BIO103 DISCUSSION ASSIGNMENT #12

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DISCUSSION  ASSIGNMENT  #12:  Lecture  20-21

 Lecture  20:  Translation and  protein  modifications  translation overview https://www.youtube.com/watch?v=Vu6LBOQSqiE (Andrew Vinal) https://www.youtube.com/watch?v=5bLEDd-PSTQ (ndsuVirtualCell)

3-D mols in real-time

https://www.youtube.com/watch?v=TfYf_rPWUdY (DNA Learning Center)

Prokaryotic Translation:

Initiation          https://www.youtube.com/watch?v=glsrY4dJzh8 (Biology animation videos) Elongation      https://www.youtube.com/watch?v=PpAg2K_7ID4 (Biology animation videos) Termination    https://www.youtube.com/watch?v=MNMc28EEkK0 (Biology animation videos)

Aminoacyl tRNA synthetase mechanism https://www.youtube.com/watch?v=igVWV8vzxYo (McGraw-Hill Animations)

Articles to read (Khan Academy):

overview:

https://www.khanacademy.org/science/biology/gene-expression-central-dogma/translation-po lypeptides/a/translation-overview

stages:

https://www.khanacademy.org/science/biology/gene-expression-central-dogma/translation-po lypeptides/a/the-stages-of-translation

1. The following are (some of the) molecules involved in translation. Describe the structure and function of each: mRNA, start and stop codon, 5’UTR, ribosomal A-, P- E-sites, rRNA, tRNA, release factor.

2. Address the following with regard to ribosomes.

a.) Compare/contrast ribosomes in prokaryotes/eukaryotes: structure, function, all places where located, where assembled, what do subunits do?

b.) Define what a ribozyme is. What are some ribozymes of the ribosome?  What is their function?

3. For the first tRNA that associates with (almost) all mRNAs and the small ribosomal subunit

(during translation initiation), indicate:

a.) What the anticodon of the tRNA is, (written in 3’ to 5’ and 5’ to 3’) and

b.) With which amino acid is that tRNA “charged” with

c.) What is meant by “charged”, how does a tRNA become charged? Explain the mechanism, in detail.

d.) Remember that every RNA, whether it becomes translated or acts as a functioning RNA, is coded for by DNA. “tRNA synthetases” are a family of enzymes, how many different tRNA’s synthetase genes exist in the genome? Describe why this number differs than the total number of tRNA’s. Hint: Can a single tRNA synthetase bind more than one amino acid? Why/not?

4. Describe, in detail, the steps of translation, including relevant nucleic acid sequences and enz/proteins involved, location, energy needs etc.

a.) initiation b.) elongation c.) termination

5. Given the info below, use the genetic code to determine the following (don’t attempt to do this in your head, draw it out and always make sure directionality “is correct”):

a.) tRNA with anti-codon 3’AUG 5’ will bind to which mRNA codon? It will be charged with which aa? What was the original dsDNA sequence, which was the sense strand? antisense?

b.) tRNA with anti-codon 3’ UAA 5’ will bind to which mRNA codon? It will be charged with which aa? What was the original dsDNA sequence, which was the sense strand? antisense?

c.) tRNA with anti-codon 5’ AUA 3’ will bind to which mRNA codon? It will be charged with which aa? What was the original dsDNA sequence, which was the sense strand? antisense?

d.) mRNA with codon 5’ CAC 3’ will be bound by which tRNA anti-codon? This tRNA will be charged with which aa? What is the dsDNA sequence and identify sense/antisense.

e.) mRNA with codon 5’ UGA 3’, What is tRNA anti-codon that will bind it? What aa is the tRNA

charged with?)

f.) the codon from the coding strand of DNA is 5’ TGG 3’, what is the mRNA codon?  What is the tRNA codon that will bind to the mRNA? What amino acid will it be charged with?

g.) the codon from the template strand of DNA is 5’ TGG 3’, what is the mRNA codon? What is the tRNA codon that will bind to the mRNA? What amino acid will it be charged with?

6. Don’t allow your eyes to JUST look for specific codon letters.  Also make sure directionality is correct!  List  the  stop  codons  (5’  to  3’)  of  mRNA with their corresponding template DNA sequences, write them in both 5’ to 3’ and 3’ to 5’. Do the same for coding DNA sequences.

STOP CODON mRNA 5’ to 3’Template DNA strandCoding DNA strand
3’ to 5’5’ to 3’3’ to 5’5’ to 3’
     
     
     

 Lecture  20:  Determining  effects of DNA mutations on  proteins

Resources:

Resources: Types of mutations (DNA and effects on proteins)- Bioninja https://ib.bioninja.com.au/standard-level/topic-3-genetics/31-genes/types-of-mutations.html

Genetic Code: https://www.genome.gov/genetics-glossary/Genetic-Code (NIH-NHG)

impact of mutations on translation (Khan Academy) https://youtu.be/1Dk0_-fyyDI https://youtu.be/JxC2phkdY8Y

(optional, but you should do for your own benefit)- Refresh your memory on the different types of DNA mutations. Define the following:

●    point mutation, substitution, transversion, transition, point deletion, point insertion, frameshift mutation

2. Mutations can also be categorized by their affects on the protein sequence.  Fill in the table below: Define silent, missense, nonsense mutations and provide an example (different from class) from DNA(coding)-> RNA-> protein for each (Just one codon is fine), note: WT = wildtype

Mutation: Silent missense nonsense   Definition:       WT DNA       RNA       Amino acid       Mut DNA       RNA       Amino acid       Cons or non- cons? Explain          

= non-mutated

3. For this question, use your combined knowledge of transcription, DNA mutations/damage and translation (Lectures 16,18, 19) to discuss which types of mutations/DNA damage would result

in conservative protein effects vs those that result in non-conservative effects. Do the same for causes of mutations). To do so, fill in the table below

 ConservativeNon-conservative
Definition:  
List of specific mutations types that would result in a       effect:  
List of specific causes that would result in a      effect:  

4. The questions below ask you to analyze the mutation, and predict the results (given which strand the mutation is on, and where in the gene the mutation occurred.) Unless indicated, assume the mutation occurred after replication and the cell is about to transcribe and translate the gene. Also assume the mutation was not repaired. Your answer should indicate specific

effects on transcription (or post txn modifications), effects on sequence of the mRNA, specific effects on translation, effects on protein sequence, predict if conservative or non and why.

a) What would be the immediate effect if there were any type of mutation in the TATA box of the promoter in euks?

b) What would happen if the +1 nucleotide of the coding strand was mutated from an A to C?

c) What would happen if the +1 nucleotide of the template strand was mutation from a T to A?

d) What would happen if there were a 2 bp deletion in an intron? The mutation was not in a splice site nor the branch point A.

e) What would happen if there was a 2 bp deletion in an intron at the 5’ splice site of between exon 2 and intron 2?

f) What would happen if there was a point substitution in the template strand from a 5’ATA3’ to a 5’ TTA3’ very early on in the ORF? What if the point substitution was at the same locus (aka place) but was on the coding strand?

h) What would happen if there was a point substitution in the template strand from a 5’ATA3’

to a 5’ TTA3’ in one of the last codons of the ORF?

i) What if there was a mutation in the polyA signal in the template strand?

5. Below is the double-stranded DNA sequence of part of a hypothetical yeast genome, which happens to contain a very small gene. Reminder: Transcription starts at the Transcription Start Site (TSS) also known as (+1 nt) after the promoter (shown in yellow), and proceeds in the direction of the arrow. Transcription stops at the end of the Transcription Terminator (shown in blue).

a) which strand is template (top or bottom)? coding? sense? antisense? how do you know?

b) What is the sequence of the mRNA produced from this gene? Label the 5’ and 3’ ends. c) What is the sequence of the protein produced from the mRNA in (b)? Label the N and C

termini.

d) If a mutation were found where a T/A (top/bottom) base pair were added immediately after the T/A base pair shown in bold, what would be the sequence of the mRNA? What would be the sequence of the protein?

6. A mutation is found in DNA, in a locus that encodes for a tRNA (aka when that part of the

DNA is transcribed, it makes a functional tRNA.) The wildtype (non-mutant) tRNA would typically be charged with Glu.

a) What mRNA codons code for glu?

b) What tRNA anticodons would bind to the mRNA codons (in (a)).

c) What if the mutant tRNA is still made and is still charged but recognizes and binds to the codon GAC? How would aa sequence be affected? predict if conservative or not and why

d) continuing (c), what if the mutant tRNA recognizes and binds to GUG? How would aa sequence be affected? predict if conservative or not and why

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