7.2 Flashcards
A gene is a sequence of DNA which is transcribed into RNA and contains three main parts:
promoter, coding sequence, terminator
Promoter
- The non-coding sequence responsible for the initiation of transcription
- The core promoter is typically located immediately upstream of the gene’s coding sequence
- The promoter functions as a binding site for RNA polymerase (the enzyme responsible for transcription)
- The binding of RNA polymerase to the promoter is mediated and controlled by an array of transcription factors in eukaryotes
- These transcription factors bind to either proximal control elements (near the promoter) or distal control elements (at a distance)
Coding Sequence
- After RNA polymerase has bound to the promoter, it causes the DNA strands to unwind and separate
- The region of DNA that is transcribed by RNA polymerase is called the coding sequence
Terminator
- RNA polymerase will continue to transcribe the DNA until it reaches a terminator sequence
- The mechanism for transcriptional termination differs between prokaryotes and eukaryotes
Antisense vs Sense
A gene (DNA) consists of two polynucleotide strands, but only one is transcribed into RNA
Either of the 2 polynucleotide strands may contain a gene, and hence the determination of sense and antisense is gene specific
Antisense strand
- The antisense strand is the strand that is transcribed into RNA
- Its sequence is complementary to the RNA sequence and will be the “DNA version” of the tRNA anticodon sequence
- The antisense strand is also referred to as the template strand
Sense strand
- The sense strand is the strand that is not transcribed into RNA
- Its sequence will be the “DNA version” of the RNA sequence (i.e. identical except for T instead of U)
- The sense strand is also referred to as the coding strand (because it is a DNA copy of the RNA sequence)
Transcription is the process by which a DNA sequence (gene) is copied into a complementary RNA sequence by RNA polymerase
- Free nucleotides exist in the cell as nucleoside triphosphates (NTPs), which line up opposite their complementary base partner
- RNA polymerase covalently binds the NTPs together in a reaction that involves the release of the two additional phosphates
- The 5’-phosphate is linked to the 3’-end of the growing mRNA strand, hence transcription occurs in a 5’ → 3’ direction
The process of transcription can be divided into three main steps: initiation, elongation and termination
- In initiation, RNA polymerase binds to the promoter and causes the unwinding and separating of the DNA strands
- Elongation occurs as the RNA polymerase moves along the coding sequence, synthesising RNA in a 5’ → 3’ direction
- When RNA polymerase reaches the terminator, both the enzyme and nascent RNA strand detach and the DNA rewinds
In eukaryotes, there are three post-transcriptional events that must occur in order to form mature messenger RNA:
capping, polyadenylation, splicing
Capping
- Capping involves the addition of a methyl group to the 5’-end of the transcribed RNA
- The methylated cap provides protection against degradation by exonucleases
- It also allows the transcript to be recognised by the cell’s translational machinery (e.g. nuclear export proteins and ribosome)
Polyadenylation
- Polyadenylation describes the addition of a long chain of adenine nucleotides (a poly-A tail) to the 3’-end of the transcript
- The poly-A tail improves the stability of the RNA transcript and facilitates its export from the nucleus
Splicing
- Within eukaryotic genes are non-coding sequences called introns, which must be removed prior to forming mature mRNA
- The coding regions are called exons and these are fused together when introns are removed to form a continuous sequence
- Introns are intruding sequences whereas exons are expressing sequences
- The process by which introns are removed is called splicing
Splicing of mRNA increases the number of different proteins an organism can produce
Splicing can also result in the removal of exons – a process known as alternative splicing
The selective removal of specific exons will result in the formation of different polypeptides from a single gene sequence
- For example, a particular protein may be membrane-bound or cytosolic depending on the presence of an anchoring motif
Transcriptional activity is regulated by two groups of proteins that mediate binding of RNA polymerase to the promoter
transcription factors and regulatory proteins
- The presence of certain transcription factors or regulatory proteins may be tissue-specific
- Additionally, chemical signals (e.g. hormones) can moderate protein levels and hence mediate a change in gene expression
transcription factors
Transcription factors form a complex with RNA polymerase at the promoter
- RNA polymerase cannot initiate transcription without these factors and hence their levels regulate gene expression
regulatory proteins
Regulatory proteins bind to DNA sequences outside of the promoter and interact with the transcription factors
- Activator proteins bind to enhancer sites and increase the rate of transcription (by mediating complex formation)
Repressor proteins bind to silencer sequences and decrease the rate of transcription (by preventing complex formation)
Control Elements
The DNA sequences that regulatory proteins bind to are called control elements
- Some control elements are located close to the promoter (proximal elements) while others are more distant (distal elements)
- Regulatory proteins typically bind to distal control elements, whereas transcription factors usually bind to proximal elements
- Most genes have multiple control elements and hence gene expression is a tightly controlled and coordinated process
Changes in the external or internal environment can result in changes to gene expression patterns
- Chemical signals within the cell can trigger changes in levels of regulatory proteins or transcription factors in response to stimuli
- This allows gene expression to change in response to alterations in intracellular and extracellular conditions
There are a number of examples of organisms changing their gene expression patterns in response to environmental changes:
- Hydrangeas change colour depending on the pH of the soil (acidic soil = blue flower ; alkaline soil = pink flower)
- The Himalayan rabbit produces a different fur pigment depending on the temperature (>35ºC = white fur ; <30ºC = black fur)
- Humans produce different amounts of melanin (skin pigment) depending on light exposure
- Certain species of fish, reptile and amphibian can even change gender in response to social cues (e.g. mate availability)
Eukaryotic DNA is wrapped around…
histone proteins to form compact nucleosomes
- These histone proteins have protruding tails that determine how tightly the DNA is packaged
Typically the histone tails have a positive charge and hence associate tightly with the negatively charged DNA
- Adding an acetyl group to the tail (acetylation) neutralises the charge, making DNA less tightly coiled and increasing transcription
- Adding a methyl group to the tail (methylation) maintains the positive charge, making DNA more coiled and reducing transcription
When DNA is supercoiled and not accessible for transcription, it exists as
condensed heterochromatin
When the DNA is loosely packed and therefore accessible to the transcription machinery, it exists as
euchromatin
Types of chromatin…
- Different cell types will have varying segments of DNA packaged as heterochromatin and euchromatin
- Some segments of DNA may be permanently supercoiled, while other segments may change over the life cycle of the cell
Direct methylation of DNA (as opposed to the histone tails) can also affect gene expression patterns
- Increased methylation of DNA decreases gene expression (by preventing the binding of transcription factors)
- Consequently, genes that are not transcribed tend to exhibit more DNA methylation than genes that are actively transcribed
Epigenetics is the study of changes in phenotype as a result of variations in gene expression levels
- Epigenetic analysis shows that DNA methylation patterns may change over the course of a lifetime
- It is influenced by heritability but is not genetically pre-determined (identical twins may have different DNA methylation patterns)
- Different cell types in the same organism may have markedly different DNA methylation patterns
- Environmental factors (e.g. diet, pathogen exposure, etc.) may influence the level of DNA methylation within cells
