Transcription, RNA Processing, and Gene Regulation in Eukaryotes and Prokaryotes

Transcription Steps

Discriminate between transcription (initiation, elongation, termination) in both prokaryotes and eukaryotes, including the unique factors involved in each system.

RNA polymerase

Understand what RNA polymerase looks like and key features of the enzyme such as: active site (with Mg2+); RNA-DNA hybrid in active site; NTP channel for substrate; dsDNA entry point; extrusion of RNA stably.

Transcription Factors (TFs)

These are proteins (like Sp1, an example mentioned in the slides) that bind to specific DNA sequences to regulate gene transcription. The Mediator complex is an example of a factor involved in eukaryotic transcription regulation.

Bacterial sigma factors

Enable RNA polymerase to recognize and bind to the correct promoter region which initiate transcription.

Eukaryotic Gene Structure

Eukaryotic genes contain non-coding segments (introns) interspersed with coding segments (exons), which necessitates extensive processing.

RNA Processing

Complex RNA processing steps, including the removal of introns via splicing, evolved in eukaryotes.

5' Cap

Eukaryotic mRNAs are 'capped' at the 5' end, but usually not in bacteria.

Polyadenylation

Eukaryotic mRNAs are modified with a poly(A) tail at the 3' end, usually not in prokaryotic mRNAs.

Operons

Eukaryotes generally do not transcribe operons (coordinated gene clusters regulated by a single promoter), unlike prokaryotes.

RNA Instability

RNA is inherently less stable than DNA, which makes it prone to degradation; it is often converted to complementary DNA (cDNA) for storage, manipulation, and amplification.

cDNA Synthesis (Reverse Transcription)

Reverse transcriptase enzyme is used along with a primer (e.g., a poly-T primer to bind the poly(A) tail of mRNA, or a random hexamer) to create a DNA strand from an mRNA template.

Blotting

To find a specific RNA (or DNA) sequence on a blot (e.g., a Northern Blot), a complementary sequence called a probe is required.

Probes

Can be radiolabeled or labeled non-radioactively, such as with digoxigenin (DIG), which is then detected by an antibody.

Reverse Transcriptase PCR (RT-PCR)

This technique combines cDNA synthesis with standard PCR to amplify the transcript of a gene of interest.

Quantitative RT-PCR (qRT-PCR)

This is an application of RT-PCR used to precisely measure the starting quantity of mRNA (i.e., the level of gene expression) by monitoring the reaction in real-time.

Fluorescence threshold in qRT-PCR

Fewer PCR cycles needed to reach a specific fluorescence threshold indicate a higher initial concentration of mRNA template.

Processing for Stability and Function

Eukaryotic post-transcriptional modifications including 5' cap, splicing and poly(A) tail can contribute to regulating mRNA stability, degradation, and to promote translation.

Gene Structure Drives Processing

The presence of introns in eukaryotic genes is the evolutionary rationale for the complex machinery of RNA splicing.

cDNA

Complementary DNA. A DNA molecule synthesized from an RNA template (usually mRNA) using the enzyme reverse transcriptase.

Digoxigenin (DIG)

A non-radioactive marker used to label nucleic acid probes. The labeled probe is detected by an antibody conjugated to a detection molecule.

Exon

A segment of a eukaryotic gene's DNA that is represented in the final mature RNA molecule (e.g., mature mRNA).

Intron

A non-coding segment of a eukaryotic gene's DNA that is initially transcribed but is later removed from the primary RNA transcript by splicing.

Probe

A single-stranded nucleic acid (DNA or RNA) with a detectable label (e.g., radioisotope or DIG) that is used to locate a specific complementary target sequence on a gel or blot.

qRT-PCR

Quantitative Reverse Transcriptase PCR. A technique that couples cDNA synthesis with real-time PCR to precisely measure the amount of starting RNA template (gene expression level).

Reverse Transcriptase

An enzyme, originally found in retroviruses, that can synthesize a DNA strand using an RNA template (RNA-dependent DNA polymerase activity).

RNA Splicing

The process in which introns are removed from the primary RNA transcript and exons are joined together to form the mature mRNA.

Sigma Factor

A protein required by bacterial RNA polymerase that helps the enzyme recognize and bind to the specific promoter sequences on the DNA template.

Transcription Factor (TF)

A protein that binds to specific DNA sequences to influence the rate of transcription (i.e., turning genes 'on' or 'off').

Alternative Splicing

A process that allows for a diverse set of proteins to be encoded from a single gene using different combinations of exons.

cDNA Synthesis

A core technique in recombinant DNA technology that involves creating a DNA copy of a mature mRNA.

Gene Structure and Function

The presence of introns and exons in eukaryotes, and RNA splicing, is a critical component of gene structure and provides a mechanism for functional diversity.

Regulation of Transcription

The roles of Transcription Factors (TFs) and the Mediator complex in regulating transcription and gene expression in eukaryotes.

Recombinant DNA Technology

A field that involves obtaining a cDNA clone to express eukaryotic genes in bacterial or mammalian cell culture systems.

Gene Expression Quantification

The qRT-PCR technique used for quantitative assessment of gene expression, regardless of RNA origin.