Gene Expression & Transcription in Eukaryotes

Gene expression is the process of converting genetic information into functional molecules like proteins or RNA.
Gene expression involves multiple steps, controlled at various points in the sequence of protein synthesis.
Eukaryotic genes are complex, composed of coding sequences (exons) and non-coding sequences (introns).
Each gene has a promoter region that initiates transcription and a coding region providing the blueprint for the protein.
The average gene spans 10-16 kb of DNA.

Objectives

Gene structure, gene expression.
Types of genes (constitutive and regulated).
Post-transcriptional modifications (RNA splicing, 3' polyadenylation, 5' capping).

Genes

Genes are segments of DNA that code for specific proteins.
The human genome contains an estimated 30,000 to 120,000 genes.
Not all genes are active in every cell at any given time.

Gene Structure

Genes consist of multiple functional components, each involved in gene expression.
Eukaryotic genes often have introns and exons.
Genes must have regulatory elements (exons, start signals, stop signals).

Gene Expression

Process converting a gene's information into cellular structures and functions.
Involves producing a biologically functional molecule of either protein or RNA.

Central Dogma

Explains the flow of genetic information: DNA → RNA → Protein.
Transcription: DNA is copied into RNA.
Translation: RNA is used by ribosomes to synthesize proteins.

Types of Genes

Constitutive Genes (Housekeeping Genes): Expressed at a constant rate, essential for basic cell function (e.g., glycolysis enzymes).
Regulated Genes: Expressed only under specific conditions or in specific cells (e.g., insulin gene in pancreatic cells).

Flow of Genetic Information in Prokaryotes vs. Eukaryotes

Prokaryotes have simpler gene expression: transcription and translation occur in the cytoplasm.
Eukaryotes have complex gene expression: transcription occurs in the nucleus, and then the resultant transcript (pre-mRNA) undergoes modifications before translation in the cytoplasm.

Eukaryotic Promoter

Promoter is a DNA region initiating transcription of a particular gene.
Promoters are upstream of the gene and consist of various elements, such as the CAAT box, TATA box, and GC box.
Promoters are typically 100-1000 base pairs long.

Eukaryotic Gene Promoter Sequences

Promoter sequences are important regulatory elements that help RNA polymerase bind to DNA.
Specific sequences like the TATA box are recognized by proteins that facilitate transcription.
These are usually 45-55 bases apart from the start of transcription, and 25 bases from the transcription start site.

Enhancer Sequences

Enhancers are regulatory DNA sequences that can increase the chances of a gene being transcribed.
Enhancers can be far away from the gene or even within the coding region, in contrast to the promoter region.

Silencers

Silencers are regulatory DNA sequences that decrease the chances a gene will be transcribed, in contrast to enhancers.

Transcription

Is a process where genetic information in a gene is copied into messenger RNA (mRNA).
DNA is copied into mRNA, and the resulting single strand of mRNA is a reversal of the DNA bases.
The resulting mRNA sequence carries the information to make proteins.

Messenger RNA (mRNA)

mRNA is a type of RNA that carries the information needed to make proteins.
mRNA is transcribed from DNA and carries the genetic code from the DNA to the ribosomes in the cytoplasm.
It's the messenger that takes the genetic information from DNA to the protein assembling site.

Reverse Transcription

Reverse transcription is the process where RNA is used as a template to make DNA.
Retroviruses contain their genetic material as RNA, which serves as the template for DNA synthesis using reverse transcriptase enzyme.
This new DNA can be inserted into the host cell's genome and transcribed to produce new viral proteins.

Transcription in Eukaryotes (Second Step)

Transcription in eukaryotes has two main steps:
- First step: RNA polymerase acts in conjunction with transcription factors to create an mRNA transcript.
- Second step: Post-transcriptional modifications like capping, polyadenylation, and splicing are performed on the pre-mRNA to mature RNA.

Capping

The 5' end of the pre-mRNA is capped with a 7-methylguanosine, protecting it from degradation and assisting in its export from the nucleus.
This process involves the removal of phosphate groups and the addition of the methylguanosine molecule.

Cleavage and Polyadenylation

The 3' end of pre-mRNA is cleaved and then polyadenylated (with addition of adenine residues) to create a poly(A) tail.
This protects the mRNA from degradation once it enters the cytoplasm.

RNA Splicing

The process removes non-coding regions (introns) from the pre-mRNA to create the final messenger RNA (mRNA) molecule.
Introns are noncoding sequences, while exons are sequences that code for proteins.
Exons are the parts of the pre-mRNA molecule that ultimately become the mature mRNA molecule.

RNA Polymerase II

RNA polymerase II is a large, complex protein that is responsible for using a template DNA strand to synthesize an mRNA transcript.
It has a globular portion to which regulatory proteins must bind to initiate transcription.
This enzyme is essential for creating the correct mRNA transcript.

Transcription Factors

Transcription factors are proteins that bind to specific DNA sequences and regulate transcription of genes.
Polymerases need TF’s to recognize and bind to transcription start sites.

The General Transcription Factors Needed for Transcription Initiation by Eukaryotic RNA Polymerase II

Several general transcription factors work together to initiate transcription.
The general transcription factors include proteins, like TFIID (which recognizes the TATA box in the promoter region), to facilitate the successful binding of RNA polymerase II and beginning of transcription.

Synthesis of Ribosomal RNA (rRNA)

rRNA is synthesized, processed, and combined with proteins to form ribosomes, the protein synthesis factories in the cytoplasm.

Synthesis of Transfer RNA (tRNA)

tRNA is synthesized by RNA polymerase III, and the resulting tRNA transcript goes through a post-transcriptional modification process to remove introns and add CCA sequence to the 3' end.
tRNA modifications include nucleotide modifications (like pseudouridine and dihydrouridine).

Clinical Correlations

An example regarding defects in mRNA splicing leading to β-thalassemia.

Synthesis of mRNA in Eukaryotes

Details the synthesis of mRNA, including the involvement of RNA polymerase, and the various steps of processing from pre-mRNA to mature mRNA, including the details about capping, polyadenylation, and splicing.