variable numbers of tandem repeats. Regions of DNA that are highly variable. Tandem repeats are short sequences of DNA that are repeated at a specific chromosomal locus. The number of repeats at a given chromosomal locus is highly variable (different) between unrelated persons.