Study guidelines for Test 4 (Fall, 2017)

Chapter 11
1. How does genetic information flow? Transcription and translation.
a. Central dogma of biology: DNA à RNA à Protein
incytosold b.
innodevs Transcription: synthesis of RNA from a DNA template
c. Translation: synthesis of a protein from a mRNA molecule

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2. How many different kinds of RNAs a cell has (remember there are other RNAs besides the three groups
we covered in the class)? What is their function?

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g¥efF a. Messenger RNAs (mRNAs) à RNAs that include nucleotide code information for polypeptide synthesis
8

b. Ribosomal RNAs (rRNAs) à RNA components of ribosomes

c. Transfer RNAs (tRNAs) à RNAs that translate the information in the mRNA nucleotide code into AA
~

Shfifftypes
order of a polypeptide
of tRNA

d. Small Nuclear RNAs (snRNAs) → in nucleus

e. Small Nucleolar RNAs (snoRNAs) nucleolus → in

f. Small Interfering RNAs (siRNAs) nucleusnfgenl →

in
involved
,

expression
g. microRNAs (miRNAs) cytoplasm →

h. Many RNAs fold into complex 3D shape à markedly diff from one type of RNA to another à folding
driven by formation of regions having complementary pairs à RNAs often contain nonstandard base
pairs & modified nitrogenous bases à unorthodox regions serve as recognition sites for proteins & other
RNAs, promote RNA folding, and help stabilize the structure of molecule à can also serve as ribozymes
and self-modify
3. Components for transcription. Transcription direction and why the polymerization is irreversible? Which
strand of DNA is used for transcription template?
a. RNA polymerases in transcription: (are processive (very quick), ~20-50 nt/second à transcription rate
highly regulated) #
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i. DNA-dependent RNA polymerases (RNA polymerases) à enzymes responsible for transcription
in both prokaryotic and eukaryotic cells
ii. Template à DNA strand whose sequence is complementary to the RNA molecule synthesized

=-
from Promoter à site on RNA to which an RNA polymerase molecule binds prior to initiating
required transcription
for RNA L

polymerase
iii. Transcription factors à proteins that are required to help RNA polymerases to recognize
r

manydifytpesof promoters (on genes) aka complementary

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transcript

b. Transcription Direction à 5’ to 3’ (same with DNA synthesis) à so template must be 3’ to 5’ (antisense)

i. Pyrophosphate à catalyzes NTP hydrolysis to release lots of energy and a couple reaction (PPi
- phosphate ↳ exetgonic nucleotide
à 2 Pi)àexergonic but high energy ↳ phosphates NThave3 ,

c. Can have several RNA polymerase molecules on a phage DNA template to speed up the transcription
rate
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4. Transcription in prokaryotes.
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required
HOIOCNZYME
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A single type of RNA polymerase composed of 5 subunits that are tightly associated to form a core enzyme. trusedtoform
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What components an E.coli promoter has?

a
called
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+1 nucleotide: at which transcription is initiated. -1 nucleotide: preceding the +1 nucleotide. Upstream: those
iinaesefiarminate
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specific is
reached portions of DNA preceding the initiation site. Downstream: those portions of DNA succeeding the initiation site.
Consensus sequence: present in the promoters of many genes. -35 element: the TTGACA consensus sequence
around -35 site. Pribnow box (-10 element): the TATAAT consensus sequence around -10 site.
5. RNA polymerases and their functions? ↳ polymerase where RNA
will bind

RNA polymerase I, II, III. Plants have two additional RNA polymerases that are not essential. The transcription in
eukaryotes requires a large variety of accessory proteins (transcription factors). RNAs (mRNAs, rRNAs, and
tRNAs) are transcribed as a considerably longer precusor RNA (primary transcript or pre-RNA). The
corresponding segment of DNA is a transcription unit. The primary transcript is processed into smaller, functional 't3' ends
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ii. the large subunit à joins amino acids to form a polypeptide chain.
ribosomal
b. Each subunit is composed of one or more ribosomal RNA (rRNA) molecules and a variety of ribosomal
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proteins (r-protein or rProtein).

7. What is nucleolus? → ribosomal
assembly
occurs here

The DNA sequences encoding rRNA are called rDNA, which are normally repeated hundreds of times and are
clustered in one or a few regions of genome. .

What genes are inside the nucleoli?

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 The clusters of rDNA are gathered together as part of one or more irregular shaped nuclear structures
(nucleolus/nucleoli).
The components of nucleoli.
The nucleoli function in producing ribosomes. The bulk of a nucleolus is composed of ribosomal subunits that give
the nucleolus a granular appearance. fc contains rDNA. dfc contains the nascent pre-rRNA transcripts and
associated proteins. gc contains ribosomal subunits in various stages of assembly
8. How are rDNA genes arranged in the nucleoli? What is included in an rRNA transcription unit?
a. The tandem arrangement of the repeated rDNA genes.
b. Multiple (about 100) rRNAs are transcribed at the same time.
c. Besides RNA polymerase at the base of each fibril, the RNA fibrils contains clumps and associated
particles (RNA and protein) that work together to convert the pre-rRNAs to their final rRNAs and
assemble them into ribosomal subunits.
d. Nontranscribed spacer. promoter gene
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Three rRNAs (the 28S, 18S, and 5.8S) are synthesized from a single primary transcript. The 5S rRNA is
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re synthesized from a separated pre-RNA outside the nucleolus.
10. Transcription and processing of 5S rRNA.
a. In eukaryotes, the 5S rRNA molecules are encoded by a large number of identical genes: separated from
other rRNA genes; located outside the nucleolus; organized in tandem array; alternating with
nontranscribed spacers; the promoter sequence lies within the coding section of the gene.
b. The 5S RNA genes are transcribed by RNA polymerase III. RNA polymerase III can bind to a promoter
site located within the transcribed portion of the target gene.
c. The 5’end of the primary transcript is identical with that of the mature 5S RNA, but the 3’end usually
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11. Transcription and processing of tRNAs.
a. There are about 50 different species of tRNA, each encoded by a repeated DNA sequence: tRNA genes
are found in small clusters scattered around the genome; a single cluster contains multiple copies of
different tRNA genes; the DNA sequence encoding a given tRNA is found in more than one cluster; in a
cluster, tRNA genes are separated by nontranscribed spacers; the promoter sequence lies within the
coding section of the gene.
b. tRNAs are transcribed by RNA polymerase III. ( SSRRNA ) also

c. The primary transcript of a tRNA is larger than the final product, and pieces on both the 5’and 3’sides of
the pre-tRNA (and a small interior piece in some cases) are trimmed away.
d. All mature tRNAs have the triplet sequence CCA at their 3’end, which are encoded in the tRNA gene in
prokaryotes and added enzymatically after the tRNA is processed in eukaryotes.
12. Properties of heterogeneous nuclear RNAs (hnRNAs).
a. Large molecular weights (up to 80S, 50,000 nt)
b. RNAs of diverse (heterogeneous) nucleotidesequence
c. Only in the nucleus.
13. The dynamic nature of rRNAs, tRNAs, hnRNAs, and mRNAs.
a. Most of the RNA in the cell is present as 18S and 28S rRNA (and small RNAs)
b. Neither hnRNA nor mRNA constitute a significant fraction of the RNA in the cell
c. mRNA and its precursor hnRNA constitute a large percentage of the RNA that is being synthesized by the
cell at any given time
d. hnRNAs and mRNA are degraded after brief periods of time. The half-lives of mRNA range from ~15 min
to a period of days
e. tRNAs and rRNAs have half-lives ranging from days to weeks.
14. Synthesis and processing of mRNA.
a. `All eukaryotic mRNA precursors are synthesized by RNA polymerase II, a complex composed of 12
different conserved subunits.
b. Besides RNA polymerase II, a number of general transcription factors (GTFs) required to initiate
transcription.
c. Promoters for RNA polymerase II lies to the 5’end of each transcription unit.
d. A TATA box (very similar to 5’-TATAAA-3’) lies between 24 and 32 bases from the transcription start site.
The TATA box is the of assembly of a preinitiation complex that contains the GTFs and RNA polymerase
II.
15. Specific transcription factors and their functions.
a. A variety of specific transcription factors are required to bind at numerous sites in the regulatory regions
of the promoter DNA to determine:
i. whether or not a preinitiation complex assembles at a promoter
 ii. the rate at which the polymerase initiates new rounds of transcription from that promoter.
16. What is a split gene? Introns and exons?
a. Split genes à genes with intervening sequences.
b. Exon à those sequences of a split that are present in the mature RNA.
c. Introns à the intervening sequences à found in all types of eukaryotic genes, including tRNA, rRNA, and
mRNA genes
17. Structure of mRNA, 5’ caps, 3’ poly(A), and their functions.
a. mRNA:
i. Contain a continuous sequence of nucleotides encoding a specific polypeptide.
ii. Found in the cytoplasm.
iii. Attached to ribosomes when they are translated.
iv. Most mRNAs contain 5’and 3’untranslated regions (UTRs), which play important regulatory
functions.
v. Eukaryotic mRNAs have special modifications at their 5’and 3’termini. The 3’end has a string of
50 to 250 adenosine residues that form a poly(A)tail, whereas the 5’end has a methylated
guanosine cap.
b. 5’caps (methylguanosine caps) and function
i. The last of the three phosphates is removed, converting the 5’terminus to a diphosphate.
ii. A GMP is added in an inverted orientation so that the 5’end of the guanosine is facing the 5’end
of the RNA chain. The first two nucleosidesare joined by an unusual 5’-5’triphosphate bridge.
iii. The terminal, inverted guanosine is methylated at the 7 position on its guanine base, while the
nucleotide on the internal side of the triphosphate bridge is methylated at the 2’position of the
ribose.
c. Functions of the 5’methylguanosine caps
i. Prevent the 5’end of the mRNA from being digested by exonucleases.
ii. Aid in transport of the mRNA out of the nucleus.
iii. Play an important role in the initiation of mRNA translation.
d. The poly(A) tail and function
i. The poly(A) tail begins about 20 ntdownstream from the sequence AAUAAA in the primary
transcipt, which serves as a recognition site for a complex that carry out the processing reactions
at the 3’end.
ii. An endonuclease in the processing complex cleaves the pre-mRNA downstream from the
recognition site.
iii. An poly(A) polymerase adds 250 or so adenosines without the need of a template. The poly(A)
tail together with an associated protein protects the mRNA from premature degradation by
exonucleases.
18. Splicing of pre-mRNA: conserved sequences for RNA splicing.
a. The 5’splice site: G/GU
b. The 3’ splice site: AG/G
c. The polypyrimidine tract
d. The preferred nucleotides in the adjacent regions of the intron.
e. Exonic splicing enhancers (ESEs): specific sequences situated in the exons.
19. What are the properties of genetic code?
a. Codons specify the same AA are clustered within a particular portion of the chart.
b. .Codon assignment are such that similar AAs tend to be specified by similar codons.
c. The greatest similarities between AA-related codons occur in the first two nucleotides of the triplet,
whereas the greatest variability occurs in the third nucleotide.
d. The genetic code is degenerate.
20. Generality of tRNAs: modification, 3’ end, two-dimensional and tertiary structure.
a. Roughly the same length: between 73 and 93 nucleotides;
b. A significant percentage of the bases are enzymatically modified posttranscriptionally
c. All tRNAs have sequences of nucleotides in one part of the molecule that are complementary to
sequences located in other parts of the molecule. Because of the base-pairing between the
complementary sequences, tRNAs have a two-dimensional structure as a cloverleaf.
d. The 3’end of the tRNA molecule is always a adenosine (A).
e. Tertiary structure: two double helices arranged in the shape of an L. One end of the L-shaped tRNA
molecule is the anticodon loop, the other end is the place where the AA is attached.
21. Anticodon: direction, wobble hypothesis.
a. Anticodon à a stretch of three sequential nucleotides in the tRNA that participates in the specific
interaction with the codon of the mRNA. The anticodon is located in the middle loop of the tRNA
molecule. This loop is invariably composed of 7 nucleotides, the middle 3 of which constitute the
anticodon.
 b. Wobble Hypothesis: The steric requirement between the anticodon of the tRNA and the codon of the
mRNA is very strict for the first two positions but is more flexible at the third position. I can't M
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23. Components/factors required for protein synthesis. Three distinct activities (stages).
The most complicated synthetic activity in a cell, involving: various “charged” tRNAs ribosomes a mRNA
numerous proteins with different functions cations GTP The synthesis of polypeptide chain includes three distinct
activities: Initiation, elongation, termination
24. Structure of ribosome: A (aminoacyl), P (peptidyl), and E (exit) sites.
a. Step 1: Aminoacyl-tRNA selection à The initiator tRNA was placed in the P site. An aminoacyl-tRNA-
initiation Tu-GTPcomplex enter the A site. Once proper aminoacyl-tRNA-Tu-GTP is bound to the mRNA codon, the
GTP is hydrolyzed and the Tu-GDP is released, leaving the aa-tRNA bound in the A Site.
b. Step 2: Peptide bond formation à The amino group of the aa-tRNA in the A site reacts with the
elongation
carbonyl group of the AA bound to the tRNA of the P site, displacing the P-site tRNA. The reaction is
catalyzed by a ribozyme peptidyl transferase.
c. Step 3: Releasing the deacylated tRNA à The deacylated tRNA leaves the ribosome, emptying the E
terminationsite.

Where and how peptide is formed at these three sites?

When a ribosome reaches one of the stop codons (UAA, UAG, or UGA), the signal is read to stop further
elongation and release the polypeptide.
25. Polyribosome (polysome).
As soon as each ribosome moves a sufficient distance along the mRNA from the initiation codon, another
ribosome attaches to the mRNA and begins its translation activity. A complex in which a number of ribosomes are
attached to one mRNA molecule is a polyribosome. The simultaneous translation of the same mRNA by
numerous ribosomes greatly increases the rate of protein synthesis within the cell.
26. Understand the relationship between transcription and translation in prokaryotes and eukaryotes (see
Figure 11.51).

E. coli and Cells of silk gland

Chapter 12
1. The structure of nucleus: the nuclear envelope, nuclear pore complex and other contents inside the
nucleus: nuclear lamina, nucleoplasm, nucleolus, chromatin, and nuclear matrix.
a. Nuclear envelope à a boundary between the nucleus and cytoplasm.
b. Chromosomes à highly extended nucleoprotein fibers (chromatin).
c. One or more nucleoli à irregular shaped electron-dense structures that function in the synthesis of
rRNA and the assembly of ribosomes.
d. Nucleoplasm à fluid substance in which the solutes of nucleus are dissolved.
e. Nuclear matrix à a protein-containing fibrillar network. The nuclear lamina is a thin filamentous
meshwork, which provides mechanical support to the nuclear envelope and serves as a site of
attachment for chromatin fibers at the nuclear periphery. The nuclear pores are the gateways across
the nuclear envelope barrier, which allow passage of macromolecules between the nucleus and
cytoplasm.
2. Nucleocytoplasmic exchange: nuclear localization signal (NLS) and nuclear export signal (NES)
 a. NLSs consist of one or two short stretches of positively charged Aas, such as the T-antigen encoded
by the virus SV40 contains an NLS identified as –Pro-Lys-Lys-Lys-Arg-Lys-Val-.

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of the nucleus where it docks with the cytoplasmic filaments that extend from the outer ring of
the NPC à The receptor-cargo complex moves through the nuclear pore
b. Proteins exported from the nucleolus contain AA sequences (nuclear exporting signals, or NESs) that
are recognized by transport receptors that carry them through the nuclear envelope to the cytoplasm.
i. A pre-mRNA containing 3 exons and 2 introns à The pre-mRNA is spliced to form a mature
mRNA that contains a EJC. One of the subunits of the EJC is the protein Aly à Another
protein TAP is added to the mRNP molecule à After passage through the NPC, Aly and TAP
are shed from the mRNP and the mRNA is translated à The mRNP is transported through
the NPC à The EJCs are displaced from the mRNA during the first round of translation
3. How is genomic DNA packaged into chromosomes? The ratio of each packing level.
Nucleosome filament (10 nm) Packing ratio: ~7:1. 30 nm fiber Packing ratio: 6:1 (~40:1 in total). 80-100 nm fiber.
Chromosome Packing ratio: >10,000:1 in total
a. Nucleosome: core histones and their combinations inside a nucleosome à Nucelosome (core
ret nucleosome, nucleosome core particle): 146 bp of supercoiled DNA wrapped almost twice around a
referee
histone octamer containing all four core histones (H2A, H2B, H3, and H4)
b. Linker histone à H1 histone (linker histone) binds to part of the linker DNA that connect one
nucleosome core particle to the next. H1 histone and the histone octamer interact with about 168 bp
of DNA.
c. Features of histones à Histones are a group of proteins that possess an unusual high content of
the basic AAs arginine and lysine. Histones are divided into 5 classes which can be distinguished by
their Arg/Lys ratio. The AA sequences of histones, especially H3 and H4, are very conserved.
However, there are several alternate versions of the core histones in most cells, which may have
specialized functions. 168 for
the
core
+

nonspecific
→ core ,

d. DNA fragment length: what is 200bp? What is 146bp? What is 168bp? 146
Hli the
for
200
for

i. Nonspecific nucleases resulted in fragments of 200 bp in length.

e. How are DNA and histones held together? à DNA and core histones are held together by
noncovalent bonds, including ionic bonds between negatively charged phosphates of the DNA
backbone and positively charged residues of the histones.
4. Chromatin, euchromatin, and heterochromatin à Chromatin: the substance of chromosome, including DNA,
chromosomal proteins, and chromosomal RNA.Euchromatin: chromatin that returns to dispersed state after
mitosis. Euchromatin is relatively extended and open and at least potentially active. Heterochromatin: chromatin
that remains compacted during interphase, ~10% of the total chromatin. Heterochromatin is very condensed and
its DNA is inaccessible.
5. Histone modification and histone code.
a. The AA residues in the core histone tails are subject to modification, mainly methylation, acetylation,
or phosphorylation
b. Histone code is the pattern (combinations) which contains encoded information governing the
properties of the chromatin. 1. The degree of compaction; 2. If a gene or cluster of genes will be
transcribed or not.
6. What is karyotype? à Chromosomes are cut out of a photograph of metaphase chromosomes, matched up into
homologous pairs, and ordered according to decreasing size, forming a karyotype.
7. Telomere structure and function.
a. The tips of each chromosome DNA molecule consist of an unusual stretch of repeated sequences,
which forms a cap at the end of the chromosome à The stretch of repeated sequences is a telomere.
b. The human telomeres contain the sequence (TTAGGG) which is the same throughout the
vertebrates, and similar sequences are found in most other organisms.
8. What is “the end-replication problem” and how is it solved?
a. DNA polymerases do not initiate the synthesis of a strand of DNA, but only add nt to the 3’end of an
existing strand à If cells could not replicate the ends of the DNA, the chromosomes would become
shorter and shorter with each round of cell division.
9. Telomerase structure and function.
a. Structure: Telomerase is a reverse transcriptase that uses a RNA molecule inside the enzyme as a
template to add new repeat unit to the 3’end of the overhanging strand.
b. Functions:
i. complete the replication of chromosomes
ii. form caps that protect the chromosomes from nucleases and other destabilizing factors
iii. preventing the ends of chromosomes from fusing with one another.
 10. Centromere à Centromere is a site where the outer surfaces are markedly indented. 1. Centromere contains
tandemly repeated sequences, which vary from one species to another species; 2. Centromeric DNA associates
with specific proteins, such as protein that serve as attached sites (kinetochores) for the microtubules that
separate chromosomes during cell division.
11. Why is nucleus an organized organelle?
a. Chromosomes are concentrated into a distinct territory that does not overlap extensively with the
territories of other chromosomes.
b. The portions of chromosomes that are not attached to the nuclear envelope are capable of moving
randomly within a restricted zone of the nucleoplasm.
c. The RNA processing machinery is not spread uniformly throughout the nucleus, instead, it is
concentrated within irregular “speckles” domains.

Chapter 13
1. DNA replication is semiconservative. What is semiconservative replication and how to demonstrate it?
a. Semiconservative replication would produce two copies that each contained one of the original strands
and one new strand.
b. Conservative replication would leave the two original template DNA strands together in a double helix and
would produce a copy composed of two new strands containing all of the new DNA base pairs.
2. Replication origin, bidirectional replication, and replication fork.
a. Replication origin à a specific site (oriC in E. coli) where a number of proteins bind to initiate the
process of replication
b. Bidirectional replication à once initiated, replication proceeds outward from the origin in both
directions.
c. Replication fork à the sites where the pair of replicated segments come together and join the
nonreplicated segments.
3. What is unwinding problem? How is it solved? DNA gyrase.
a. The unwinding problem à Unwinding strands cause torsional stress: unseparated portion becomes
more tightly wound. DNA ahead of the replication machinery becomes overwound and accumulates
positive supercoils.
b. How is it solved? DNA gyrase à DNA gyrase relieves the mechanical strain that builds up during
replication: 1. cleaving both strands of the DAN duplex; 2. passing a segment of DNA through the double-
stranded break to the other side; 3. sealing the cuts. The process is driven by the energy released by
ATP hydrolysis.
4. DNA replication direction: 5’ to 3’. DNA replication requires a template and primer (provides 3’ OH).
a. A template DNA strand to copy
b. A primer to which nucleotides can be added (Primer is a DNA strand provides the necessary 3’OH
terminus)
c. DNA polymerase can only synthesize DNA in a 5’-to-3’(5’-->3’) direction. Both newly synthesized strands
are assembled in a 5’--->3’direction. One grows toward the replication fork, while the other grows away
from the replication fork.
5. Leading strand and lagging strand. How are they replicated? What is Okazaki fragment?
a. Leading strand à the strand that is synthesized continuously.
b. Lagging strand à the strand that is synthesized discontinuously.
c. What is Okazaki fragment? Okazaki fragments: the small segments (1000-2000 nt in bacteria, ~150 nt
in eukaryotes) synthesized during DNA replication.
6. How is DNA synthesis initiated? Primase: a RNA polymerase.
The initiation of Okazaki fragments is accomplished by a distinct type of RNA polymerase (primase) that
constructs a short RNA primer. A short RNA primer is also synthesized at the 5’end of the leading strand by
primase. The RNA primers are subsequently removed, and the resulting gaps are filled with DNA and then sealed
by DNA ligase.
7. Helicase, single-stranded DNA-binding proteins, and DNA polymerases.
Helicase (DNA unwinding enzyme): uses energy from ATP hydrolysis to unwind a DNA duplex and exposes the
single-stranded DNA templates.Helicase (such as DnaB) has six subunits forming a ring-shaped complex that
encircles the lagging-strand template and moves in a 5’--->3’direction. Single-stranded DNA-binding (SSB)
proteins: selectively bind to single-stranded DNA, keeping it in an extended state and preventing it from becoming
rewound. Three DNA polymerase;; DNA polymerase III: polymerizing activity and 3’--->5’ exonuclease activity
part of a large protein complex called DNA polymerase III holoenzyme (replisome), synthesizing DNA strands
during DNA replication. DNA polymerase II: polymerizing activity and 3’---->5’exonuclease activity Unknown
functions DNA polymerase I: polymerizing activity, 5’--->3’exonuclease activity, and 3’--->5’exonuclease activity a
single polypeptide, having three activities in different domains of the polypeptide, functioning in DNA repair and
removing the RNA primers of Okazaki fragments and replacing them with DNA.
 8. Compare prokaryotic DNA polymerases with eukaryotic DNA polymerases. What is the function of each
DNA polymerase?
a. DNA repair (exonuclease) à removes RNA primers and replaces them with DNA.
b. Polymerase alpha is tightly associated with primase, and together they initiate the synthesis of each
Okazaki fragment.
c. Polymerase beta functions in DNA repair.
d. Polymerase Y replicates mitochondrial DNA.
e. Polymerase 8 is the primary DNA-synthesizing enzyme during replication.
f. Polymerase E is unclear, maybe involved in nuclear DNA replication.
g. Other DNA polymerases: n, k, and l have a specialized function that allows cells to replicate damaged
DNA.
9. Difference between prokaryotic and eukaryotic DNA replication.
Eukaryotic cells replicate their genome in small portions called replicons. Each replicon has its own origin from
which replication forks proceed outward in both directions. The timing of replication of a chromosomal region is
roughly correlated with the activity of the genes in the region and/or its state of compaction. Autonomous
replicating sequences (ARSs): sequences promote replication of the DNA in which they are contained. Origin
recognition complex (ORC): a multisubunit complex specifically binds to the core element of an ARS.
10. Mechanism restricting replication once per cell cycle.
a. The origin of replication is bound by an ORC complex
b. “Licensing factors” bind to the ORC to assemble a prereplication complex.
c. Activation of key protein kinases leads to the initiation of replication. The activity of Cdk, a cyclin-
dependent kinase, remains high from S phase through mitosis, which suppresses the formation of new
prereplication complex.
d. The “licensing factors” move with the replication fork to help complete replication of a replicon.
11. What are replication foci? à In eukaryotes, replication forks that are active at a given time are not distributed
randomly throughout the cell nucleus, but instead are localized within 50 to 250 sites, called replication foci.
12. Replication of chromatin structure (histones).
The assembly of DNA onto nucleosomes is a very rapid event. The nucleosomes on both daughter duplexes are
very near the replication fork. The (H3H4)2 tetramers present prior to replication remain intact and are distributed
randomly between the two daughter duplexes. The two H2A/H2B dimers of a nucleosome separate from one
another and bind randomly to the new and old (H3H4)2 tetramers present on the daughter duplexes.

Chapter 14
1. What is cell cycle? à the cycle of cell growth, replication of the genetic material and nuclear and cytoplasmic
division.
2. How many stages are in a cell cycle? How are these stages defined?
a. Interphase à the period between cell divisions, which is a time when the cell grows and engages in
diverse metabolic activities
b. G1 à the period following mitosis and preceding DNA synthesis. Cell grows and carries out normal
metabolism; organelles duplicate.
c. S phase à the period in which DNA is replicated and additional histones are also synthesized. DNA
replication and chromosome duplication.
d. G2 à the period between the end of DNA synthesis and beginning of M phase. Cell grows and prepares
for mitosis.
e. M phase à mitosis
3. How did cell fusion experiments demonstrate the presence of key factors that regulate the transition from
G1 to S and G2 to M? What is premature chromosomal compaction?
a. Cell fusion experiments à The cytoplasm of a replicating cell contains diffusible factors that stimulate
initiation of DNA synthesis in G1-phase nuclei; When G 2-phase and S-phase cells were fused, the G2-
phase nuclei did not initiate another round of DNA synthesis. 2. the G2-phase nuclei can no longer
respond to initiation factors present in the S-phase cell cytoplasm. suggesting that the transition from G1
to S was induced by cytoplasmic factors.
b. what is premature chromosomal compaction à When G1-phase and M-phase cells were fused, the
G1-phase chromatin underwent premature chromosomal compaction (PCC) to form a set of elongated
compacted chromosomes. The cytoplasm of an M-phase cell contains diffusible factors that stimulate
premature chromosomal compaction of G1-phase, G2-phase, and S-phase chromosomes, suggesting
that the transition from G2 to M was induced by the cytoplasmic factors.
4. Definition of mitosis and cytokinesis.
a. Mitosis à a process of nuclear division in which the replicated molecules of each chromosome are
faithfully partitioned into two nuclei.
b. Cytokinesis à a process by which a dividing cell splits in two, partitioning the cytoplasm into two cellular
packages.
5. What are the five stages of mitosis? How are they defined? Remember events in each stage.
a. The stages à Prophase, prometaphase, metaphase, anaphase, telophase
b. How are they defined? Remember events in each stage. à
i. Prophase à
1. Formation of the mitotic chromosome. Chromosome compaction
(condensation).Chromosome scaffold. Condensin. Cohensin.
2. Formation of the mitotic spindle.
3. The dissolution of the nuclear envelope.
ii. Prometaphase àThe dissolution of the nuclear envelope marks the start of prometaphase.
1. The microtubules of the spindle penetrate into the central region of the cell
2. The free ends of the microtubules grow and shrink in a highly dynamic fashion, searching
for chromosomes
3. The two sister chromatids become connected by their kinetochores to microtubules that
extend from opposite poles
4. The chromosomes are moved by a process called congression toward the center of the
mitotic spindle
iii. Metaphase à Alignment of chromosomes to the center of the mitotic spindle marks the start of
metaphase.
iv. Anaphase à the sister chromatins of each chromosome split apart and start their movement
toward opposite poles
v. Telophase à As the chromosomes reach their respective poles, they tend to collect in a mass,
which marks the beginning of telophase. Chromosomes become dispersed; Nuclear envelope
reforms; Golgi complex and ER reform.
6. Chromosome scaffold, codensin and cohesion. Kinetochore, its structure and function.
a. Chromosome scaffold à Scaffold proteins: a structural framework of nonhistone proteins that maintain
the basic shape of the intact chromosome. During interphase, the scaffold proteins are dispersed within
the nucleus, forming part of the nuclear matrix.
b. Condensin à a multisubunit protein complex that binds to DNA and helps DNA to form positively
supercoiled loops.
c. Cohesion à a multisubunit protein complex that associates with DNA along its length and holds the two
sister chromatids together in G2. Most of the cohesin dissociates from the chromosome arms as they
become compacted during prophase. The cohesin at the centrosomes is released at anaphase
d. Kinetochore (structure & function) à Kinetochore is a proteinaceous, button-like structure at the outer
surface of the centromere of each chromatid.
i. The site of attachment of the chromosome to the dynamic microtubules of the mitotic spindle
↳ pull chromatin
ii. The residence of several motor proteins involved in chromosome motility apart
sisters

iii. A key component in the signaling pathway of an important checkpoint.

7. How is the mitotic spindle formed? What is chromosome congression?
a. Mitotic spindle formation à
i. Replication of centrosome: initiated at the G1-S transition.
1. the two centrioles move apart within the centrosome
2. A daughter centriole is assembled next to each maternal centriole
3. The centrosome splits into two adjacent centrosomes.
ii. Formation of astral microtubules around each centrosome during early prophase.
iii. The two centrosomes move toward opposite ends of the cell through elongation of polar
microtubules, establishing the two poles of a bipolar mitotic spindle.
b. Chromosome congression à Lack of a kinesin-like protein (Kid) along the chromosomes results in an
alignment failure at the center of the spindle.
8. Metaphase plate, astral microtubules, chromosomal microtubules, and polar microtubules.
a. Metaphase plate à imaginary plane perpendicular to the spindle fibers of a dividing cell, along which
chromosomes align during metaphase
b. Astral microtubules à subpopulation of microtubules which only exist during & immediately before
mitosis à defined as any microtubule originating from the centrosome which does not connect to a
kinetochore
c. Chromosomal (kinetochore) microtubules à any of the spindle microtubules that attach to the
kinetochores of chromosomes by their plus ends, and maneuver the chromosomes during mitotic or
meiotic chromosome segregation
d. Polar (interpolar) microtubules à interdigitate (definition: the locking of two/more things) at the spindle
midzone and push the spindle poles apart via motor proteins
9. What is treadmill? When does it happen? à“Treadmill”of the microtubules of the metaphase spindle. A net
addition of subunits at the plus end ands a net loss of subunits at the minus ends cause a pole-ward flux of tubulin
subunits in a mitotic spindle.
10. Anaphase A and B.
a. Anaphase A à the movement of the chromosomes towards the poles à the net loss of subunits at the
minus ends and losses of subunits at the plus ends of the chromosomal microtubules result in shortening
of the chromosomal fibers
b. Anaphase B à the two spindle poles move further apart à net addition of subunits to the plus ends of
the polar microtubules elongates the mitotic spindle.
11. Which molecular motors are involved in mitotic movement and how?
All of the motors involved in mitotic movements are microtubule motors, including a number of different kinesin-
related proteins and cytoplasmic dynein. Motor proteins located along the polar microtubules: keeping the poles
apart and elongating the spindle during anaphase B. Motor proteins residing on the chromosomal microtubules:
moving the chromosomes during prometaphase, maintaining the chromosomes at the metaphase plate, and
separating chromosomes during anaphase.
12. Spindle checkpoint à operates at the transition between metaphase and anaphase. If a chromosome fails to
become aligned properly at the metaphase, the checkpoint mechanism delays the onset of anaphase.
13. What is the difference between animal cell cytokinesis and plant cell cytokinesis?
a. Cytokinesis definition à a process dividing a cell into two daughter cells.
b. In plant cells à plant cells start cytokinesis by forming a partition called the cell plate in the center of the
cell. The position of the cell plate is determined by the preprophase band formed in late G2.
c. In animal cells à during anaphase, indentation of cell surface in narrow band formed around cell à
deepens to form furrow that completely encircles the cell (plane of furrow = same plane as metaphase
plate) à additional PM delivered to cell surface via cytoplasmic veisciles that fuse with the advancing
cleavage furrow à continues to deepen * opposite surfaces fuse in the center of the cell, splitting the cell
in two
14. Plant cell plate and its formation.
a. The vesicles fuse with each other to form an interwoven tubular network.
b. New vesicles continue the process of tubule formation and fusion, which extends the network on an
outward direction.
c. The leading edge of the growing network contacts the parent PM at the boundary of the cell.
d. The tubular network matures into a continuous, flattened partition. The membranes of the network
becomes the PMs of the two daughter cells, whereas the secretory products form the cell plate.
15. Know the difference between mitosis and meiosis.
Mitosis Meiosis
Chromosome Behavior Homologous chromosomes Homologous chromosomes pair,
independent forming bivalents until anaphase I
Chromosome Number Identical daughter cells; diploid; no reduction; daughter cells haploid
reduction
Genetic Identity of Progeny Identical daughter cells Daughter cells have new
assortment of parental
chromosomes (chromatids not
identical, crossing over)

16. Remember stages in meiosis. Fives stages of prophase I and events in each stage of prophase I.
a. stages of meiosis à
i. meiosis I à interphase, prophase I, metaphase I, anaphase I, telophase I
ii. meiosis II à prophase II, metaphase II, anaphase II, telophase II
b. Prophase I à
i. Stage 1: Leptotene à
1. Chromosomes begin to condense.
2. Near the end of leptotene, telomeres become localized at the inner surface of the nuclear
envelope at one side of the nucleus.
ii. Stage 2: Zygotene à
1. Chromosomes continue to condense and shorten.
2. Homologues associate with one another. This process of chromosome pairing is called
synapsis.
iii. Stage 3: Pachytene à The end of synapsis marks the beginning of pachytene, which is
characterized by a fully formed synaptonemal complex. Within the synaptonemal complex there
are protein complexes, called recombination nodules, that contain the enzymes required to
complete the genetic recombination. Genetic recombination is completed by the end of
pachytene.
iv. Stage 4: Diplotene à Dissolution of the synaptonemal complex leaves the chromosomes
attached to one another at a specific points by X-shaped structures, chiasmata (chiasma).
v. Stage 5: Diakinesis à
 1. The chiasmata move to the ends of the chromosomes in a process called terminalization.
2. Meiotic spindle is assembled.
3. Disappearance of nucleolus and breakdown of nuclear envelope.
4. The tetrads (bivalents) move to the metaphase plate.
17. Synapsis, structure of synaptonemal complex, and bivalent (tetrad).
a. Synapsis à Homologues associate with one another. This process of chromosome pairing is called
synapsis.
b. Structure of synaptonemal complex à a ladder-like structure, connects homologue chromosomes
together. Two lateral elements (mainly cohesin)Transverse protein filaments.
c. Bivalent (tetrad) à is the association of a pair of homologous chromosomes physically held together by
at least one DNA crossover. This physical attachment allows for alignment and segregation of the
homologous chromosomes in the first meiotic division.
18. Recombination and recombination nodule. Chiasmata.
a. Recombination & recombination nodule à Within the synaptonemal complex there are protein
complexes, called recombination nodules, that contain the enzymes required to complete the genetic
recombination.
b. Chiasmata à At mid diakinesis, the circular structures are located in chromatin bridges chiasmata.
19. How does meiosis increase the genetic variability in a population of organisms from one generation to
the next?
a. Independent assortment of maternal and paternal chromosomes.
b. Genetic recombination (crossing-over).
20. Holliday junction and how is it resolved?
a. Definition à is a key intermediate in homologous recombination, a biological process that increases
genetic diversity by shifting genes between two chromosomes, as well as site-specific recombination
events involving integrases
b. Resolution à recombination