From DNA to Protein – The Middle Molecule

One Gene-One Enzyme Hypothesis
Initially suggested that each gene corresponds to one enzyme, proposing a direct relationship between genes and the functional proteins they encode. However, this concept has evolved, since some genes encode multiple polypeptides or proteins that assemble into complex structures, as seen in proteins like hemoglobin, which consists of multiple polypeptide chains that work together to carry oxygen in the blood.

Central Dogma of Molecular Biology
The Central Dogma describes the flow of genetic information in biological systems, encompassing two fundamental processes:

• Transcription: The process by which a particular segment of DNA is copied into RNA (specifically, messenger RNA or mRNA). This involves enzymes such as RNA polymerase that synthesize a complementary RNA strand from a DNA template.

• Translation: The subsequent step where mRNA directs the synthesis of polypeptides (the building blocks of proteins) at ribosomes, utilizing transfer RNA (tRNA) to decode the mRNA sequence into amino acids, ultimately forming a functional protein.

The sequence of events can be summarized as: DNA → RNA → Protein.

RNA Types
There are several crucial types of RNA involved in gene expression and regulation:

• mRNA (messenger RNA): Carries the genetic information transcribed from DNA to the ribosome for protein synthesis.

• tRNA (transfer RNA): Functions as an adapter molecule that translates the mRNA codon sequences into corresponding amino acids during protein synthesis.

• rRNA (ribosomal RNA): A structural and functional component of ribosomes, crucial for the assembly of amino acids into polypeptides.

• Non-coding RNAs (ncRNA): Comprising various types such as microRNAs (miRNAs) and long non-coding RNAs (lncRNAs), play essential roles in the regulation of gene expression, chromatin structure, and other cellular functions without coding for proteins.

RNA Processing in Eukaryotes
In eukaryotic cells, pre-mRNA undergoes several modifications before it can be translated:

• Addition of a 5' cap which protects the mRNA from degradation and assists in ribosome binding.

• Addition of a poly-A tail at the 3' end, enhancing mRNA stability and export from the nucleus.

• Splicing: Introns (non-coding regions) are removed, and exons (coding regions) are spliced together to form mature mRNA, ready for translation.

Prokaryotic vs. Eukaryotic Transcription

• Prokaryotes: Utilize a single RNA polymerase enzyme for all transcription needs. The resulting mRNA is polycistronic, meaning it can encode multiple proteins, often organized in operons that are expressed together.

• Eukaryotes: Involve three different RNA polymerases (I, II, and III) for synthesizing various RNA types. The mRNA produced is monocistronic, generally encoding one protein, and undergoes complex processing before translation.

Transcription Steps

1. Initiation: RNA Polymerase (RNAP) binds to the promoter region of the gene, creating an open complex that unwinds the DNA double helix.

2. Elongation: RNAP synthesizes RNA in the 5' to 3' direction, complementary to the template DNA strand, incorporating ribonucleotides into the growing RNA chain.

3. Termination: RNAP encounters a termination signal in the DNA, resulting in the release of the newly synthesized RNA transcript from the enzyme and the DNA template.

Gene Regulation
Gene expression is tightly regulated at multiple levels, including:

• Transcriptional regulation: Controlling the transcription of genes via various mechanisms, including the binding of transcription factors.

• Translational regulation: Modulating the efficiency of translation through interactions with components of the translation machinery.

• Post-translational modifications: Altering the protein after synthesis through processes such as phosphorylation, glycosylation, or proteolytic cleavage, affecting protein function and activity.
  In prokaryotes, regulation often occurs through operon systems, while in eukaryotes, it involves intricate interactions among multiple transcription factors and regulatory elements.

Lac Operon Example
The lac operon in E. coli is a classic example of gene regulation. It is regulated by a repressor protein (LacI) and an inducer (lactose).

• Inducible Genes: In the presence of lactose, the repressor is inactivated, allowing transcription of the genes required for lactose metabolism, thus facilitating the expression of the operon's enzymes.

Tryptophan Operon Example
The tryptophan operon functions as a repressible operon, where the presence of tryptophan inhibits gene expression.

• Repressible Genes: When levels of tryptophan are high, it binds to the repressor, activating it and preventing further production of enzymes involved in tryptophan biosynthesis.

Transcription Factors
Transcription factors are proteins that can either enhance or inhibit the transcription of specific genes by binding to nearby DNA sequences. They play vital roles in orchestrating the complex processes that control gene expression.

• They interact not only with the RNA polymerase but also with other regulatory proteins such as enhancers and silencers, effectively modulating transcriptional activity.

Chromatin Structure
The packing of DNA into chromatin can greatly influence gene expression.

• Euchromatin: Loosely packed chromatin that is generally accessible to transcription machinery and thus actively transcribed.

• Heterochromatin: Tightly packed regions that are usually transcriptionally inactive.
  Additionally, DNA methylation, especially in regions known as CpG islands, typically inhibits transcription by adding methyl groups to cytosine bases, impacting the accessibility of the DNA for transcription.

ncRNA Functions
Non-coding RNAs, such as miRNAs and siRNAs, play crucial roles in the regulation of gene expression and chromatin dynamics.

• miRNAs: Function in post-transcriptional regulation by binding to mRNA molecules and inhibiting their translation or promoting their degradation.

• siRNAs: Similar to miRNAs, they are involved in silencing specific mRNA sequences and can also play a role in gene regulation through RNA interference pathways.

• CRISPR-Cas System: An adaptive immune response mechanism found in bacteria that uses RNA to recognize and cut foreign DNA, showcasing the intricacies of RNA in cellular defense mechanisms and biotechnology applications.