Lecture 4 Controlling gene expression in Prokaryotes Flashcards
Gene expression in Prokaryotes
Involves RNA polymerase and sigma factors for progression from the promoter.
Gene expression in Prokaryotes- Polycistronic mRNA
Polycistronic mRNAs generated from a gene.
Usually proteins/enzymes involved in regulation of a process.
Cis-regulatory regions and operator regions control initiation of transcription.
Repressors of transcription
Binding to the repressor to a ligand enables binding to the operator region and then displaces initiation complexes containing RNA polymerase
Summary
Prokaryotic genomes are more simplepartially due to polycistronic mRNAs.
Prokaryotes have less “non-coding” DNA and less elaborate control of gene transcription.
Common features of Eukaryotic and Prokaryotic gene control mechanisms include:
◦Consensus sequences
◦Transcription factors (trans-activators)
◦Ability to inhibit transcription with binding of repressors
Controlling gene expression in Eukaryotes- RNA Polymerase II regulation
Post translational modification of the largest subunit (RBP1) contains many repeats of a phosphorylation site.
Phosphorylation causes a shape change and enables transcription to be initiated.
Non coding DNA is as important as coding sequences- why?
on-coding DNA, which makes up a large portion of the genome in many organisms, was once thought to be “junk” because it does not directly code for proteins. However, research has shown that non-coding DNA plays crucial roles in gene regulation, genome stability, and other biological processes. Here are several reasons why non-coding DNA is as important as coding sequences:
- Regulation of Gene Expression
Promoters: Regions of non-coding DNA located near genes that help initiate transcription. Transcription factors and other proteins bind to promoters to control when, where, and how much a gene is expressed.
Enhancers and Silencers: Non-coding DNA regions that interact with promoters to enhance or suppress the transcription of specific genes. Enhancers can be located far from the gene they regulate and can increase the level of gene expression.
Insulators: Regions that can block the influence of enhancers or silencers, ensuring that genes are regulated correctly within certain domains.
Why it’s important: Without these regulatory regions, genes wouldn’t be expressed in a controlled manner, leading to problems like incorrect development, disease, or unregulated cell growth (as in cancer). - Non-Coding RNAs (ncRNAs)
MicroRNAs (miRNAs): Small, non-coding RNAs that regulate gene expression by binding to messenger RNAs (mRNAs), preventing their translation into proteins or marking them for degradation.
Long Non-Coding RNAs (lncRNAs): Involved in regulating chromatin structure, gene expression, and epigenetic modifications. LncRNAs help orchestrate complex cellular processes, such as differentiation and development.
Small interfering RNAs (siRNAs): Involved in RNA interference, where they degrade mRNAs to silence gene expression post-transcriptionally.
Why it’s important: These ncRNAs play essential roles in controlling gene expression, maintaining cellular homeostasis, and protecting the cell from viral RNA and other harmful elements. - Structural Roles in Chromosome Maintenance
Telomeres: Non-coding DNA sequences at the ends of chromosomes that protect them from degradation. Telomeres shorten with each cell division, and their length is critical for cellular aging and genome stability.
Centromeres: Non-coding DNA that forms the central part of chromosomes, ensuring proper segregation during cell division. The structure of centromeric DNA helps in the attachment of spindle fibers, which pull chromosomes apart.
Satellite DNA: Non-coding, repetitive sequences that contribute to chromosome structure and are involved in the organization of chromatin.
Why it’s important: Non-coding DNA helps maintain the integrity of chromosomes and ensures that they are properly replicated and segregated during cell division. Without telomeres or centromeres, chromosomes would be unstable and lead to genetic disorders. - Epigenetic Regulation
DNA Methylation: Non-coding DNA regions are often targets for methylation, a chemical modification that can silence gene expression without altering the DNA sequence. Methylation patterns are crucial for normal development and are often altered in diseases such as cancer.
Histone Modifications: Non-coding regions play a role in determining how histones (proteins that help package DNA into chromatin) are modified, affecting the accessibility of genes for transcription.
Why it’s important: Epigenetic modifications regulate gene expression in response to environmental stimuli and during development, ensuring that different cells express the right genes at the right time. - Alternative Splicing
Introns: Non-coding regions within genes that are removed from the pre-mRNA during splicing. However, introns can influence alternative splicing, a process that allows a single gene to produce multiple different protein isoforms.
Regulatory Sequences in Introns: These sequences help control the splicing machinery and determine which exons are included in the final mRNA.
Why it’s important: Alternative splicing increases the diversity of the proteins a single gene can produce, allowing organisms to adapt and perform more complex functions with fewer genes. - Mobile Genetic Elements and Evolution
Transposons: Segments of non-coding DNA that can move around within the genome. While they can sometimes disrupt gene function, they also contribute to genetic diversity and evolution by creating new regulatory elements or influencing nearby genes.
Retrotransposons: These elements copy themselves and insert into new locations in the genome, affecting genome structure and potentially regulating gene expression.
Why it’s important: Transposons and other mobile elements have contributed to genome evolution by reshuffling genes, creating new gene regulatory networks, and even serving as raw material for the evolution of new genes. - Genome Organization
Topologically Associating Domains (TADs): Regions of non-coding DNA that organize the genome into functional units, ensuring that regulatory elements like enhancers interact with the correct genes.
Non-coding RNA Interaction: Non-coding RNAs often help bring together various parts of the genome or maintain the structure of particular chromatin regions, enabling efficient gene regulation.
Why it’s important: Proper genome organization ensures that genes are expressed in the right context, which is essential for maintaining the health and function of cells and tissues. - Protection from Harmful Mutations
Non-coding DNA can act as a buffer against mutations. If mutations occur in non-coding regions, they are less likely to have immediate harmful effects than mutations in coding sequences. However, mutations in regulatory regions can still lead to diseases, so non-coding regions aren’t entirely “safe.”
Why it’s important: This buffering capacity helps reduce the impact of mutations and contributes to genetic diversity, which is important for evolution and adaptation. - Pseudogenes
Pseudogenes are non-functional sequences that resemble functional genes. While they don’t code for proteins, some pseudogenes can regulate their functional counterparts by acting as decoys for miRNAs or by other mechanisms.
Why it’s important: Pseudogenes can influence the expression of real genes by interacting with the same regulatory networks, contributing to the overall complexity of gene regulation. - Conservation Across Species
Many non-coding regions are highly conserved across different species, which suggests that they have important biological functions. Conserved non-coding sequences often regulate genes that are critical for basic cellular functions and development.
Why it’s important: The conservation of non-coding regions implies that they play fundamental roles in maintaining vital processes, as evolutionary pressure has preserved their sequences.
Conclusion
Non-coding DNA is not simply “junk” but plays critical roles in the regulation of gene expression, genome organization, and evolutionary processes. It contributes to the overall complexity of an organism by enabling precise control of when and where genes are expressed, ensuring genome stability, and allowing for greater protein diversity. Without non-coding DNA, complex life as we know it would not be possible.
The promoter- contains?
*Transcription start site (TSS).
*Consensus sequences
Consensus sequences are like signs
Eukaryotic RNA polymerase is part of a complex including general transcription factors.
General transcription factors bind to the promoter to recruit RNA polymerase
Consensus sequences are like “signs” or “signals” in DNA because they provide key instructions that help cellular machinery recognize important regions for processes like transcription, replication, and splicing. Just as road signs guide drivers on what to do at certain points, consensus sequences guide proteins and enzymes on where to bind and what actions to take within the genome. Here’s how:
- Promoters as Start Signs
Consensus sequences in promoters (such as the TATA box) are like “start” signs for transcription. They tell RNA polymerase and other transcription factors where to assemble and begin transcribing a gene.
Analogy: Just as a “START” sign at a race indicates where the runners should begin, consensus sequences in promoters indicate where transcription should initiate. - Splicing Signals as Guideposts
In eukaryotic genes, consensus sequences like the splice donor (GU) and splice acceptor (AG) sites act as guideposts for the splicing machinery. They mark where the non-coding regions (introns) should be removed and the coding regions (exons) joined together.
Analogy: These are like “cut here” signs on packaging, directing exactly where the RNA should be cut and spliced. - Replication Origins as Go Signs
Consensus sequences at origins of replication (like the OriC in bacteria) act as “go” signs, showing where DNA replication should start.
Analogy: Think of them like a traffic light turning green, signaling when and where the cell’s machinery can start duplicating the DNA. - Termination Sequences as Stop Signs
Certain consensus sequences, like polyadenylation signals or terminators, act as “stop” signs, signaling where transcription should end.
Analogy: Just like a “STOP” sign tells you to halt your car, these sequences tell RNA polymerase to stop transcription at the right location. - Enhancer and Silencer Sequences as Detour Signs
Consensus sequences found in enhancers and silencers tell regulatory proteins where to bind to either increase (enhancers) or decrease (silencers) the expression of nearby genes.
Analogy: These can be thought of as “detour” or “yield” signs, directing the cellular machinery to take alternate routes, either speeding up or slowing down gene expression. - Signal Sequences for Direction
Signal sequences in proteins, determined by consensus DNA sequences, tell the cell where a newly synthesized protein should go (e.g., to the mitochondria or outside the cell).
Analogy: Like signs that give directions on a highway, these sequences direct proteins to the correct cellular destination.
Conclusion:
Consensus sequences act as essential “signs” in the genome, guiding various processes like transcription, splicing, and replication. Just as road signs provide clear instructions for navigating the physical world, consensus sequences provide clear molecular instructions to cellular machinery, ensuring that biological processes occur correctly and efficiently.
Initiating Transcription
Transcription initiation is the first step of gene expression where the DNA is transcribed into RNA. This process is highly regulated and involves several key steps, especially in eukaryotes and prokaryotes. Here’s how transcription is initiated:
- Recognition of the Promoter
Promoter Sequence: Transcription starts when RNA polymerase (the enzyme that synthesizes RNA) recognizes and binds to a specific DNA sequence called the promoter. The promoter is located upstream (before) the start site of the gene.
Key Regions in Promoters:
In prokaryotes: The promoter typically contains consensus sequences like the -10 (TATAAT) and -35 (TTGACA) regions, which help RNA polymerase recognize the start site.
In eukaryotes: The promoter often includes a TATA box (around -25 to -30 bp upstream of the start site), which serves as a binding site for transcription factors. - Binding of Transcription Factors (Eukaryotes)
General Transcription Factors: In eukaryotes, transcription factors (TFs) must bind to the promoter region before RNA polymerase can attach. These transcription factors help position RNA polymerase at the correct site.
For example, the transcription factor TFIID binds to the TATA box, and other factors like TFIIB, TFIIE, and TFIIF help assemble the pre-initiation complex.
RNA Polymerase II Recruitment: After the transcription factors bind, RNA polymerase II (in eukaryotes) is recruited to the promoter to start transcription. - Formation of the Transcription Bubble
DNA Unwinding: Once RNA polymerase is bound to the promoter, it causes the DNA double helix to unwind. This unwinding creates a small open section of DNA called the transcription bubble.
Template Strand Exposure: The exposed single-stranded DNA serves as the template for RNA synthesis. The RNA polymerase will read the template strand to make a complementary RNA copy. - Initiation of RNA Synthesis
Start of RNA Synthesis: After the transcription bubble forms, RNA polymerase begins synthesizing the RNA strand by adding ribonucleotides (A, U, G, C) complementary to the DNA template.
Abortive Initiation: In some cases, RNA polymerase may release short RNA fragments initially before proceeding into full-length transcription. Once it passes this stage, RNA polymerase enters elongation, where the RNA chain grows longer. - Escape from the Promoter (Promoter Clearance)
Transition to Elongation: Once RNA polymerase has synthesized a small RNA strand (around 10 nucleotides long), it undergoes conformational changes that help it move away from the promoter region and continue RNA synthesis. This is called promoter clearance or escape.
Release of Transcription Factors: In eukaryotes, some transcription factors are released from the complex, allowing RNA polymerase to proceed with elongation.
Summary:
1. Recognition: RNA polymerase and/or transcription factors bind to the promoter region.
- Binding: In eukaryotes, multiple transcription factors and RNA polymerase assemble at the promoter.
- Unwinding: The DNA unwinds, exposing the template strand.
- RNA Synthesis: RNA polymerase begins synthesizing the RNA transcript.
- Promoter Escape: RNA polymerase moves beyond the promoter to elongate the RNA molecule.
The process varies slightly between prokaryotes and eukaryotes, but the fundamental steps of promoter recognition, transcription initiation, and RNA synthesis are common to both.
The enhancer
➢Non-coding DNA
➢Cis-regulatory regions and Trans-activating factors regulate this area.
➢3D effect that brings a complex of proteins together to form a “mediator” complex
RNA Pol II needs partners in crime
RNA polymerase cannot work alone, it interacts with:
➢Activators
➢Mediators
➢Chromatin modifying proteins
Summary
➢Non-coding DNA in the form of promoters and enhancers controls initiation of transcription.
➢Promoters contain consensus sequences to recruit general transcription factors.
➢The components of the mediator complex control initiation through 3D positionin
