MOLECULAR BIOLOGY MIDTERM REPORT

By:Fitra

1. The DNA Transcription Process

Pre-initiationIn eukaryotes, RNA polymerase, and therefore the initiation of transcription, requires the presence of

a core promoter sequence in the DNA. Promoters are regions of DNA that promote transcription and, in eukaryotes, are found at -30, -75, and -90 base pairs upstream from the start site of transcription. Core promoters are sequences within the promoter that are essential for transcription initiation. RNA polymerase is able to bind to core promoters in the presence of various specific transcription factors.

The most common type of core promoter in eukaryotes is a short DNA sequence known as a TATA box, found 25-30 base pairs upstream from the start site of transcription. The TATA box, as a core promoter, is the binding site for a transcription factor known as TATA-binding protein (TBP), which is itself a subunit of another transcription factor, called Transcription Factor II D (TFIID). After TFIID binds to the TATA box via the TBP, five more transcription factors and RNA polymerase combine around the TATA box in a series of stages to form a pre initiation complex. One transcription factor, DNA helicase, has helicase activity and so is involved in the separating of opposing strands of double-stranded DNA to provide access to a single-stranded DNA template.

However, only a low, or basal, rate of transcription is driven by the pre-initiation complex alone. Other proteins known as activators and repressors, along with any associated co-activators or co-repressors, are responsible for modulating transcription rate. Thus, pre-initiation complex contains: 1. Core Promoter Sequence 2. Transcription Factors 3. DNA Helicase 4. RNA Polymerase 5. Activators and Repressors

RNAP = RNA polymerase

In bacteria, transcription begins with the binding of RNA polymerase to the promoter in DNA. RNA polymerase is a core enzyme consisting of five subunits: 2 α subunits, 1 β subunit, 1 β' subunit, and 1 ω subunit. At the start of initiation, the core enzyme is associated with a sigma factor that aids in finding the appropriate -35 and -10 base pairs downstream of promoter sequences.

Transcription initiation is more complex in eukaryotes. Eukaryotic RNA polymerase does not directly recognize the core promoter sequences. Instead, a collection of proteins called transcription factors mediate the binding of RNA polymerase and the initiation of transcription. Only after certain transcription factors are attached to the promoter does the RNA polymerase bind to it. The completed

assembly of transcription factors and RNA polymerase bind to the promoter, forming a transcription initiation complex.

Promoter clearanceAfter the first bond is synthesized, the RNA polymerase must clear the promoter. During this time

there is a tendency to release the RNA transcript and produce truncated transcripts. This is called abortive initiation and is common for both eukaryotes and prokaryotes. Abortive initiation continues to occur until the σ factor rearranges, resulting in the transcription elongation complex (which gives a 35 bp moving footprint). The σ factor is released before 80 nucleotides of mRNA are synthesized. Once the transcript reaches approximately 23 nucleotides, it no longer slips and elongation can occur. This, like most of the remainder of transcription, is an energy-dependent process, consuming adenosine triphosphate (ATP).

Promoter clearance coincides with phosphorylation of serine 5 on the carboxy terminal domain of RNA Pol in eukaryotes, which is phosphorylated by TFIIH.

Elongation

One strand of the DNA, the template strand (or noncoding strand), is used as a template for RNA synthesis. As transcription proceeds, RNA polymerase traverses the template strand and uses base pairing complementarity with the DNA template to create an RNA copy. Although RNA polymerase traverses the template strand from 3' → 5', the coding (non-template) strand and newly-formed RNA can also be used as reference points, so transcription can be described as occurring 5' → 3'. This produces an RNA molecule from 5' → 3', an exact copy of the coding strand (except that thymines are replaced with uracils, and the nucleotides are composed of a ribose (5-carbon) sugar where DNA has deoxyribose (one less oxygen atom) in its sugar-phosphate backbone).

Unlike DNA replication, mRNA transcription can involve multiple RNA polymerases on a single DNA template and multiple rounds of transcription (amplification of particular mRNA), so many mRNA molecules can be rapidly produced from a single copy of a gen.

Elongation also involves a proofreading mechanism that can replace incorrectly incorporated bases. In eukaryotes, this may correspond with short pauses during transcription that allow appropriate RNA editing factors to bind. These pauses may be intrinsic to the RNA polymerase or due to chromatin structure.

Termination

Bacteria use two different strategies for transcription termination. In Rho-independent transcription termination, RNA transcription stops when the newly synthesized RNA molecule forms a G-C-rich hairpin loop followed by a run of Us. When the hairpin forms, the mechanical stress breaks the weak rU-dA bonds, now filling the DNA-RNA hybrid. This pulls the poly-U transcript out of the active site of the RNA polymerase, in effect, terminating transcription. In the "Rho-dependent" type of termination, a protein factor called "Rho" destabilizes the interaction between the template and the mRNA, thus releasing the newly synthesized mRNA from the elongation complex.

Transcription termination in eukaryotes is less understood but involves cleavage of the new transcript followed by template-independent addition of As at its new 3' end, in a process called polyadenylation.

2. The mRNA translation Process

Activation

In activation, the correct amino acid is covalently bonded to the correct transfer RNA (tRNA). The amino acid

is joined by its carboxyl group to the 3' OH of the tRNA by a peptide bond. When the tRNA has an amino acid linked

to it, it is termed "charged".

Initiation

Initiation of translation usually involves the interaction of certain key proteins with a special tag bound to the 5'

cap. The protein factors bind the small ribosomal subunit (40S), and these initiation factors hold the mRNA in place.

The eukaryotic Initiation Factor 3 (eIF3) is associated with the small ribosomal subunit, and plays a role in keeping

the large ribosomal subunit from prematurely binding. eIF3 also interacts with the eIF4F complex which consists of

three other initiation factors: eIF4A, eIF4E and eIF4G. eIF4G is a scaffolding protein which directly associates with

both eIF3 and the other two components. eIF4E is the cap-binding protein. It is the rate-limiting step of cap-

dependent initiation, and is often cleaved from the complex by some viral proteases to limit the cell's ability to

translate its own transcripts.

Elongation

With the formation of the complex containing fMet-tRNA in the peptidyl site, an aminoacyl tRNA with the

complementary anticodon sequence can bind to the mRNA passing through the acceptor site. This binding is aided

by elongation factors that are dependent upon the energy from the hydrolysis of GTP. Elongation factors go through

a cycle to regenerate GTP after its hydrolysis.

With tRNA bearing a chain of amino acids in the ribosome p site and tRNA containing a single amino acid in

the ribosome A site, the addition of a link to the chain can be made. This addition occurs through the formation of a

peptide bond, the nitrogen-carbon bond that forms between amino acid subunits to form a polypeptide chain. This

bond is catalyzed by the enzyme peptidyl transferase.

The peptide bond occurs between the carboxyl group on the lowest link in the peptide chain located at the p

site and the amine group on the amino acid in the A group. As a result, the peptide chain shifts over to the A site,

with the original amino acid on the A site as the lowest link in the chain. The tRNA in the A site becomes peptidyl

RNA, and shifts over to the P site. Meanwhile, the ribosome engages in a process called translocation: spurred by

elongation factors, the ribosome moves three nucleotides in the 3' prime direction along the mRNA. In other words,

the ribosome moves so that a new mRNA codon is accessible in the A site.

Termination

Translation ends when one of three stop codons, UAA, UAG, or UGA, enters the A site of the ribosome. There

are no aminoacyl tRNA molecules that recognize these sequences. Instead, release factors bind to the P site,

catalyzing the release of the completed polypeptide chain and separating the ribosome into its original small and

large subunits.

3. What does Helicase do ?

DNA helicase unwinds the DNA molecule by breaking hydrogen bonds.

4. What does Primase do?

Primase is an RNA polymerase that makes the RNA primer.

5. The events in the formation and resolution of Holliday structures.

Two homologous double helices are aligned,although note that they have been rotated so that

the bottom strand of the first helix has the samepolarity as the top strand of the

second helix (5 3 in this case)

Then a nuclease cleaves the two strands that have the same polarity

The free ends leave their original complementary strands and undergo hydrogen bonding with

the complementary strands in the homologous double helix

Ligation produces the structure

The Holliday structure creates a cross bridge, or branch, that can move, or migrate,

along the heteroduplex

The Holliday structure can be resolved by cutting and ligating either the two originally exchanged strands (Figur f, left) or the originally unexchanged strands (Figure f, right). The former generates a pair of duplexes that are parental, except for a stretch in the middle containing one strand from each parent. If the two parents had different alleles in this stretch, as indicated here, then the DNA will be heteroduplex. The latter resolution step generates two duplexes that are recombinant, with a stretch of heteroduplex DNA. The Holliday model also postulated that the heteroduplex DNA mismatches can be repaired by an enzymatic

correction system that recognizes mismatches and excises the mismatched base from one of the two strands, filling in the excised base with the correct complementary base. The resulting molecules will carry either the wild-type or the mutant allele, depending on which allele is excised.

6. Design a gene knockout animal model by DNA recombination.

When cells that have loxP sites in their genome express Cre, a recombination event can occur between the loxP sites. The double stranded DNA is cut at both loxP sites by the Cre protein. The strands are then rejoined with DNA ligase in a quick and efficient process. The result of recombination depends on the orientation of the loxP sites. For two lox sites on the same chromosome arm, inverted loxP sites will cause an inversion of the intervening DNA, while a direct repeat of loxP sites will cause a deletion event. If loxPsites are on different chromosomes it is possible for translocation events to be catalysed by Cre induced recombination.

polyI

creMx1

Lox P

Exon 3Exon 1

Lox P Lox P

Inducible AR knock-out system in mouse model

Exon 3Exon 1 Exon 2

Removal of lox P neogene from a 3-loxP knockout allele. Cre-mediated recombination

between loxP 2 and 3 deletes the ploxPneowhile the recombination between loxP 1 and 3 deletes

all DNA sequences between loxP 1 and 3. Different recombination events can be detected by PCR

analysis using primers a, b, and c. If this is performed in mice, it could generate conditional mutant

mice and null mice carrying the delta allele at the same time. ( b) Removal of a Frt-floxed neogene

through the expression of Flp recombinase. Because the loxP 2 is placed outside the Frt1, the Flip/

Frt-mediated recombination only deletes the Frt-foxed neo, generating a loxP fl oxed allele, which

can be used for conditional knockout