Name: 1 point Chem 465 Biochemistry II Test 3

Multiple choice 4 points each 1. RNA polymerase: A) binds tightly to a region of DNA thousands of base pairs away from the DNA to be transcribed. B) can synthesize RNA chains de novo (without a primer). C) has a subunit called ë (lambda), which acts as a proofreading ribonuclease. D) separates DNA strands throughout a long region of DNA (up to thousands of base pairs), then copies one of them. E) synthesizes RNA chains in the 3'65' direction.

2. Splicing of introns in nuclear mRNA primary transcripts requires: A) a guanine nucleoside or nucleotide. B) endoribonucleases. C) polynucleotide phosphorylase. D) RNA polymerase II. E) small nuclear ribonucleoproteins (snurps).

3. Which one of the following statements about mRNA stability is true? A) Degradation always proceeds in the 5' to 3' direction. B) Degradation of mRNA by polynucleotide phosphorylase yields 5'-nucleoside monophosphates. C) In general, bacterial mRNAs have longer half-lives than do eukaryotic mRNAs. D) Rates of mRNA degradation ared always at least 10-fold slower than rates of mRNA synthesis. E) Secondary structure in mRNA (hairpins, for example) slows the rate of degradation.

4. Which of the following statements about the tRNA that normally accepts phenylalanine is false? (mRNA codons for phenylalanine are UUU and UUC.) A) It interacts specificially with the Phe synthetase. B) It will accept only the amino acid phenylalanine. C) Its molecular weight is about 25,000. D) Phenylalanine can be specifically attached to an -OH group at the 3' end. E) The tRNA must contain the sequence UUU.

5. In bacteria the elongation stage of protein synthesis does not involve: A) aminoacyl-tRNAs. B) EF-Tu. C) GTP. D) IF-2. E) peptidyl transferase.

1 Short answer questions (5 points each) You may skip ONE 6. What is the function of the ó subunit in the E. coli RNA polymerase

The ó subunit of RNA polymerase is the unit that recognizes the promoter sequence. The ó70 is the most common one and it recognizes the seqence TTGACA in the -35 region and TATAAT in the -10 region.

7. Briefly describe the processing that goes into making a the structure of a eukaryotic mRNA. The brief part is that I wanted you just to mention: The 7-methylguanosine cap and some methylation of ribose at the 5' end by the Cap-synthesizing complex on the CTD region of the RNA polymerse The splicing our of introns in the middle of the mRNA by the snurps also on the CTD region of the RNA polymerase The cleavage of the message at the AAUAAA sequence and addition of a poly A tail of 80-250 A’s and the 3' end by polyadenylate kinase.

8. Why does a typical retrovirus particle contain both an RNA genome and a tRNA from the previous host cell? The tRNA from the previous host serves as a primer so the DNA polymerase of the reverse transcriptase can begin to make DNA from the single stranded RNA genome of the virus.

9. The eukaryotic cell has RNA polymerases, pol I, pol II, and pol III, what are the function of these three polymerases, and how are they different structurally? RNA pol I is specific for synthesizing preribosomal RNA which gets cleaved to make 18S,5.8S and 28S ribosomal RNA RNA pol II is specific for making mRNA and a few special functions RNA’s RNA pol III makes tRNAs and the 5S rRNA I thought there was a table in the text comparing the size of these polymerases, but I can’t find it, so I will forget about the part of ‘... how are they different structurally’ but one could mention how complex RNA pol II is due to all the different functions it serves.

10. There are two classes of Aminoacyl-tRNA Synthetases. How are the two classes different from each other. Both classes start by attaching an amino acid to the á phosphorous of ATP. In class I synthetases the amino acid is next transferred to the 2' OH of the 3' A of the tRNA. The AA is then transferred to the 3' OH of this ribose in a second step. I the class II synthetases the amino acid is transferred directly from the AA-AMP to the 3'OH of the tRNA. In addition class I synthetases recognize one side of the tRNA while the class II synthetases recognize the other side.

2 11.What is the function of the Shine and Dalgarno sequence. The Shine and Dalgarno sequence is the sequence on a eukaryotic mRNA that hybridizes to a segment of 16S RNA on the 30S subunit of a ribosome to initiate the binding of the mRNA to the 30S subunit and initiate the assembly of a ribosome.

Longer questions (12.5 points each) - You make skip ONE (total of 6) 12. What are the 4 types of introns? In what organisms are they found? How are they the same? How are they different?

Type I - Self splicing by the RNA itself. Found in some nuclear, mitochondrial and chloroplast. Uses the OH of the from a guanosine nucleoside or nucleotide to cleave the 5' end of the intron. The OH on the 5' exon then attacks the 3' end of the intron to fuse with the 3' exon and release the linear intron.

Type II - Self splicing by the RNA itself, Found in mitochondrial and chloroplast in fungi, algae and plants. In this mechanism the OH of residue in the intron attacks the 5' end of the intron to release the 5' exon. The OH from terminus of the 5' exon then attacks the residue at the end of the 3' exon to fuse the exons together and release a lariat shaped intron.

Type III This mechanism is used for most mRNA’s for most eukaryotic organisms. In this mechanism a spliceosome composed of several snRNPs is used to splice out lariat shaped introns

Type IV is used only for certain tRNAs requires splicing endonucleases to cleave the intron at its 5' and 3' ends, and then ATP is used to join the exons together in a reaction similar to that used in DNA ligases.

13. Since the last test we have seen lots of RNA molecules that have important functions in the eukaryotic cell Identify each of the following RNA’s and what they do gRNA -guide RNA - used in mitochondria and chloroplasts to guide additions or deletions from some RNA messages mRNA - message RNA - the RNA that is used to carry a DNA sequence to a ribosome to translation to a protein sequence. miRNA -microRNA short pieces of RNA used in degradation and suppression of mRNA’s rRNA - ribosomal RNA - the RNA that has large structural and catalytic functions in ribosomes snRNA - small nuclear RNA’s - the RNA part of the spliceosomal small nuceoprotein complexs. snoRNA - small nucleolar RNA’s - guide nucleoside modifications in rRNA.

] tRNA - transfer RNA’s - the RNA molecule that an amino acid gets attached to and used in protein synthesis tmRNA (2 bonus points) - a specialized RNA used in ribosome rescue to release a ribosome from an mRNA that has lost its termination codon.

3 14. In the chapter on RNA metabolism you saw several different kinds of enzymes that make RNA. Tell me about the different enzymes, how are they different and what are their functions in the cell. DNA dependent RNA polymerase - makes RNA from a DNA template Polyadenylate polymerase - adds a polyA tail to mRNA. Primease - make a short sequence of RNA on DNA before the DNA polymerase takes over RNA dependent RNA polymerase (RNA replicase)- makes RNA from RNA. This activity is see in certain RNA viruses that do not copy RNA to DNA in their life-cycle. Interestingly the active replicase only has one subunit encoded on the virus genome. The other three subunits are host proteins normally used in protein synthesis

It wasn’t till I was making this key that I realized that when I wrote this question I was thinking about reverse-transcriptases and telomerases, but they make DNA from RNA so the question I asked did not include them.

15. What is the tie between transposons, retroviruses and introns. Transposons are pieces of DNA that move from one place to another. Many transposons use a mechanism that involves an RNA intermediate, and that RNA intermediate resembles a retrovirus in structure and mechanism. The major difference being that the transposon is missing the sequences that would correspond to the viral coat proteins so it can’t make a virus particle. Group I and group II introns are also ‘mobile’ in that they can move around like a transposon. In addition to self-splicing the intron also contains DNA endonucleases that allow the intron to insert into DNA like a transposon. In particular the group II introns use an RNA intermediate in this homing process so it also looks like a retrovirus like mechanism.

16. What are ADAR’s and APOBEC’s. Describe how they functions and why they are important in eukaryotic cells. ADAR - Adenosine deaminases that act on RNA - changes adenosine to inosine. Common in primates and works primarily in Alu elements. These element are usually located in introns or in untranslated parts of the mRNA on the 3' and 5' ends of the message. While this activity would seem to have minimal effects since the modifications are usually not in coding regions of the message, defect in this system are associated with ALS, epilepsy and major depression. APOBEC - apoB mRNA catalytic peptide changes Cytidine to Uridine. The APOBEC family of proteins are implicated in a wide range of cellular function and some will modify DNA as well as RNA. The one example of APOBEC activity mentioned in class was to change a C to a U in the mRNA of a LDL in the small intestine. In this tissue the C to U changes a Gln codon to a stop codon, so the protein synthesized in the small intestine roughly half the size of the protein synthesized in the liver which does not have the APOBEC activity.

4 17. Compare the initiation step of protein synthesis between prokaryotes and eukaryotes. Initiation of protein synthesis in prokaryotes involves three factor, IF1,IF2, and IF3. IF1 and IF3 first bind to the 30S subunit of the ribosome. The mRNA next binds to the complex and a portion of the 16S rRNA in the 30S subunit hybridizes to the Shine- Delgarno sequence of the mRNA to position the initiation AUG codon in the P site of the complex. The IF2 factor next binds GTP and tRNAfMet and guides it into the P site of the complex. At this point the GTP hydrolyzes, the initiation factors are released and the 50S unit binds to complete the ribosome. Initiation in eukaryotic systems is a bit more complex. To begin with there are at least 12 initiation factors: eIF1 ,eIF1A, eIF2, eIF2B, eIF3, eIF4A, eIF4B, eIF4E, eIF4F, eIF4G, eIF5 and eIF5b ( I don’t expect you to know all of these). eIF1, eIF1A, and eIF3 start things off by binding to the 4 0S ribosomal subunit. The initiation Met tRNA is next bound to an eIF2 that contains GTP. This tRNA complex plus eIF5B that also has a bound GTP next bind to the 40S ribosomal complex. Concurrently eIF4F has bound both 3' and 5' ends of an mRNA. And this now binds to the growing 40S complex. At this time the eIF4F starts hydrolyzing ATP and moving down from the 5' cap on the mRNA until it finds the first AUG codon. Once this happens the GTP’s hydrolyze to GDP, and the initiation factors are released as the 60S ribosomal subunit binds to complete the initiation complex.

5 1B 2E 3E 4E 5D

6