ADN Replication

* DNA ligase joins 3' end of one Okazaki fragment to 5' end of downstream Okazaki
fragment (Fig 11.7).
* As helicase unwinds DNA ahead of replication fork, positive supercoils form elsewhere
in the molecule. For replication fork to move, the helix must rotate (estimated at 50
revolutions/sec). The problem of supercoiling is solved by the action of topoisomerases
(specifically a Gyrase) which introduce negative supercoils to counteract positive
supercoils intoduce by helicases.
Rolling circle replication
* For many viral DNAs and some plasmids (e.g. F plasmid in E. coli), rolling circle
replication has been demonstrated.
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* Synthesis usually continues beyond a single chromosomal unit. This results in many
head-to-tail copies of the plasmid, which is then cut and rejoined into new circular
molecules.
Replication of telomeres in eukaryotes
* There are special problems associated with replication of the ends of linear
chromosomes (called telomeres). Recall that DNA polymerases only add nucleotides to
the 3' end of a growing chain. When the linear chromosomes of eukaryotes replicate, the
resulting daughter molecules will each have an RNA primer left over at the 5'end (Fig
11.14). This RNA primer is removed, leaving a single stranded DNA segment. If not fixed,
this single-stranded DNA region will get degraded, and the linear chromosomes will get
shorter with each round of DNA replication.
* In most eukaryotes, an enzyme called telomerase, maintains the ends of chromosomes
by adding telomere repeats to chromosome ends. The mechanism is shown below (and in
Fig 11.5).
* Telomerase is a ribonucleoprotein (has RNA molecule as part of its structure) which
adds tandem repeats to the 3' end of chromosomes using an RNA molecule as a
template. After is has added many telomeric repeats and has left, a new DNA molecule is
made starting from a new RNA primer, which is again is removed, but by this time the
chromosme has already been extended.
* The absence of telomerase activity in cells is correlated with senescence of cells (i.e.
die after certain number of cell divisions). Conversely, enhanced telomerase activity
correlated with cell immortalily (i.e. cells divide indefinately).
o cells with short telomerse undergo fewer doublings than ones with long telomerase.
o fibroblasts form individuals with progeria (rare disease characterized by premature
aging) have short telomeres.
o most somatic cells have no active telomerase (divide only 20-60 times)
o sperm cells, stem cells and unicellular eukaryotes (essentially immortal ) have active
telomerase and stable telomeres.
o cancer cells, which are also essentially immortal, have active telomerase (promising
target for drug design)
o Elimination of telomerase activity in somatic cells may be a cellular senescence
mechanism that protects multicellular organisms from cancer
Chapter 13 : Transcription
* Outline
o Genes and RNA
o Properties of RNA
o Classes of RNA
o Making functional transcripts
+ RNA polymerases
+ Initiation
+ Elongation
+ Termination
o RNA processing in eukaryotes
Genes and RNA
Biological information flow from DNA to protein requires an RNA intermediate. RNA is
produced by a process that copies the nucleotide sequence in DNA to produce a
transcript. This process is called transcription.
Properties of RNA
1. Single stranded, but can undergo intramolecular base-pairing
- forms variety of 3D structures specified by sequence.
2. Ribose sugar (not deoxyribose)
3. Uracyl in place of thymine
Classes of RNA
* There are a variety of different RNAs that can be classified into two classes.
o 1. Informational RNAs (e.g. messenger RNA)
+ intermediate which is later translated into protein.
+ most genes encode mRNA
o 2. Functional RNAs
+ never translated
+ diverse roles in cell
+ main classes of functional RNAs play critical roles in various steps in the information
processing of DNA to protein:
# rRNA - components of ribosome
# tRNA - bring amino acids to mRNA during translation
# snRNA (small nucleolar RNAs) - involved in splicing of introns
# scRNAs (small cytoplasmic RNAs) - protein trafficking
* All DNA and RNA function is based on two key elements:
o 1. Complementary bases in single stranded nucleotide chains can H-bond to form
double stranded structures.
o 2. Specific sequences can be recognized by specific nucleic-acid binding proteins.
Making functional transcripts
* Transcription uses one DNA strand as template
o Strands of double helix must be separated, so that one of these strands (template
strand) can serve as template to direct the synthesis of transcript.
* Either strand along the chromosome can serve as template, but for a given gene, its
always the same strand.
* RNA polymerase catalyzes the synthesis of RNA using DNA template (Fig 13.1).
o RNA grows in 5' to 3' direction, and the template is read in the 3' to 5' direction.
o sequence of RNA is complementary to template strand (noncoding strand), but the
same as nontemplate strand (coding strand) except T replaced with U.
* A typical prokaryotic gene has the folowing features:
RNA Polymerases
* Prokaryotes have only one RNA Polymerase but eukaryotes have 3:
1. RNA Pol I: transcribes rRNA genes
2. RNA Pol II: transcribes protein encoding genes
3. RNA Pol III: transcribes other functional RNAs (tRNAs, snoRNAs etc )
* In eukaryotes, transription takes place in nucleus.
* In prokaryotes, transcription and translation are coupled.
* Transcription involves 3 distinct stages: initiation, elongation, and termination.
Initiation
* In E. coli, transcription requires a complex of RNA polymerase and the sigma factor (s)
which binds to a promoter. The RNA polymerase core enzyme (4 has four subunits, two
a, one b and one b') complexed with the sigma factor is known as the holoenzyme. Once
transcription is initiated, the sigma factor dissociates.
* promoter = DNA sequence to which RNA Pol binds to initiate transcription.
o note that by convention, gene is labelled the same way as RNA transcript. So promoter
is at 5' end of gene (Fig 13.3).
* RNA pol + sigma factor scans DNA for promoter sequence, binds DNA at the promoter
sequence (- 10 region and -35 region), unwinds it, and begins synthesis of a transcript at
transcription initiation site. Promoter sequences are not transcribed. NOTE: RNA pol does
not need a primer to initiate RNA synthesis not does it need a helicase.
o there are consensus sequences for all promoters in E. coli. A consensus sequence is the
sequence found most frequently at each position. E.g consensus sequence at -10 position
is 5'-TATAAT-3'
o The more similar the promoter sequence is to the consensus, the higher the rate of
transcription.
o It is the sigma factor that binds the promoter. Different sigma factors bind different
promoters.
* What is described above is the minimum required for transcription initiation. In chapter
19 we will study how genes are regulated in prokaryotes in more detail.
Elongation
* RNA pol moves along DNA, maintaining transcription "bubble" to expose template
strand, and catalyzes the 3' elongation of transcript.
o energy for reaction derived from splitting high-energy triphosphates into
monophosphates.
o rate of transcription is about 30-50 nt/sec
Termination
* Results from different mechanisms signalled by termination sequences at 3' end of a
gene. Two mechanisms known:
o Rho-independent termination
+ involves formation of hairpin loop (Fig 13.5) in nascent transcript causing RNA strand
and RNA Pol to be released from DNA template.
o Rho-dependent termination
+ Rho is a protein that binds RNA terminator sequence and then uses energy from ATP
hydrolysis to separate transcript from RNA polymerase.
+ Rho-dependent terminators lack hairpin loop.
* The transcript that is made is called mRNA and in prokaryotes it has the following
structure (note that it's always larger than what is needed to encode the polypeptide):
* Prokaryotes have coupled transcription and translation, so that even as mRNA is being
transcribed, ribosomes attach to 5' end and begin translation.
* Many mRNAs in prokaryotes are polycistronic, i.e. they can encode more than one
polypeptide (E.g. lac operon)
RNA processing in Eukaryotes
* Transcription in eukaryotes is more complicated, in that there are more regulatory
sequences involved, and there is a sequential assembly of many different transcription
factors at the promoter before RNA polymerase binds and initiates trancription. We will
study transcription in eukaryotes in more detail in chapter 20.
* In this chapter, we will focus on processing of the initial transcript .
* In eukaryotes, the initial product of transcription (primary transcript) is processed in
several ways before transport to cytosol. In prokaryotes, there is no such processing.
* Processing performed by RNA binding proteins.
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* Processing involves (Fig 13.9 and Fig 13.11):
o 1. addition of 5' cap
+ Guanyltransferase adds 7-methylguanosine using a 5'-to-5' triphosphate linkage
+ protects transcript aginst degradation by exonucleases.
+ also important for binding of ribosome during translation
o 2. addition poly(A+) tail
+ transcript cleaved 20 bases downstream of AAUAAA sequence near 3' end by an
endonuclease, then 50-250 adenine nucleotides added by Poly(A) polymerase.
+ Poly(A+) tail required for efficient transport out of the nucleus into cytoplasm. Once in
cytoplasm, polyA tail also protects against early degradation by exonulceases.
o 3. RNA splicing to remove introns (Fig 13.13)
+ the GU-AG rule
+ spliceosome
Chapter 10: DNA as a genetic material
Search for genetic material
* Scientists reasoned early on that whatever the genetic material turned out to be, it had
to have 3 important characteristics:
1. Store information (about structure, function, development, reproduction)
2. Replicate accurately (so progeny can receive information from parents)
3. Capable of change. Without mutation there is no variation and adaptation. Evolution
does not occur.
Griffith’s transformation experiment[/b]
* In 1928, Frederick Griffith was working with two different strains of Streptococcus
pneumoniae (causes pneumonia).
1. S strain: forms smooth colonies; highly infectious because it forms a capsule which
allows bacteria to evade immune system of host (virulent).
2. R strain: forms rough colonies; harmless because it lacks a capsule, therefore gets
detected by immune system early and effectively (avirulent).
* Griffith injected mice with the different strains and checked for virulence. The
experiment was as follows:
* The experiment showed that bacteria need to be alive and to have a polysaccharide
capsule to be infectious and kill the host. More importantly, it also showed that bacteria
could uptake genetic material from their surroundings. Griffith called this material the
transforming principle, which he believed was protein. The real importance of Griffith’s
experiment is that it provided the experimental basis for further experiments on the
chemical nature of the transforming principle.
Transformation experiments of Avery, MacLeod and McCarty
* In the 1930’s and 40’s these researchers followed up on Griffiths experiments, by
fractionating the heat killed cell extract into proteins, and nucleic acids. Only the nucleic
acid fraction was capable of causing transformation of a rough strain into a virulent S
strain. This indicated that proteins were not the transforming principle, and so could not
be the genetic material.
* Since there are two types of nucleic acids, DNA and RNA, a further fractionation and
transformation experiment was carried out as shown below:
* These results strongly suggested that DNA was the genetic material. However, too
many scientists were still too predisposed to thinking that proteins were the genetic
material. Further proof was necessary.
Hershey-Chase experiment with labeled phage
* In 1953, Alfred Hershey and Martha Chase finally proved to the satisfaction of most
scientists that DNA was the genetic material.
* Hershey and Chase studied bacteriophage T2, which was known to be made entirely of
protein and DNA. T2 phage replicates by invading E. coli, taking over its replication
machinery to make progeny phage. The host cell then lyses and releases progeny phage
capable of infecting new cells.
* Their experiment started with the radioactive-labeling of either DNA (with 32P, not
found in proteins) or proteins (with 35S, not found in DNA) in phage as shown below:

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