Exam 2 - Translation

Question 1

mRNA

Answer 1

protein coding genes transcribed and used as template to make protein

Question 2

rRNA

Answer 2

pair with ribosomal proteins to make ribosomes

Question 3

tRNA

Answer 3

Each aa covalently bound to subset of tRNA containing 3-nucleotide anticodon sequence

Question 4

Protein synthesis in ribosome

Answer 4

tRNA loaded with aa floating in

need to decode message

Question 5

Codons

Answer 5

64 codons
20 amino acids
Multiple different codes for same aa; except Met and Try

Question 6

Reading frames

Answer 6

5’ AUG start codon

Question 7

Mutations

Answer 7

Frameshift: addition/deletion of a base causing a shift in the frame

Nonsense: aa changes to stop codon

Missense: one aa changed
Silent: no change in sequence

Question 8

RNA molecules can bp and fold into structure

Answer 8

Conventional: UA, GC
Unconventional: CU,UC,GA
tRNA: folded into 3D stem and loop structure

Question 9

tRNA structure

Answer 9

deamination of adenine to make inosine

wobble position of tRNA to bp with more codons

Question 10

wobble position

Answer 10

First nucleotide of anticodon

Question 11

Inosine

Answer 11

can base pair with, G, C, U

Question 12

Loading tRNA

Answer 12

20 different amino-acyl synthetases

Couples specific aa via high E ester bond to the tRNA that has the proper anticodon (cognate tRNA)

2 pocket to fit aa for double checking

Question 13

Ribosome during translation

Answer 13

rRNA: 50% mass

Question 14

Translational mechanism

Answer 14

1.) Pre-initiation complex comes together to recognize mRNA

2.) Small subunit pre-bundled with initiator tRNA and eIF2 GTP

3.) Small subunit scans for start codon (ATP required)

4. ) AUG identified. Conformational change in tRNA recognized by eIF2 which hydrolyzes its ATP

5.) eIF2 leaves. Large subunit binds

6.) Elongation can happen but requires proof reading.
Codon anticodon matching
Ribosomal RNA hugging
eF1alpha-GTP
Time delays after GTP hydrolysis

7.) peptidyl transfer catalyzed and peptide bonds formed

8.) eF2alpha moves ribosome along mRNA by one codon

9.) Stop codon. tRNA cannot match, eRF1 and eRF3 bind to stop codon.
Water added and protein translation terminates, ribosome falls apart.

Question 15

eIF

Answer 15

eukaryote initiation factor

initiation complex interacts with both 5’ cap and poly A BP

Question 16

Elongation

Answer 16

amino-acyl tRNA float into A site.

Need more than codon/anticodon matching to present mistakes

Proofreading

Question 17

Subunits of Ribosomal RNA

Answer 17

Small: provides mRNA binding site and 3 binding sites for tRNA

Large: catalytic activity of peptidyl transfer

Question 18

Peptidyl transer

Answer 18

peptide bond forms between adjacent acids in ribosome

tRNA kicked out with OH group before leaving ribosome

Question 19

Analysis of Gene expression at different levels

Answer 19

RNA expression:
RNA seq
scRNAseq (what cell expressing what)
In situ hybridization (where in tissue)

Proteins
Western blotting
Immunochemistry, florescence, confocal microscopy (where)

DNA status
Epigenetic marks and maps (ChIP seq)

Question 20

RNA seq

Answer 20

Compare transcriptome of normal neurons vs mutated neurons

Problem: total RNA is mixture of all types; only want mRNA
Use PolyA tail to separate
Oligo dT binds to PolyA tails

mRNA isolated
Fragment into pieces
Convert to cDNA
Add linkers
Amplify PCR

Question 21

Single Cell RNA

Answer 21

can make library for each cell

Investigate cellular plasticity, different patterns of cellular differentiation

increased resolution

increased cost

Question 22

scRNAseq

Answer 22

Identify cell types within heterogenous mixtures

Question 23

scRNAseq

Answer 23

composite library based on transcript

Info not provided:
Where in the tissue such genes are expressed (RNA and protein level)

transcribed genes become functional proteins

The PTMs to make the protein active

How expression of the gene changes over time

Detection of genes transcribed at very low levels

Question 24

CRISPR-Cas9