CELS191 Lecture 11: Gene Expression - Transcription

• The content and delivery of resources for CELS191 are copyrighted and for educational use only.
• Annika Bokor works in the Department of Biochemistry at the University of Otago.
• Annika's research interests include biochemistry, genetics, and forensics.
• Lecture 11 for CELS191 2025 covers gene expression, specifically transcription.
• After revising Lecture 11, you should be able to outline the Central Dogma of Molecular Biology, the process of transcription, and the structure of a eukaryotic gene, focusing on non-coding regions.

Central Dogma of Molecular Biology

• In 1955, Crick proposed the "Central Dogma of Molecular Biology"
• The central dogma explains how the information content of a gene is expressed.
• DNA (information) -> Transcription -> RNA (messenger) -> Translation -> Protein (worker)
• DNA replication, reverse transcription, and RNA replication add complexity to the central dogma

What is Gene Expression?

• It is the process by which information from a gene is used to synthesize a functional gene product, which can be protein or non-coding RNA.
• A gene is defined as a region of DNA that produces a type of RNA molecule with some function.
• Genes may contain sequences responsible for the regulation, production, and further processing of RNA.
• There are approximately 20,000 protein-coding genes.

Transcription

• DNA-dependent RNA synthesis involves synthesizing a single-stranded RNA molecule using a double-stranded DNA molecule as a template.
• Transcription is catalyzed by RNA polymerase, also known as DNA-dependent RNA polymerase.
• RNA polymerase catalyzes the extension of the 3'-end of an RNA strand by adding one nucleotide at a time.
• RNA Polymerase uses phosphodiester bonds between ribonucleotides to form an RNA transcript
• RNA polymerase selects the correct nucleotides to incorporate into RNA based on the DNA sequence being transcribed.
• mRNA synthesis is catalyzed by RNA polymerase II (RNA pol II).
• RNA Pol II can start a chain de novo because it has a primase function, which provides a 3' OH group for further dNTP addition
• DNA has two complementary strands, one is the coding strand (5' → 3') and the other is the template strand (3' → 5').
• mRNA is transcribed from the template strand.
• Transcription can be broken down into three stages: initiation, elongation, and termination.

Initiation

• Transcription factors bind to the TATA box and other regions of the promoter.
• RNA polymerase II (RNA pol II) then binds, forming a transcriptional initiation complex along with the transcription factors.
• The two DNA strands separate, and RNA pol II starts mRNA synthesis.
• RNA polymerase II recruits helicase, an enzyme that 'unzips' DNA by breaking hydrogen bonds and binds to the AT-rich region of the promoter to start 'unzipping’.

Chromatin structure

• RNA polymerase II gains physical access to DNA in the loosely packed euchromatin, which is transcriptionally active.
• Heterochromatin, which is tightly packed, is transcriptionally inactive.
• DNA accessibility also depends on the sliding of histone proteins along the DNA strands.

Elongation

• RNA polymerase II uses the template strand, which runs in the 3' → 5' direction.
• RNA polymerase II inserts complementary RNA nucleotides in the 5' → 3' direction.
• Topoisomerase II releases the tension (supercoil) that builds up ahead of RNA pol II.

Gene Structure

• The gene contains the necessary elements for this complex process.
• A gene sequence may be transcribed or translated.

Anatomy of Eukaryotic Genes

• Eukaryotic genes have regulatory sequences, promoters, 5' UTRs, coding sequences, 3' UTRs, and transcriptional/translational start and stop sites.
• Additional regulatory sequences are positioned upstream; a transcriptional start site (+1), and a translational start codon (ATG).
• An AT-rich region (aka TATA box) is positioned at (-25), as well as a 5' G cap sequence and ribosome binding site
• A Polyadenylation signal and cleavage site is positioned at the 3' end, as well as translational and transcriptional stop sites
• Splicing is when transcribed region is made of up mRNA, as well as the promoter.

Functions of Gene Structure Elements

• Non coding DNA regions can be transcribed but are not translated.
• Coding sequences are the portions of a gene's DNA that are translated into a protein.
• Promoters are DNA segments recognized by RNA polymerase to initiate transcription.
• UTRs (untranslated regions) contain regulatory elements that influence gene expression at the transcriptional and/or translational level:
  • 5' UTR facilities the addition of the 5' G cap
  • 3' UTR facilitates the addition of the poly-A tail
• A 5' G cap prevents mRNA degradation, promotes intron excision, and provides a binding site for the small ribosomal subunit.
• The poly-A tail prevents mRNA degradation and facilitates export of the mRNA from the nucleus to the cytoplasm.
• Eukaryotic genes contain non-coding DNA regions upstream and downstream of the coding sequence, as well as within it, and are transcribed but not translated, these regulate gene expression.
• Changes (mutations) in these non-coding gene sequences may disrupt normal gene expression.
• Prokaryotes- Transcription and translation occur in the cytoplasm and are coupled.
• Eukaryotes- Transcription occurs in the nucleus, and translation occurs in the cytoplasm and are NOT coupled.

Summary

• Transcription is the synthesis of a ssRNA molecule from a dsDNA molecule by an RNA polymerase.
• During transcriptional initiation, RNA polymerase recruits a helicase and topoisomerase enzyme.
• RNA polymerase II gains physical access to DNA in the loosely packed heterochromatin, and can therefore transcribe it.
• RNA polymerase II reads the DNA template strand in the 3' to 5' direction, synthesizing an RNA transcript in the 5' to 3' direction, and makes an RNA copy of the DNA coding strand
• There are multiple non-coding DNA sequences within a gene, which all have regulatory functions.