The 'Central Dogma'

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. The ‘Central Dogma’ is the process by which the instructions in DNA are converted into a functional product.

. The central dogma of molecular biology explains the flow of genetic information, from DNA to RNA,making a functional product, the protein.

. The central dogma suggests that DNA contains the information needed to make all of our proteins, and that RNA is a messenger that carries this information to the ribosomes.

. The ribosomes serve as factories in the cell where the information is ‘translated’ from a code into the functional product.

. The process by which the DNA instructions are converted into the functional product is called gene expression.

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• Gene expression has two key stages - transcription and translation.

In transcription, the information in the DNA of every cell is converted into small, portable RNA messages.

During translation, these messages travel from where the DNA is in the cell nucleus to the ribosomes where they are ‘read’ to make specific proteins.

The central dogma states that the pattern of information that occurs most frequently in our cells is:

   *From existing DNA to make new DNA (DNA replication)

   *From DNA to make new RNA (transcription)

   *From RNA to make new proteins (translation).

Reverse transcription is the transfer of information from RNA to make new DNA, this occurs in the case of retroviruses. It is the process by which the genetic information from RNA is assembled into new DNA.

How does the ‘Central Dogma’ always apply?

1.With modern research it is becoming clear that some aspects of the central dogma are not entirely accurate.

2.Current research is focusing on investigating the function of non-coding RNA?.

3.Although this does not follow the central dogma it still has a functional role in the cell. 

DNA Replication

DNA replication is a biological process that occurs in all living organisms acting as the most essential part of biological inheritance. It is the process by which DNA makes a copy of itself during cell division.

Step 1: Replication Fork Formation

Before DNA can be replicated, the double stranded molecule must be “unzipped” into two single strands.

In order to unwind DNA, these interactions between base pairs must be broken.

• This is performed by an enzyme known as DNA helicase.

• DNA helicase disrupts the hydrogen bonding between base pairs to separate the strands into a Y shape known as the replication fork.

• This area will be the template for replication to begin.

• DNA is directional in both strands, signified by a 5' and 3' end.

• This notation signifies which side group is attached the DNA backbone.

• This directionality is important for replication as it only progresses in the 5' to 3' direction.

• However, the replication fork is bi-directional; one strand is oriented in the 3' to 5' direction (leading strand) while the other is oriented 5' to 3' (lagging strand).

• The two sides are therefore replicated with two different processes to accommodate the directional difference. Replication Begins

Step 2: Primer Binding

• The leading strand is the simplest to replicate.

• Once the DNA strands have been separated, a short piece of RNA called a primer binds to the 3' end of the strand.

• The primer always binds as the starting point for replication. Primers are generated by the enzyme DNA primase.

• DNA Replication: Elongation

• DNA polymerases attach themselves to the DNA and elongate the new strands by adding nucleotide bases.

Step 3: Elongation

• Enzymes known as DNA polymerases are responsible creating the new strand by a process called elongation.

• There are five different known types of DNA polymerases in bacteria and human cells. In bacteria such as E. coli, polymerase III is the main replication enzyme, while polymerase I, II, IV and V are responsible for error checking and repair.

• DNA polymerase III binds to the strand at the site of the primer and begins adding new base pairs complementary to the strand during replication.

• In eukaryotic cells, polymerases alpha, delta, and epsilon are the primary polymerases involved in DNA replication.

• Because replication proceeds in the 5' to 3' direction on the leading strand, the newly formed strand is continuous.

• The lagging strand begins replication by binding with multiple primers.

• Each primer is only several bases apart. DNA polymerase then adds pieces of DNA, called Okazaki fragments, to the strand between primers.

• This process of replication is discontinuous as the newly created fragments are disjointed.

Step 4: Termination

• Once both the continuous and discontinuous strands are formed, an enzyme called exonuclease removes all RNA primers from the original strands.

• These primers are then replaced with appropriate bases. Another exonuclease “proofreads” the newly formed DNA to check, remove and replace any errors.

• Another enzyme called DNA ligase joins Okazaki fragments together forming a single unified strand.

• The ends of the linear DNA present a problem as DNA polymerase can only add nucleotides in the 5′ to 3′ direction.

• The ends of the parent strands consist of repeated DNA sequences called telomeres.

• Telomeres act as protective caps at the end of chromosomes to prevent nearby chromosomes from fusing.

• A special type of DNA polymerase enzyme called telomerase catalyzes the synthesis of telomere sequences at the ends of the DNA.

• Once completed, the parent strand and its complementary DNA strand coils into the familiar double helix shape.

• In the end, replication produces two DNA molecules, each with one strand from the parent molecule and one new strand.

DNA Transcription

• Transcription is the first step of gene expression, in which a particular segment of DNA is copied into RNA (mRNA) by the enzyme RNA polymerase. It is the process by which the information in DNA is copied into messenger RNA (mRNA) for protein production.

• Transcription has three distinct stages: initiation, elongation, and termination.

1. Initiation

• The regions of the DNA that signal initiation of transcription in prokaryotes are termed promoters.

• In the E. coli promoters appear in the form of two regions of partial homology appear in the genome.

• These regions have been termed the −35 and −10 regions because of their locations relative to the transcription initiation point.

• The dissociative subunit of RNA polymerase, the σ factor, allows RNA polymerase to recognize and bind specifically to promoter regions.

• First, the holoenzyme searches for a promoter and initially binds loosely to it, recognizing the −35 and −10 regions. The resulting structure is termed a closed promoter complex.

*Then, the enzyme binds more tightly, unwinding bases near the −10 region. When the bound polymerase causes this local denaturation of the DNA duplex, it is said to form an open promoter complex. This initiation step, the formation of an open complex, requires the sigma factor.

Illustration showing the flow of information between DNA, RNA and protein. Genome Research Limited

2. Elongation

• Shortly after initiating transcription, the sigma factor dissociates from the RNA polymerase.

• The RNA is always synthesized in the 5′→3′ direction, with nucleoside triphosphates (NTPs) acting as substrates for the enzyme.

• The sequential addition of nucleotides takes place one at a time in the 5′-to-3′ direction.

• The chain grows by the formation of a bond between the 3′ hydroxyl end of the growing strand and a nucleoside triphosphate, releasing one pyrophosphate ion (PPi). This results in the net addition of one phosphate, which is incorporated into the backbone of the new strand.

• DNA grows by reaction with deoxyribonucleoside triphosphates, and RNA grows by reaction with ribonucleoside triphosphates. Certain sequences may cause stalling or pausing, which becomes critical for termination of transcription.

3. Termination

• RNA polymerase also recognizes signals for chain termination, which includes the release of the nascent RNA and the enzyme from the template.

*Bacteria use two different strategies for transcription termination: Rho-independent termination and Rho-dependent termination.

• In Rho-independent transcription termination, also called intrinsic 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.

Translation

• In translation, messenger RNA (mRNA)—produced by transcription from DNA—is decoded by a ribosome to produce a specific amino acid chain, or polypeptide.

• The polypeptide later folds into an active protein and performs its functions in the cell.

• The ribosome facilitates decoding by inducing the binding of complementary tRNA anticodon sequences to mRNA codons.

• The tRNAs carry specific amino acids that are chained together into a polypeptide as the mRNA passes through and is "read" by the ribosome. The entire process is a part of gene expression.

In brief, translation proceeds in four phases:

a.Initiation: The ribosome assembles around the target mRNA. The first tRNA is attached at the start codon.


b.Elongation: The tRNA transfers an amino acid to the tRNA corresponding to the next codon.


c.Translocation: The ribosome then moves (translocates) to the next mRNA codon to continue the process, creating an amino acid chain.


d.Termination: When a stop codon is reached, the ribosome releases the polypeptide.

• Many of transcribed RNA, such as transfer RNA, ribosomal RNA, and small nuclear RNA, do not undergo translation into proteins.

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

• In bacteria, translation occurs in the cell's cytoplasm, where the large and small subunits of the ribosome bind to the mRNA.

• In eukaryotes, translation occurs in the cytosol or across the membrane of the endoplasmic reticulum in a process called vectorial synthesis.

• In many instances, the entire ribosome/mRNA complex binds to the outer membrane of the rough endoplasmic reticulum (ER); the newly created polypeptide is stored inside the ER for later vesicle transport and secretion outside of the cell.