Fundmental Processes. DNA to RNA to Protein

Contents

Chapter 1 Biological process

Chapter 2 DNA replication

Chapter 3 Transcription

Chapter 4 RNA processing/Splicing

Chapter 5 Translation

Chapter 1 Biological process

The biological living systems contain large number of fundamental processes that control the system. The components present in the system are interlinked and forms network of interactions. The molecules in the systems perform functional relationships that process the mechanisms based on the structural and functional aspects.

Genome organization

The simple relationship between chromosome number, genome size, and species depends on c-value paradox that are not been present similar. The average size of gene in E. coli is 950 bp and in eukaryotes is 27,000 bp due to introns and gene desert stretches DNA, where there is no gene.

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Genome Organization in Eukaryotes

Genome sizes

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Nucleosome is the basic unit of chromosome organization where DNA is competed around 6 fold. Histone proteins have side chains with basic amino acids like lysine, arginine, etc, that neutralizes the net negative charge of DNA.

The interactions between DNA and histones includes

I. Hydrogen bond between protein and the O2 atom of phosphate backbone
II. Hydrophobic interactions
III. Salt linkage

The modifications in histone tail protein (N-terminal) regulate chromosome structure, usually phosphorylation and acetylation that promote nucleosome assembly which is required for gene expression.

The eukaryotic chromosome based on increase in Compaction can be discuss in following manner

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Eukaryotic chromosome organization

Binding of H1 protein results in a zig-zag appearance of DNA because of its nucleosomes that are organized with 30mm fiber. The 30mm fiber has 6 nucleosomes per turn. The H1 Protein protects DNA and additional 20bp stretch of DNA (in total 165bp) results for action of nucleases. The solenoids are organized in loop, helped by a proteinaceous structure known as the nuclear scaffold. The loops are finally arranged specifically in chromosomes.

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Twist remove enzyme are Topoisomerases.

Chapter 2 DNA Replication

DNA replication is the process of producing DNA from other DNA molecule.

Key features of DNA replication

Semi conservative replication (each parental strand function as template)

Given by Meselson and stahl in 1958.

➔Experiment on E.coli
➔N15
➔CsCl2 density gradient centrifugation (apply technique)

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1) The CsCl2 equilibrium density gradient centrifuge is used to separate molecule based on their density.

Eg: the N15 DNA content molecule and N14.

2) Sucrose velocity density gradient centrifuge is used to separate the molecules on the basis of size.

Eg: DNA molecules from phage λ and from φ29.

3) Taylor, woods et al. (1957) - vicia paba 5

- H3 tymidine

- Auto radiography (tech used)

AT rich sequence
→ Have 2 hydrogen bonds
→ Utility is termed as origin of replication
→ Transcription termination
→ Transcription starts from adimine (AuG)

Bidirectional replication

It was first demonstrated in phage λ by inman and schnos. They used the technique denaturation mapping. Exceptionally coli phage p2, mitochondria, chloroplast DNA seems to replicate semi directionally. With the bi-directional replication, the circular DNA produced from eye shaped structure while the linear DNA produced from eye shaped structures. The € each replication fork is 4 – shaped.

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Bidirectional Replication

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Bidirectional Replication DNA Synthesis

Replication proceeds in 51-31 direction occurs due to the absolute requirement of free 31 OH end. Since DNA polymerase can only elongate the 31 OH end, the polymerization (formation of phosphor di ester bonds) is an SN2 reaction. The free 31 OH end (off primer) attach the- phosphoryl group of the incoming nucleoside tri phosphate releasing the pyro phosphate.

Unit of Replication

The replicon is the unit of DNA that means capable of the independent replication of other fragments of DNA. Each replicon has “one origin of replication” and initiate replication, only one in each cell cycle. In bacterial and viral chromosomes usually there is one unicyte of origin of replication but eukaryotes have multiple replicon.

Origin of replication: (part of replicon)

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ARS (Autonomously Replicon sequences) is around 50bp that is having a core 11bp AT rich sequences; function has “origin of replication”

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Terminus of replication

In E.coli, there are two termini of replication, each termini contain multiple terminators. The hall terminator sequences (terminus A, terminus B,…. Terminus E) contains 23 base pair sequence which functions only in one direction.

TUS – Termination, Utilization, Substance

TUS gene

→

TUS protein (contra helicase activity)

→

Recognizes of binds at termination sequence

→

Helicase action is stopped

→

Replication stops

Replicon

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The semi discontinuous nature of replication

In this method of replication, one origin and 2 replication forks move in bi-directions. At each replication forks there is one leading strand and lagging strands that means each strand is composed of a leading stretch and many lagging stretchers.

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DNA polymerase III

(Holo enzyme) ➔ (Prominent replicase) DNA polymerase-I ➔ remove RNA primers and fill gene gaps

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DNA Polymerase I

Polymerases include the DNA polymerase I (Pol I) enzyme, encoded by the pol A gene and everywhere among prokaryotes. Polymerase is involved in elimination repair with 3'-5' and 5'-3' exonuclease activity and processing of Okazaki fragments generated during lagging strand synthesis. Pol I is most abundant polymerase accounting for >95% of polymerase activity in E. coli. Pol I added ~15-20 nucleotides each second, thus showing poor processivity. An additional Pol I starts adding nucleotides at the RNA primer, template junction known as the origin of replication (ori) around ~400 bp downstream from the origin.

DNA Polymerase II

DNA polymerase II (Pol II) is a Family B polymerase, encoded by the pol B gene. Pol II has 3'-5' exonuclease activity and participates in DNA repair mechanism. The replication restart to bypass lesions and its cell presence can move from ~30-50 copies per cell to ~200-300 through SOS induction. The main role of Pol II is thought to be the ability to direct polymerase activity at the replication fork and helped stalled Pol III bypass terminal mismatches.

DNA Polymerase III

DNA polymerase III holoenzyme is the primary enzyme involved in DNA replication in E. coli and belongs to Family C polymerases. It consists of three assemblies: the pol III core, the beta sliding clamp processivity factor and the clamp-loading complex. The core consists of three subunits - α, the polymerase activity hub, exonucleolytic proofreader, and θ, which may act as a stabilizer. The holoenzyme contains two cores, one for each strand, the lagging and leading. The beta sliding clamp processivity factor is also present in duplicate, one for each core, to create a clamp that encloses DNA allowing for high processivity. The third assembly is a seven-subunit (τ2γδδ′χψ) clamp loader complex. Recent research has classified Family C polymerases as a subcategory of Family X with no eukaryotic equivalents. The Pol III holoenzyme is assembled and takes over replication at a highly processed speed and nature.

DNA Polymerase IV

In E. coli, DNA polymerase IV (Pol 4) is an error-prone DNA polymerase involved in non-targeted mutagenesis. Pol IV is a Family Y polymerase expressed by the dinB gene that is switched on via SOS induction caused by stalled polymerases at the replication fork. During SOS induction, Pol IV production is increased 10-fold and one of the functions during this time is to interfere with Pol III holoenzyme processivity. This creates a checkpoint, stops replication, and allows time to repair DNA lesions via the appropriate repair pathway. Another function of Pol IV is to perform translation synthesis at the stalled replication fork like, for example, bypassing N2-deoxyguanine adducts at a faster rate than transversing undamaged DNA. Cells lacking dinB gene have a higher rate of mutagenesis caused by DNA damaging agents.

DNA Polymerase V

DNA polymerase V (Pol V) is a Y-family DNA polymerase that is involved in SOS response and translation synthesis DNA repair mechanisms. Transcription of Pol V via the umuDC genes is highly regulated to only produce Pol V when damaged DNA is present in the cell generating an SOS response. Stalled polymerases cause RecA to bind to the ssDNA which causes the LexA protein to autodigest LexA, then loses its ability to repress the transcription of the umuDC operon. The same RecA-ssDNA nucleoprotein post translationally modifies the UmuD protein into UmuD' protein. UmuD and UmuD' form a heterodimer that interacts with UmuC which in turn activates umuC's polymerase catalytic activity on damaged DNA

DNA polymerization – III is a multimeric enzyme

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Characteristics of the DNA polymerases: (Single active site) Steric exclusion of rNTP’s (the active site of DNA polymerization cannot accommodate the 21 OH group present in r NTP’s). The 31 exonuclease activity of epsilon unit perform proof realizing function.

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- : Processivity is process of average number of nucleotide unit that added each time the enzyme binds a primer template junction. The high processivity of DNA polymerase is due to the ability of DNA polymerase to slide along the template. A protein sliding clamp in prokaryotes is β submits and in eukaryotes it is PCNA (proliferating cell nuclear Antigen). The clamp loader ATPase activity is √, δ complex in prokaryotes and replication factor like C in Eukaryotes

DNA polymerase I: (Kornberg enzyme)

It is a monomer

1. 51 ➔ 31 – polymerase activity

2. 31 ➔ 51 – exonuclease activity

3. 51 ➔ 31 – endonuclease activity

Utility: DNA polymerase is used to excise the RNA primer (51-31 exonuclease activity) in prokaryote.

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Eukaryotic DNA polymerases

1. DNA polymerase is engaged in replication end leading a strand.
2. DNA polymerase € is engaged in replication of lagging strand.
3. DNA polymerase α acts as kinase in Eukaryotes. It is involved in the synthesis of primers.
4. The process of replacing DNA polymerase α by DNA polymerase δ or DNA polymerase is known as the polymerase switching

Helicase: It is a hexamer separates the two strands of DNA using ATP. 1ATP is used for unbinding of every base pair. It is binding to DNA polymerase III holo enzyme at (ζ) tall subunit. It stimulates the rate of strand separation.

TOPO isomerases: There are 2 classes

1. TOPO isomerase➔ linking no-1➔ss nick, need no ATP
2. TOPO isomerase➔ linking no-2➔ ds break, need ATP

Due to the helicase action DNA strand get some topological constraints. TOPO isomerase resolves those constraints in prokaryotes. The DNA gyrase is topoisomerase ii.

Single strand binding (SSB) protein: The binding of such protein stabilizers provide single strand stretch of DNA. The SSB protein show cooperative binding.

- : It is modified RNA polymerase, which is engaged in the sync (or synchronization) of oligonucleotide chain of RNA (primers). The primers are needed for DNA replication to provide initial 31 OH end on which DNA polymerase can act.

Polynucleotide ligase

Polynucleotide ligase seals the nick and finally forms the phosphodiester bond. The two different types of ligases are

1. T4 ligases (ATP users)

2. E.coli ligases (users NAD)

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A negatively supercoiled DNA molecule undergoes a B to Z transition over a segment of 180 base pairs. Calculate the effect on the writhing (supercoiling)

The twist changes from that in B-form (TB) to that in Z DNA (TZ)

TB = 180/ +10 = +18

TZ = 180/-12 = -15

DT = TZ - TB = - 15 - (+18) = -33

The molecule is not opened during this transition, so the linking number does not change

DL = 0

DW = -DT = -(-33) = +33

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