1. Write the differences among three splicing mechanisms of reaction.

Nuclear pre-mRNA: spliceosome mediated, lariat intron, A residue in the branch site.

Group II intron: self-splicing by RNA enzyme, lariat intron, A residue in the branch site

Group I intron: self-splicing by RNA enzyme, linear intron, G residue in the branch site

2. What is the role of SR proteins in RNA splicing.

SR proteins binds to the exonic splicing enhancers, which functions to make sure that the
spliceosome will bind to splicing sites with the ESEs, the SR protein will recruits the U2AF to the 3’ splice
site and U1 snRNP to the 5’ splice site.

3. Write the components of translation. How do all components work together?

mRNA, tRNA, aminoacyl-tRNA synthetase, ribosome. How do they all work together?

The mRNA provides the information to be interpreted, tRNA will be the physical interface
between the amino acid in growing polypeptide chains with the mRNA, aminoacyl-tRNA synthetase will
charge the tRNA will specific amino acids – coupling the amino acids with its respective codons, and
ribosome will be recruited to the mRNA, coordinating the recognition of mRNA by each tRNA and
catalyze peptide-bond formation.

4. Describe the role of the components of spliceosome complex to complete RNA splicing reaction.

Spliceosome complex consists of 150 proteins and 5 small nuclear ribonucleoproteins. Most of
the work is done by the 5 snRNPs (U1, U2, U4, U5, and U6). But other proteins are still involved in the
splicing process such as the BBP (SF1 in mammalian) and U2AF. Although the main order of the process
unclear, the main process include these steps:

- formation of early (E) complex:

the formation of E complex involves U1 recognizing the 5’ splice site. U2AF subunit 65 and
subunit 35 recognize and binds to polypyrimidine tract of branch site and 3’ splice site respectively. As
well as binding of BBP to the A residue of the branch site, aided by U2AF.

- formation of A complex:

Formation of A complex is when the BBP is displaced by the U2 snRNP hence extruding the A
residue of the branch site.

- Transition of A to B complex:

Transition of A to B complex happens when tri-snRNP (consist of U4-U6 snRNP) binds to the pre-
mRNA. In this transition, U6 will replace U1 as it leaves the complex

- formation of C complex:
 The C complex will form after U6 interacts with U2 and U4 leave the complex, which will form an
active site. This will also cut the 5’ splice site hence producing the 5’ exon with 3’-OH ends (first
transesterification happens). And lastly U5 will aid the second transesterification in which the 3’-OH of
the 5’ exon attacks the 3’ splice site and combines the 2 exons.

5. Provide details in RNA transcription (initiation, elongation, and termination).

Initiation: 3 main steps:

Closed complex: initial binding of pol to promoter seq, DNA remains ds

Open complex: dsDNA separates about 13 bp long and forms transcription bubble

Initial transcribing complex: first 2 rNT brought to active site, happens multiple times
until it reaches about 10 rNT, promoter escapes

Elongation:

After 10 rNTs

DNA passes thru elongation enzyme, RNA pol will unwinds the DNA strand infront of it
while reanneals the strand behind it

Separate growing RNA chain with the dna template allowing proofreading

Termination: after finding stop codon

6. Significance of poly-adenylation in mRNA molecules.

Stabilize and guards the mRNA from endonuclease so it will not be degraded, and it recruits key
translation initiation factors

7. Ribosome structures and functions – attention to detail.

2 subunit, small and large subunit, does not work simultaneously. Small subunit binds to mRNA
first, followed by the large subunit.

8. Different transposons and their functions.

Transposon function:

Virus integration into the host, v(D)J recombination

3 types of transposons:

DNA transposons

Virus-like retrotransposons
 Poly-A retrotransposons

CSSR: phage insertion of DNA to host chromosome, alter gene expr, maintain structural integrity
of circular DNA

9. Translation – different phases and factors associated with each phase.

Initiation: ribosome must be recruited to the mRNA, charged tRNA must be placed to the P-site,
and ribosome must be precisely positioned in the start codon.

In prokaryotes, initiation is helped by 3 key factors, IF1 to prevents binding of tRNA to the part of
small subunit that will later become the A-site, IF2 to make sure that the initiator tRNA binds to the P-
site, and IF3 to prevents binding with the large subunits. After the initiator tRNA, mRNA, and all the Ifs
binds to the small subunits, IF3 will leave, allowing the recruitment of large subunits, followed by the
release of IF2 and IF1 from the complex. Hence, the final result is sandwiched mRNA between the two
ribosome subunits with initiator tRNA in the P site.

In Eukaryotes, however, the small subunit is already associated with the initiator tRNA when it is
recruited to the capped 5’ end. It then scans the mRNA from 5’ to 3’ direction until it reaches the first
start codon. First, the initiation factors (eIF1, eIF1A, eIF3, and eIF5) binds to the small subunit, they
perform similar functions as the prokaryotes IF3 and IF1, to prevent binding of large subunit and block
the A-site. Initiator tRNA (charged with methionine and not N-formyl methionine) will then be escorted
by eIF2 to the P-site, forming the 43S PIC (pre-initiation complex).

In separate reaction, eIF4E recognize and binds to the 5’ cap of mRNA. Other members of the IF4
families are then recruited such as eIF4A, eIF4G, and eIF4B, where eIF4B will unwinds any secondary
structure of the mRNA to ensure that the mRNA is single-stranded and ready to be translated. The eIF4-
mRNA complex will then recruits the 43S PIC forming 48S PIC. The 48S PIC, facilitated by eIF4A/B-
associated RNA helicase will scans the mRNA for start codon. After the initiator tRNA’s anticodon loop
meets with the AUG, a conformation change will happen. eIF1, eIF2, and eIF5 will be released and now
the 60S large subunit can be recruited.

Factors in initiation: ribosome subunits, initiator tRNA (charged with N-formyl

methionine/methionine in prokaryotes/eukaryotes), initiator factors (IF1/eIF1 blocks the A-site, IF3/eIF2
and eIF5B to recruit initiator tRNA and large subunit recruitment, IF3/eIF1 prevents binding of large
subunit)

Elongation: loading of correct aminoacyl-tRNA to the A site (by: EF-Tu ), formation of peptide
bond between aminoacyl-tRNA in A-stie and peptidyl-tRNA in P-site, translocation of the tRNA from P-
site to E-site and A-site to P-site (by: EF-G)

Termination: once it reaches stop codon it will be recognize by the release factors. Class 1 RF will
hydrolyze the peptide chain from the tRNA in P-site upon recognizing the stop codon. Class II RF will
stimulate dissociation of class I RF after the release of PP chain.
10. Key attributes in translation with specific functions.

11. Homologous, nonhomologous, and site-specific recombination – specific differences.

Homologous: occurs during meiosis, exchange between homologous protein

Non-homologous: chromosomal DNA DSB repair mechanism in somatic cell, directly ligate break
ends

Site-specific recombination: use restriction enzyme to cut at specific site and insert at desired
location, recombination DNA technology

12. Significance of the homologous recombination in eukaryotes

Genetic integrity, meiotic recombination ensure genetic diversity by gene reshuffling, DNA break
repair, chromosome pairing during meiosis (ensures no disalignment), restart stalled or damaged dna
replication, regulation of gene expression

13. Causes for the insertion of transposon in a gene.

Can happen because the presence of transposase/integrase, it can occurs due to a viral infection
or randomly. The transposase/integrase will cut the transposable element from the initial location and
then the transposon DNA element will nicks the target DNA and recombine.

14. Compare and contrast replication vs. transcription.

Replication: process that passes on genetic material, any mistakes (if not repaired) will be
permanent and passed on to the next generation, copies entire genome once during each cell division

Transcription: Produces transient copies and several copies of transcribed region, mistakes are
not permanent and does not have a big consequences, only selective part of the genome, occurs variedly
across the gene (regulated how much and how intensely a region is transcribed)

15. Major cause of mutations that are presence in the genome of all organisms? Provide specific

examples.

Transposons, accounts for 45% of duplicated sequence, transposon have little to no selectivity
which means it can randomly enter the genome.

16. Does mRNA bind two ribosomal subunits together? Why and why not?
 No, the small subunit binds to the mRNA first, initiating translation.

The large and small subunit associate with each other and the mRNA, translate the mRNA, and
dissociate after. At the very first step of the initiation process, the mRNA and initiating tRNA binds to free
small subunit of the ribosome. This complex will then recruits the large subunit of the ribosome and
sandwiches the mRNA together.

In prokaryotes, the small subunit will bind to the RBS via base pairing with the 16S rRNA. After
that, initiator tRNA will enter the P-site, skipping over the A-site, this tRNA will is charged with a
modified methionine known as N-formyl methionine.

The ribosome has 3 tRNA binding sites, the A-, P-, E- sites. A-site for aminoacylated-tRNA, P-site
for peptidyl-tRNA, and e_site for released tRNA after the polypeptide chain has been transferred to the
aminoacyl-tRNA.

17. Multiple ribosomes can translate the same mRNA at the same time. Describe how this is

advantageous to the cell.

Binding two ribosomal subunits together can happens, it can be beneficial especially when the
cell needs a huge amount of protein. More subunit bound to the mRNA means more protein will be
made.

18. What initiates recombination? Explain.

DSB, in a nutshell:

1. two double stranded dna

2. DSB happen in one of them, while the other unzips (opened)

3. the DSB strand will invade the unzipped dsDNA, so will the unzippend DNA invade the DSB-ed DNA.

4. holiday junction will be formed bcs of the invasion

5. holiday junction will move by branch migration elongating the exchange

6. holiday junction is cut

7. recombination complete

19. Why crossing over and why it is important?

Crossing over ensure genetic integrity, variability especially in meiosis where we need haploid
cells.
20. Homologous recombination can be used to fix DNA double strand breaks. What is involved in

this process?

RecBCD helicase/nuclease: unwound dsDNA, and process the ssDNA (MRX-complex in eu)

RecA strand invasion (Rad51 or Dmc1 in Eu)

RuvAB, recognize holiday junction and branch migration

RuvC, holiday junction resolution (Rad51C, RecQ, XRCC3 in eu)

21. What is the organization of nucleotide sequence information in mRNA?

Mono- and poly-cistronic?

22. What is the structure of tRNAs? How do tRNA structures contribute to the protein translation?

tRNA’s 3d structure offer stability due to the fact that it has base stacking, non-conventional
Watson-Crick pairing, and hydrogen bonds between the base pairing. The secondary structure consist of
parts that will help the translation such as the acceptor stem that will act as the site of amino acid
attachment, pseudo D loop contains modified base, D loop, contains dihydrouridines, anticodon loop
that is used for recognizing the codon by base pairing with the mRNA, and variable loop that is located
between the anticodon loop and pseudo U loop

23. How do aminoacyl-tRNA synthetases recognize and attach the correct amino acids to each

tRNA?

The aminoacyl-tRNA syhnthetase will charge tRNA will correct amino acid in 2 steps:
adenylylation of amino acid and transfer of adenylylated amino acid to tRNA. In the first step, the amino
acid will be adenylylated by hydrolysing pyrophosphate out of ATP resulting in a transfer of AMP from
ATP to the amino acid. The adenylylated aa will react to tRNA following a reaction that will results in
transfer of amino acid to the 3’ end of tRNA via the 2’- or 3’-OH and release of AMP. Aminoacyl-tRNA
synthetase is a highly specific components, meaning that one tRNA synthetase is dedicated of charging
just one specific amino acid to the tRNA. This specificity is due to some determinants found in the tRNA
including acceptor stem and anticodon loop. Other than that, aminoacyl-tRNA will more likely bind to
their respective amino acid, even when two similar amino acids present (i.e. valine and isoleucyl can
both fit into isoleucyl-tRNA synthetase, but binding of the isoleucyl-tRNA synthetase with valine will
produce extra -2 to -3 kcal.mol free energy hence the synthetase will bind to isoleucyl more
preferentially). Another mechanism to increase the accuracy is using an editing pocket to proofread the
charged products. Again, this editing pocket can be entered by numerous AMP-amino acids molecule,
but their respective AMP-protein molecule cannot enter this pocket. For example, isoleucyl-tRNA
synthetase’s editing pocket can be entered by AMP-valine, when this happened, the AMP-valine will be
hydrolyzed producing free valine and AMP. Meanwhile, AMP-isoleucyl will be too big hence it will not be
able to enter the editing pocket and won’t be hydrolyzed.
24. How does the ribosome orchestrate decoding of nucleotide sequence information and addition of

amino acids to the growing polypeptide chain?

Ribosome cannot distinguish between correctly and incorrectly charged tRNAs. The two subunits
of ribosome have different function, with the large subunit functions as peptidyl transferase center nd
the small subunit as decoding center. The vacant A-site of ribosome is placed in a distinctive position that
ensures the incoming aminoacyl-tRNA does not have access to bases immediately adjacent to the codon.
The ribosome also uses codon-anticodon interactions, exploits minor groove and 2 phases proofreading
to ensure that the correct aminoacyl-tRNA binds to the A-site.,

25. Differentiate different structure of typical prokaryotic and eukaryotic mRNAs.

Prokaryot: less to no intron, polycistronic, start codon sometimes GUG or UUG, contain RBS
(ribosome binding site) to recruit ribosome,

Eukaryote: a lot of intron, monocistronic, start codong always AUG, uses 5’ cap to recruit
ribosome,

RNA Splicing:

- important sequences:

-- 5' splice site:

exon-intron, consencus sequence: GU

-- 3' splice site

intron-exon, consencus sequence: AG

-- branch site

polypyrimidine near 3' end of intron, consencus sequence: A