Assignment 11-key-1 - MBB 222 Assignement 11 PDF

Preview

Summary

MBB 222 Assignement 11...

Description

Assignment 11

MBB 222 Fall 2020 (Paetzel)

Answer the questions in your own words and to the best of your ability. Upload as WORD or PDF files to Canvas by Mon. Nov. 27, 2020, 11:30 pm. This assignment will be graded for completion only. Max. 1% of final grade. Marks are only included to give you an idea of how exams will be marked. 1. For the coding strand DNA sequences listed below write out the sequences of the corresponding template strand and mRNA. Use the correct convention for polarity and label the template and mRNA strands. (Use Courier font to get nucleotides to line up.) (3) Eg.

coding strand 5’-AATTCGGCTCAAGTAGCTTCTAGGGGCCA-3’ template strand: 3’-TTAAGCCGAGTTCATCGAAGATCCCCGGT-5’ mRNA: 5’-AAUUCGGCUCAAGUAGCUUCUAGGGGCCA -3’

(a) 5’-TTCGCGACAATTATTCG-3’ (DNA coding strand) 3’-AAGCGCTGTTAATAAGC-5’ (DNA template strand) 5’-UUCGCGACAAUUAUUCG-3’ (mRNA) (b) 5’-GGCGATCGATAT-3’ (DNA coding strand) 3’-CCGCTAGCTATA-5’ (DNA template strand) 5’-GGCGAUCGAUAU-3’ (mRNA) (c) 5’-ATATACGTGCCAGGGCTTTA-3’ (DNA coding strand) 3’-TATATGCACGGTCCCGAAAT-5’ (DNA template strand) 5’-AUAUACGUGCCAGGGCUUUA -3’ (mRNA) (mRNA sequences match those of the coding strands with Us in place of Ts) Note: the convention is to write sequences 5’-3’; often the ends are not labeled. 2. For the mRNA sequences listed below, write out the corresponding DNA duplex. Use the correct convention for polarity and label the coding and the template strands. (Use Courier font to get nucleotides to line up.) (3) Eg. mRNA: 5’-AUGGCUUAUGACCGCAUGAUC-3’ DNA duplex: 5’-ATGGCTTATGACCGCATGATC-3’ – coding strand 3’-TACCGAATACTGGCGTACTAG-5’ – template strand (a) 5’-AUUCAGAGUGCAACU-3’ (mRNA) 5’-ATTCAGAGTGCAACT-3’ (DNA coding strand) 3’-TAAGTCTCACGTTGA-5’ (DNA template) (b) 5’-CCUGGUCACGUGUUUCAUGCGAAUGUAACUA-3’ (mRNA) 5’-CCTGGTCACGTGTTTCATGCGAATGTAACTA-3’ (DNA coding strand) 3’-GGACCAGTGCACAAAGTACGCTTACATTGAT-5’ (DNA template) (c) 5’-CGUACAUGCCUAAGUCUGAC-3’ (mRNA) 5’-CGTACATGCCTAAGTCTGAC-3’ (DNA coding strand) 3’-GCATGTACGGATTCAGACTG-5’ (DNA template) 3. Suggest why DNA is a better molecule than RNA for the purpose of long-term storage of genetic information? (1) RNA is very reactive because of its 2’OH, so it is not as stable as DNA. (FYI: The 3’ OH can react with other species – as a nucleophile. It can also “autohydrolyse”. The single stranded nature of RNA 1

Assignment 11

MBB 222 Fall 2020 (Paetzel)

als makes is less stable than ds DNA because its hydrophobic bases are exposed, and ss nucleic acid is more susceptible to nucleases than ds nucleic acid.) 4. List 3 similarities and 3 differences for polymerization reactions catalyzed by DNA pol III vs. RNA polymerase. (6) Similarities: - both synthesize a polynucleotide chain in the 5’ to 3’ direction - both require a DNA template strand and synthesize a strand complementary to the template - in both processes (replication and transcription) incoming nucleotides are selected by base pairing to the template DNA strand - both use nucleoside triphosphates (either ribo- or deoxyribo- ) as substrates and - both catalyze the addition of one nucleotide at a time, use nucleophilic attack by the 3’ OH of the last nucleotide (nt) added on the 5’ PO4 of the incoming nt, form - both are Differences - DNA pol III has two active polymerase subunits so two daughter strands are replicated simultaneously; RNAP has multiple subunits but a single polymerase subunit ~1000 nt/sec) than RNA pol (20-50 nt/sec) - RNA pol has helicase activity, DNA pol III does not (requires a separate helicase to unwind DNA) - dNTPs are used for DNA synthesis, NTPs are used for RNA synthesis - DNA replication requires a primer; ually ATP, in its active site by coordination with a bound Mg2+ - the 3’OH of this first nt is the nucleophile for the next NTP to be added (so the first nucleotide in the RNA strand is a triphosphate; PPi is not released) - DNA replicatio – more robust proof-reading, 3’-5’ nuclease activity to remove mismatched nucleotides (RNA proofreading not well-understood – “back-translocation to remove mismatched nts but no discrete nuclease active site) 5. For the new RNA strand shown at the right being synthesized, and draw an incoming NTP and show which atom executes . Draw the base as a box and the phosphates as circles. Label the a, g฀ phosphates ฀฀฀฀฀฀฀฀attacking 3’ OH. (6)

2

Assignment 11

MBB 222 Fall 2020 (Paetzel)

6. List three

. (6) bacteria

human

• transcription th translation (i.e. they occur simultaneously on the same mRNA transcript)

• transcription and translation oth temporally and spatially because transcription occurs in th d translation occurs in the cytoplasm

• transcription produces mRNA that is ready to be used

• the RNA produced from transcription is : - acquire a 5’ cap - acquire a 3’ poly(A) tail - introns are excised out

• gene expression is controlled mainly at the level of transcription, in particular at initiation of transcription

• gene expression is controlled at many levels – transcription, RNA processing, transport of RNA to cytosol, translation

• a single polymerase,

• use 3 different polymerases to make rRNA, tRNA, mRNA and other RNAs

• use s f • is both (

, makes all of the

cognize dif

• use • is packing in chromatin (30 nm fiber), , activators, histone acetylases, chromatin remodeling complexes

)

7. Match the transcription component on the left with the correct definition/role in the middle column. Indicate which or these components are prokaryotic, eukaryotic or both. (24) Reg. component

Definition/role in transcription regulation

Prokaryotic/eukaryotic?

operon

a cluster of genes controlled by a single promoter

prokaryotic

promoter

DNA sequence bound by RNA polymerase to initiate transcription

both (is upstream of gene in prokaryotes; can s

operator

)

inducer

binds to repressor, reduces its a

prokaryotic

operator

site o

mediator

links

repressor

binds to th

prokaryotic

enhancer

DNA sequence bound by activator

eukaryotic

promoter

DNA sequence that RNA pol (RNAP) binds

both (is upstream of gene in prokaryotes; can overlap w/ coding sequences in eukaryotes)

prokaryotic at enhancers

3

eukaryotic

Assignment 11 activator

MBB 222 Fall 2020 (Paetzel) protein that binds at/near promoters to enhance RNAP binding and initiation of transcription (prokaryotes); binds to enhancer sequences to enhance transcription (eukaryotes – enhancer seqs can be quite far from the promoter brought to the promoter by “looping” of the DNA)

both (but activators promoter; activators bi

co-repressor

enhances affinity of repressor for operator

prokaryotic

transcription factor

binds t

eukaryotic

TATA box

a DNA sequence within the promoter that is bound by the TATA binding protein (TBP) of TFIID

eukaryotic

Sigma factor

part of RNAP holoenzyme, binds to promoto

prokaryotic

s RNAP II

8. Explain the difference between a

)

-repressor. (3)

-repressors are both effector molecules (ligands) that bind to repressors and alter their activity but they have opposite effects. ind to repressors and cause them to dissociate from the operator, allowing transcription to proceed. Coind to repressors and increase their affinity for the operator, resulting in inhibition of transcription. 9. List the RNA products of the prokaryotic RNA polyermase and each of the three eukaryotic RNAPs (4). prokaryotic RNAP: mRNA, rRNA, tRNA RNAP I: 45S RNA (the precursor rRNA, is cleaved to form rRNAs for the ribosome) RNAP II: mRNAs RNAP III: tRNAs, 5S rRNA (the other component of rRNA) 10. Fill in the blanks regarding eukaryotic transcription. (7) (a) The TATA binding protein (TBP) is part of the TFIID complex and binds to the TATA box. (b) The 5S rRNA is synthesized by RNAP III in the nucleus, whereas the 45S rRNA is synthesized by RNAP I in the nucleolus. Ribosomes are assembled in the nucleolus using ribosomal proteins synthesized in and imported from the cytoplasm. (c) RNAP II synthesizes all of the mRNA in the cell. (d) TFIIH is the transcription factor for RNAP II that has both helicase and kinase activity. 11. List the processing events that occur in the ;

. (3)

,s

12. Explain what happens at

. (4)

Soon after RNAP II transcribes through the cleavage signal sequence, a protein complex containing an endonuclease and a polyadenylate polymerase (among other proteins) complex and c . The polymerase will continue to transcribe for a while 4

Assignment 11

MBB 222 Fall 2020 (Paetzel)

before t this polymerase (associated with the CTD of RNAP I

. The polyadenylate -mRNA.

-

13. What are the two regulatory sequences o located relative to the gene? (2)

and where are they

The promoter s located just upstream (5’) of the gene and t as 50,000 bp away from the gene or even downstream of the gene.

can be located upstream as far

14. In addition to the proteins that bind to regulatory sequences on DNA, such as RNA polymerase II, transcription factors, and other regulatory proteins, what e necessary to allow . (2) Chromatin remodeling complexes and histone modifying enzymes (esp. acetylases/acetyltransferases) are required to expose the DNA to be accessed by the binding proteins. 15. Briefly explain why n help to s thousands of base pairs away. Why does a eukaryotic gene have so many different proteins in addition to the RNAP II transcription factors regulating its expression? (4) Activators bind to (sometimes distant) enhance sequences and are connected to transcription factors via a mediator protein complex, help TFs to bind to the promoter, facilitating binding and activation of RNAP II. Each regulatory protein can bind to sequences on DNA that are present in multiple locations on the chromosomes. Having many proteins that have to bind these sequences and interact with each other ensures that genes are not expressed randomly in an unregulated way. The set of proteins that regulate expression of a given gene are regulated themselves by ligand binding, which allows the cell to sense environmental changes and respond. The ligands, like bacterial effectors, alter the DNA binding affinity of the regulatory proteins. 16. Where and when do mRNA processing (5’ capping, splicing and 3’ polyadenylation” occur in the eukaryotic cell? (2) They occur in the nucleus as the mRNA is being synthesized. The proteins/enzymes/factors involved in each of these processes associate with the C-terminal domain of RNAP II. 17. What do the loops represent in the EM image in slide 18 of Lecture 24? (1) Introns in the DNA that have been excised from the mRNA by splicing. 18. Generally and briefly (1 sentence) explain the role of snRNAs in spliceosome-mediated splicing? (1) They allow the snRNPs to recognize precise sites on mRNA via base complementarity; they catalyze the splicing reaction (U6, FYI). 19. Compare and contrast

-s

Group

-

ns. (6)

S : Both involve two trans-esterification reactions to precisely cut out the intron and join the exons; both involve nucleophilic attacks by ribose OHs. :S se a large protein:RNA complex (the spliceosome) to catalyze excision of the intron and splicing together of the exons whereas atalyze their own excision and exon ligation (self-splicing); the first nucleophilic attack on the upstream splice site of 5

Assignment 11

MBB 222 Fall 2020 (Paetzel)

spliceosome introns comes from the 2 ’OH of a branch-site A within the intron; it comes from the 3’ OH of a free G (guanosine, GMP, GDP, GTP) for Group I introns; a lariat structure is formed for spliceosome introns whereas a linear intron is released for Group I introns 20. Could the g

n splicing? (1)

No, it lacks

k.

21. On the drawing below of raw in the branch site A as a box with its nucleophile. Use arrows to shown the nucleophilic attacks that occur to release exons. Number them in order of occurrence. Be precise about your arrow placement. (5)

the

22.

How does alternative splicing increase the diversity of the genome? (1) One gene can encode for more than one protein if the exons as well as introns can be excised in different ways.

6...