Chapter 34A

1. Describe the different histone proteins and their functions.

There are five major classes of these proteins, histones H1,H2A,H2B, H3, and
H4, all of which have a large proportion of positively charged residues. These
proteins therefore ionically bind DNA's negatively charged phosphate groups. The
amino acid sequences of histones H2A, H2B, H3, and H4 have remarkably high
evolutionary
histones.
stability. The fifth histone, histone H1, is more variable than the other
With micrococcal nuclease digestion of nucleosomes, 200 bp DNA is digested to
167 bp, Hl is released next from chromatosomes before DNA is further degraded
to 147 bp. H1 binds to nucleosomal DNA in a cavity formed by a central segment
of DNA and DNA that enter and exit. Where Hl is present, DNA enters and leaves
nucleosome on same side, in contrast if Hl is absent the entry and exit points are
more random. Length of linker DNA is controlled by subspecies linker histones
(H1). H5 is a variant of Hl and binds chromatin more tightly.
2. Describe the levels of DNA packaging in eukaryotic cells from B-DNA to
methaphase chromosomes.
Nucleosomes are the first level of chromatin organization. Chromatin consists of
an equal number of H2A, H2B, H3 &H4, half of which is H1. EM images of
chromatin show beads on a string, presumably 10 nm diameter particles
connected with naked DNA. Abrief digestion of chromatin with micrococcal
nuclease (cleaves dsDNA), cleaves chromatin between particles. In the chemical
cross-linking experiment showed that H3 and H4 associate to form tetramers:
(H3)2(H4)2. Kornberg proposed that chromatin particles, called nucleosomes,
consists of octamer of histones (H2A)2(H2B)2(H3)2(H4)2 associated with 200 bp
of DNA
The second level of chromatin organization are 30 nm fibers, at physiological salt
concentration, chromatin forms 30-nm-diameter fiber and nucleosomes are
visible.
Radial loops: The third level of chromatin organization is the formation of radial
loops. EMs of chromosome cross-sections suggest that chromatin fibers are
radially arranged.
3. Explain the functions of cohesin and condensin.
During the Gi phase of the cell cycle, cohesin loads onto the dispersed chromatin
fibers in an ATP-dependent manner. When the DNA is subsequently replicated
during Sphase, the replisome passes through the cohesin rings, thus keeping the
resulting sister chromatids together. As the cell enters Mphase, condensin
organizes the radial loops of the metaphase chromosomes.

Chapter 30 - Tutorial Questions

1. Summarize the functions of the following proteins in E. coli DNA replication:
DNA polymerase I, DNA polymerase ll, DnaA, helicase, SSB, primase, the
sliding clamp, clamp loader, DNA ligase, Tus and topoisomerases.
DNA polymerase I An E. coli DNA polymerase that plays a role in primer removal
and gap filling between Okazaki fragments and in the núcleotide excision repair
pathway.
DNA polymerase Il The E. coli replicative polymerase that catalyzes genome
replication

1
 DnaA Contains four domains: a helicase interaction domain, a linker, an ATPase
domain (AAA+ family), and a DNA binding domain. In the presence of ATP, DnaA
recruits subunits and generates local positive supercoils.
Helicase An enzyme that unwinds a polynucleotide double helix (either DNA or
RNA).
SSB (single-stranded DNA-binding protein) Binds to single-stranded DNA and
keeps it from base pairing with a complementary strand, used during DNA
replication.
Primase The enzyme that catalyzes the formation of an RNA primer during DNA
replication.
clampaHelical movement of a DNA-binding protein along the DNA due to
tracking along aroove of the DNA over short distances.
Clamp loader must tightly bind sliding clamp before template loading.
DNA ligase An enzyme that joins two double-stranded DNAs end to end.
Tus required to arrest replication fork, inhibits helicase action of DnaB, and
prevents unwinding of DNA from one side of Tus but not the other.
Topoisomerase An enzyme that converts (isomerizes) one topoisomer of DNA to
another.

2. What is the role of metal ions in the polymerase active site?

Metal ion Aactivates primer's 3'-OH for nucleophilic attack on incoming dNTP's a
phosphate
group group. Metal ion Borient &stabilize negatively charged triphosphate
of dNTP.

3. Why are there no Pol I mutants that completely lack 5'»3' exonuclease
activity?
RNA cannot be removed or replaced. Fatal for cell. It's not found because should
this mutation occur, the cell dies.
4. Describe how pol a, pol 5, PCNA, RNase H1, FENI and DNA ligase participate
in eukaryotic DNA synthesis.
Pol a Participates in replication of chromosomal DNA, replicates DNA by extending
primer in 5'+3direction with ssDNAas template, but lacks exonuclease activity.
Forms a heterotetramer with a polymerase subunit, a primase subunit that is
required for primase activity, and regulates initiation.
Pol d Is a heterotrimer that lacks primase, but contains proofreading 3'5
exonuclease activity, also has high processivity.
PCNA Proliferating cell nuclear antigen, or PCNA, forms a ring structure similar to
the sliding clamp in E. coli.
RNaseH RNA degraded by RNaseH activity
FEN1 (flap endonuclease-1) removes RNaseH
DNA ligase An enzyme that joins two double-stranded DNAs end to end.
5. In what ways does replication differ between eukaryotes and prokaryotes?
In prokaryotic cells, there is only one point of origin, replication occurs in two
opposing directions at the same time, and takes place in the cell cytoplasm.
Eukaryotic cells on the other hand, have multiple points of origin, and use
unidirectional replication within the nucleus of the cell. Initiation at a single ori
site, primer synthesis by primase and RNAP, primer removal by POL Itermination
at Ter sites and Tus protein. Euk: Multiple sites, Primer synthesis and initial
elongation by Pol a/primase, primer removal by RNase H and FEN I. Replication
continues till replication forks meet.
6. Describe the structure, function and synthesis of
telomeres.

2
 Alimitation of homologous end-joining, particularly in haploid cells, is that a
homologous chromosomal segment may not be available.
12. Epigenetics is the study of heritable changes in gene activity that are not
caused by changes in the DNA sequence. Explain this statement.
? Not covered in syllabus.
Chapter 31 - Tutorial Questions
1. What is the experimental advantage of using IPTG instead of 1,6-allolactose
as an inducer of the lac operon?
It structurally resembles allolactose, but is not degraded by B-galactosidase.
2. Why does promoter efficiency tend to decrease with the number of G·C base
pairs in the -10 region of a prokaryotic gene?
The need to form an open complex explains why promoter efficiency tends to
decrease with the number of GC base pairs in the -10 region; this presumably
increases the difficulty in opening the double helix as is required for chain
initiation. Recall that GC pairs are more stable than AT base pairs.
3. Describe how transcription is terminated with and without Rho factor in
prokaryotes.
" Without Around half the transcriptional terminators in Ecoli induce termination
without assistance. The sequences of these terminators share two common
features: 1) A tract of 7-10 consecutive AT's with the A's on the template strand.
The transcribed RNA is terminated in or just past this sequence. 2) AGC rich
segment with a palindromic sequence that is immediately upstream of the series
of AT's. The RNA transcript of this region can therefore form a self-complementary
hairpin structure that is terminated by several U residues. The stability of a
terminator's GC rich hairpin and the weak base pairing of its oligo (U) tail to
template DNA are important factors in ensuring proper chain termination. The
formation of the GC-rich hairpin causes RNAP to pause for several seconds at the
termination site.
With Half of termination sites lack similarities, and are therefore unable to fom
strong hairpin structures, these require a protein known as a Rho factor. The Rho
Factor is from the
RecA family of hexameric helicase. It enhances termination efficiency of
spontaneous termination, while also inducing nonspontaneous termination. It
requires specific recognition sequence on newly transcribed RNA upstream of
termination site. The Rho Factor binds to the recognition sequence (rut), a C-rich
segment of about 40 nt, and translocates along ssRNA in a 53 direction. This
reaction is energy dependent until it encounters RNAP. RNAP pauses at the
termination site. The Rho Factor unwinds RNA-DNA hybrid, and pushes the RNAP
forward, which partially rewinds dsDNA helix at the transcription bubble while
unwinding RNA-DNA hybrid. RNA is released.
4. Describe the regulation of the lac operon by lac repressor and CAP.
The lacrepressor binds to a region on the operon known as an operator. In the
absence of the inducer the lacrepressor will bind to the operator, thereby
preventing the transcription of mRNA.
CAP (catabolite gene activator complex.
protein), undergoes a large conformational
This CAP-CAMP Compley binds to the
when binding cAMP, forming a
lacoperon and stimulates transcription in the absence of the repressor. Structure
shows that CAP-CAMP complex binding results in DNA bending around protein

4
 Therefore. CAP-CAMP is a positive regulator, and the lacrepressor is a negative
regulator.
5. What effect would a mutant Lac repressor that cannot bind to allolactose
have on the function of the lac operon (assuming no glucose is present)?
Allolactose is the real inducer that binds the Lac repressor and causes it to
dissociate from the lac operator. A mutant Lac repressor that cannot bind
allolactose would remain bound to the lac operator in both the presence and
absence of allolactose, thereby inhibiting transcription of the lac operon. The lac
operon would not be induced. No expression.
6. Describe the transcription of the trp operon in the absence of active
ribosomes and tryptophan.
There are 5trpbinds
(end product) operon genes
to the under regulation
repressor and forms of the trp repressor.
a complex, L-tryptophan
which binds to the trp
operator and reduces the rate of trp operon transcription by 70 fold. Figure 31-44
and 45 Formation of 1 2 hairpin structure.
7. Why can't eukaryotic transcription be regulated by attenuation?
Transcription and translation are not coupled and occur in different compartments.
8. Predict the effect of deleting the leader peptide sequence on regulation of
the trp operon.
The entire trp operon will be transcribed no matter what the level of tryptophan is
in the celland the ribosome will never prevent the binding of region 2with region
3 and the terminator loop.
9. Explain how riboswitches can turn gene expression on and off.
. Binding of the metabolite usually serves as an "off" switch, decreasing the
expression of the gene products used to make the metabolite. Repression occurs
either by terminating transcription or by preventing translation initiation.
10. Explain the functions of RNAP Il's CTD, clamp, wall, funnel, rudder, bridge
and trigger.
CTD required for mRNA processing in vivo and has been shown to bind to
processing t in vitro. During the transcription cycle, the CTD undergoes
dynamic phosphorylation of serine residues at positions 2 and 5 in the repeat.
cíamp Big part of Rpbl &Rpb2 "clamp" that traps DNA
Wall Part of Rpb2 ("wall") directs template strand out in -90°turn.
Funnel NTP enters active site through funnel
Rudder Aloop extending from clamp ("rudder") that separates RNA and DNA
Bridge connects transcriptional activators bound at enhancers, or other long
range regulatory elements, with RNA pol I|
Trigger conversion of RNAP Ilinitiation complex to elongation complex