Bio 351
Tuesday’s Lab Section
Extra Credit
Dr. Read
#51: Compare and contrast the processes of transcription and translation in prokaryotes and eukaryotes.
Transcription:
Prokaryote
Eukaryote
 One type of RNA polymerase
 3 types of RNA polymerase: I, II, and III
 RNA polymerase forms holoenzyme by
 I & III transcribe tRNA, rRNA, and
binding with sigma factor
snRNA. II transcribes proteins.
 Multiple types of sigma factors allow the
 General transcription factors (TF**) must
binding of different types of promoters
assemble at the promoter with polymerase
before transcription can begin (no sigma
 RNA polymerase/sigma sub-unit
factor)
holoenzyme bind to promoter region
 TFIID binds the TATA box, distorting the
 DNA is unwound by RNA polymerase (no
DNA and creating a landmark for other
ATP required)
proteins which, with polymerase II, forms
 When transcription starts, sigma factor
the transcription initiation complex
dissociates after ~10 bases, allowing
 TFIIH contains helicase, and protein kinase
conformational changes by RNA
to phosphorylate the RNA polymerase II
polymerase and a tightening of the jaws on
tail, which also receives capping factors,
the DNA
splicing factors, and polyadenylation
 Elongation speeds up to 50
factors. Conformational changes allow
nucleotides/second
tight binding of the polymerase, releasing
 Terminator halts RNA polymerase by
the TFs and getting transcription going.
destabilizing its hold on the RNA
 Other proteins present at start of
 New RNA strand is released and RNA
transcription: mediator, chromatin
polymerase detaches from DNA
remodeling complexes, histone acetylases.
 Polymerase is released and seeks another
 Elongation factors may assist with
sigma factor
transcription around the nucleosomes.
 Termination signal consists of string of A Supercoiling is resolved by topoisomerase.
T pairs, which, when transcribed to RNA,
 When about 25 nucleotides of the new
form the hairpin loop, possibly forcing
RNA strand have been completed, the
open the jaws of the polymerase
RNA polymerase tail caps the 5’ end with
 Promoters are asymmetric—transcription
a modified guanine nucleotide: a
usually occurs on only one strand of DNA
phosphatase is removed from the 5’ end, a
 Supercoiling is resolved by gyrase which
guanyl transmethyl transferase adds a
produces negative supercoils in plasmid
GMP, and methyl transferase adds a
DNA
methyl group to the guanosine (CBC cap
 Transcriptions are polycistronic
binding complex).
 Translation begins before translation has

CstF (cleavage stimulation factor) and
completed (no introns)
CPSF (cleavage and polyadenylation
factor) are transferred from the RNA
polymerase tail to the 3’ end of the
emerging RNA molecule, cleaving it and
adding the poly-A tail.
 Polymerase I synthesizes rRNA. No Cterminal tail because it doesn’t cap or tail
the rRNA.
 rRNA precursor is chemically modified
into: 16S, 5.8S, 28S rRNAs. Polymerase
III actually makes the 5S rRNA.
 The nucleolus is where ribosomal subunits
are assembled.
 Transcriptions are monocistronic
 Transcripts are composed of coding (exon)
Bio 351 – Tuesday’s Lab Section – Extra Credit –Page 1 of 4
Prokaryote
Translation:
Prokaryote
 In prokaryotes, there is no 5’ cap, so the
small ribosomal subunit seeks out the
Shine-Delgarno sequence a few
nucleotides up from the AUG start codon.
Because the prokaryote does not require
the 5’ cap for starting, it can utilize start
codons positioned later in the mRNA
sequence, encoding several different
proteins.
 Bacterial mRNAs are polycistronic;
eukaryotes only code for a single protein.
 Bacterial translation can also occur while
transcription is still occurring.
Eukaryote
and non-coding (intron) sequences and
must be edited prior to translation.
Translation cannot occur before
transcription ends.
Eukaryote
 snRNAs (small nuclear RNAs) complex to
form snRNP (small nuclear ribonucleoproteins)—the core of the spliceosome.
 Splicing signals on the pre-mRNA
molecule are recognized, the two ends of
the intron are brought together, a lariat is
formed, and the intron sequence is excised,
yielding the finished mRNA.
 Rearrangements during splicing allow for
translation of a variety of proteins from the
original pre-mRNA transcript.
 The nuclear pore complex recognizes the
proper set of proteins bound to the
completed mRNA molecule to allow its
transport into the cytosol.
 Aminoacyl-tRNA synthetase binds the
correct amino acid to the 3’end of the
correct tRNA, producing a high energy
bond in the process.
 The small ribosomal subunit is loaded up
with eukaryotic initiation factors and the
initiator tRNA, binds the 5’cap, and seeks
out the initial AUG start codon.
 Translation begins, the initiation factors
pop off and the large subunit assembles
with it.
 Initiator tRNA = brings methionine to the
AUG start codon
 Proteins are assembled as per the
transcript, with each codon (three bases)
indicating the next amino acid to be
attached to the polypeptide by the
ribosome.
 The stop codon at the A site stops
translation and causes release factors to
bind the A site, forcing peptidyl transferase
in the ribosome to catalyze the addition of
a water molecule to the peptidyl-tRNA
rather than an amino acid. This releases
the carboxyl end of the chain from its
tRNA, releasing the polypeptide, and
separating the ribosomal subunits and the
mRNA.
 Wobble describes the fact that the third
position of the anticodon on a tRNA can
vary and still work.
 hnRNP remain on introns, helping to
distinguish them from mature mRNA
molecules.
Bio 351 – Tuesday’s Lab Section – Extra Credit –Page 2 of 4
#52: How is it possible to package 2 meters of DNA into a nucleus with a diameter of 6 micrometers?
Five levels of DNA packaging:
1. Double helix – formed via hydrogen bonds
2. Beads on a string – describes the packaging of DNA around histones into necleosomes. Two of
each of the histone proteins (H2A, H2B, H3 and H4) form the histone octamer, around which the
DNA helix binds, forming the “beads on a string.” Necleosomes repeat about every 200
nucleotides, the DNA in between is called linker DNA. DNA binds to the nucleosome complex
via 142 hydrogen bonds between the amino acid backbone of the histones and the phosphodiester
backbone of the DNA, hydrophobic interactions, and salt linkages. Kinks do occur in the DNA.
The N-terminal amino acid tails of the 8 histones stick out from the nucleosome.
3. 30 nm fiber – fiber formation facilitated by histone tails. Tail modifications by HATs (histone
acetyl transferases) and HDACs (histone deacetylases) alter stability of the 30-nm fiber.
4. Loops and coils -- nucleosomes are further packed into the compact 30-nm chromatin fiber via
zigzag folding. Loop regions can be accessed and genes can be actively transcribed in loop
regions.
5. Coiled coil – condensation of loops and coils catalyzed by condensing. Helps protect DNA from
breakage.
#53: Sketch a replication for of bacterial DNA in which one strand is being replicated discontinuously and
the other is being replicated continuously. List 6 different enzyme activities associated with the replication
process and identify the function of each, and indicate on your sketch where each would be located on the
replication fork. In addition, identify the following DNA template, RNA primer, Okazaki fragments, and
single-stranded binding protein.
Gyrase
(releaves
superhelica
l tension)
Bio 351 – Tuesday’s Lab Section – Extra Credit –Page 3 of 4
1.
2.
3.
4.
5.
DNA polymerase: attaches complementary nucleotides forming daughter strands. In prokaryotes,
DNA primase and helicase are linked together on the lagging strand forming the primosome.
Similarly the clamp and polymerase are also linked—forming a replication machine. The lagging
strand is folded to facilitate transfer of machine parts as needed. DNA polymerase stays on the
DNA via clamp protein, with the clamp loader providing the necessary ATP hydrolysis. On the
lagging strand, the DNA polymerase pops off at the 5’ start of a new RNA primer, then hops back
on where the next clamp is assembled at the 3’ end.
DNA primase: synthesizes short RNA primers on the lagging strand. DNA polymerase can begin
creation of the Okazaki fragment from the 3’-OH terminus of the RNA primer, and then pops off
the strand when it runs into the 5’ start of the next RNA primer. In prokaryotes, DNA primase
forms the primosome with DNA helicase on lagging strand.
DNA helicase: unwinds the two strands of DNA for replication, with ATP hydrolysis propelling it
along.
DNA ligase: the DNA repair system replaces the RNA primer with DNA, and DNA ligase joins
the 3’ and 5’ ends of the new DNA fragments together.
DNA gyrase: reduces helical tension in prokaryote replication. (In eukaryotes, DNA
topoisomerases break the DNA phosphodiester bond during replication, allowing it to swivel and
avoid binding of the helices.
a. topoisomerase I = breaks one strand for swivel
b. topoisomerase II = double breaks one strand allowing other strand to “pass through”)
Bio 351 – Tuesday’s Lab Section – Extra Credit –Page 4 of 4