Gene organisation, transcription and regulation

Question 1

DNA versus RNA: explain the basic differences between DNA and RNA, explain what is meant by transcription

Answer 1

Protein Synthesis: Overview

Central dogma of biology:

DNA —transcription–> RNA —translation–> Protein

Initial product of gene expression: RiboNucleic Acid

• RNA is a single-stranded nucleic acid species
• The pentose sugar in RNA is Ribose
• Thymine is not a base used in RNA. It is replaced by Uracil

Gene Expression:

• occurs in the cell nucleus.
• RNA is exported to the cell cytoplasm, to be used in “Protein Translation”

TRANSCRIPTION: The process in which nucleotide information in the DNA is copied into RNA

Question 2

RNA classes: List the major classes of RNA and the classes of RNA polymerases involved in synthesising each of these

Answer 2

Types of RNA, and the EUKARYOTIC enzyme responsible for their synthesis:

1. mRNA (messenger RNA, involved in Transcription) synthesised by RNA POLYMERASE II
2. tRNA (transfer RNA, involved in Translation) synthesised by RNA POLYMERASE III
3. rRNA (ribosomal RNA, involved in ribosome structure) synthesised by RNA POLYMERASE I
4. snRNA (small nuclear RNA, involved in RNA splicing) synthesised by RNA POLYMERASE II or III

Question 3

Gene transcription: explain what is meant by the terms “gene promoter” and “transcription factor”, explain with the aid of diagrams the processes involved in transcribing a eukaryotic gene

Answer 3

Transcription: Making an RNA copy of a DNA strand

Ribonucleotide bases are joined by phosphodiester bonds. The RNA chain grows one base at a time in a 5’ ->3’ direction

5’ ->3’ direction: sense strand
3’ ->5’ direction: anti-sense strand (new RNA is complementary to this)

Proteins involved in Transcription:

• Gene transcription is carried out by enzymes called “RNA Polymerases” (RNA Polymerase II - Transcribes genes encoding proteins into mRNA)
• Gene transcription also involves special gene regulatory proteins called “Transcription Factors”
• The “start” of a gene contains DNA sequences that are important in bringing about Transcription
• The DNA sequences at which the initiation complex assembles is called a gene promoter.
• The amount (level) of transcription from a given gene is regulated by the activity of DNA binding proteins known as Transcription Factors.

Anatomy Of A Gene Promoter

1. TATA sequence: This is the starting point for Transcription of a gene
2. Transcription Factor Binding Site: TFs can bind or unbind, to upregulate or downregulate transcription of the corresponding TATA sequence
3. GENE PROMOTER: TATA sequence + TF Binding Site. This is where the initiation complex assembles

Mechanism: Components of the Basal Transcription Complex

1. TF II D binds to TATA sequence

• Contains TATA Binding Protein (TBP) + TBP Accessory Factors (TAFs)
• Partially unwinds DNA helix by widening minor groove to allow extensive contact with bases within the DNA
• Unwinding is asymmetric, thereby assuring transcription is unidirectional 5’ to 3’

2. TF II A and B bind to TF II D

3. TF II F binds to RNA Polymerase II

• This complex then binds to TF II B

4. TF II E and H and J bind to RNA Polymerase II

• TF II H further unwinds DNA helix to allow RNA synthesis by RNA Polymerase II.

The whole complex is called the BASAL TRANSCRIPTION COMPLEX, as it contains the essential TFs needed to achieve transcription

• The Basal Transcription Complex allows RNA polymerase II to be phosphorylated and then engage in transcription.
• In the absence of binding of other Transcription Factors this produces a Basal (low) level of transcription.
• Transcription factors “bend DNA” on binding. They can interact with each other and the Basal Transcription Complex to modulate transcription
• Transcription factors also facilitate transcription by helping to remodel “chromatin”. They do this by recruiting proteins with enzymatic activities that modify histones
• Histone tails extend from nucleosome => subject to post-translational modification
  • HYPERACETYLATION CORRELATES WITH GENE EXPRESSION
    Acetylated histones “OPEN”
  • HYPOACETYLATION CORRELATES WITH GENE REPRESSION
    Deacetylated histones “CLOSED”

Question 4

Explain Gene Regulation In Human Disease

Answer 4

Gene Regulation In Human Disease:

• Mutated Transcription Factors have been implicated in several human hereditary disorders
• Abnormal Transcription Factor expression is found in several human cancers
• Mutations affecting the regulation of specific genes have been described in certain human diseases

example 1 : Inflamation and Transcription Factors

• NFkB; A Transcription Factor
• IkB; An Inhibitor of NFkB
• Initiators of inflammation -> “Cytokines”; Mediators of inflamation
• Aspirin acts to inhibit the breakdown of IkB. Consequently, NFkB remains in the cytoplasm and is unable to initiate transcription of cytokine genes

Example 2; Transcription Factors in Leukaemia

• Over half of Acute Lymphoblastic Leukaemia’s (ALL) involve mutated transcription factors. These mutations are often caused by chromosomal translocations.

Question 5

mRNA processing: explain with the aid of diagrams the events that take place in pre-mRNA processing; define what is meant by the terms “splice donor site” and “splice acceptor site”, the function of the spliceosome, and explain the addition of a “cap” and “poly A tail” to pre-mRNA

Answer 5

Gene expression requires RNA processing: Primary RNA transcripts need to be processed to form mature mRNA

• A gene will be made up of 2 types of sequences: introns and exons
• The final mRNA contains only exons
• Introns will be transcribed initially, but will be spliced out of the final mRNA

SPLICE DONOR SITE: At the starting (5’) end of the intron

• The donor site ALWAYS starts with GU

SPLICE ACCEPTOR SITE: At the end (3’) end of the intron

• The acceptor site ALWAYS ends with AG
• The acceptor site is preceded by: pyrimidine-rich region (Pyr(x)), then any base (N), then cytosine (C)

The sequence of events in mRNA splicing:

Small ribonuclear proteins (snRNPs, synthesised from snRNA) are vital for forming a spliceosome that cleaves the intron:

1. The first to be used is U1 which binds to the splice donor sequence
2. The next to bind are snRNP’s: U2, U4, U5 and U6
3. U5 binds to the splice acceptor site
4. The binding of U2, U4, U5 and U6 completes the formation of the splicing complex or “spliceosome”
5. This results in cleavage of the splice donor sequence.
6. The UG sequence loops back on the intron, and the G residue approaches the A residue near the midpoint of the intron
7. An “A” residue in the intron is used as the “branchpoint” in an intermediate step in mRNA splicing
8. A phosphodiester bond forms between the 5’ phosphate of G and 2’ hydroxyl of the branchpoint
9. The phosphodiester bond between the “G” at the end of the intron and the next exon is then cleaved and the intron removed as a “lariat” structure
10. The exposed, adjacent exon sequences are finally ligated” (joined) together

Post transcriptional modification of mRNA

1. Methylated RNA Cap:

Added to 5’ end

• Formed by hydrolysis of the terminal triphosphate of the mRNA to a diphosphate.This then reacts with the a phosphate of GTP to form a 5’-5’ phosphate linkage
• The cap is a guanosine molecule that is methylated at the 7’ position by methyltransferase, so it is called 7MeG
• The cap acts to protect mRNA at the 5’ end and also greatly enhances the translation of mRNA
• POLIO: works by interfering with recognition of methylated cap during translation

2. Polyadenylation:

• Addition of a POLY A TAIL to pre-mRNA at 3’ end
• This is added 11-30 bases downstream to the sequence AAUAAA (found in ALL mRNAs)
• The poly A tail is added one base at a time
• Protects 3’ end

This final modification converts pre-mRNA into mature mRNA, ready for translation

Question 6

Give examples of transcription disorders

Answer 6

Transcription Disorders:

1. Thalassaemia:

• Imbalance in relative amounts of globin chains
• Alpha and Beta subtypes (name denotes which globin chain is deficient)
• Several types of Beta caused by splice site mutations
• Iron Overload (Hemosiderosis) elevated GI absorption of iron due to chronic anaemia results in: hepatic fibrosis & cirrhosis (by age 5 years) darkening of skin (iron-stimulated melanin production) cardiomyopathy (arrhythmias, congestive heart failure, recurrent pericarditis)
• Extramedullary Hematopoiesis/ hepatomegaly/hepatosplenomegaly

2. Duchenne Muscular Dystrophy:

• Mutation in Dystrophin gene
• Most DMD caused by deletions of one or more exons
• Causes premature end during translation
• TREATMENT: Alternative Splicing
• It is possible to make shorter, but still function forms of dystrophin by skipping certain exons in the gene
• Pharmacologically possible to re-programme spliceosomes to favour alternative splicing

Question 7

Distinguish between different types of non-coding RNAs and explain the process of lyonization

Answer 7

Non-coding RNAs: Any RNA molecule that is not translated into protein (ie Does not include mRNA)

• Includes house keeping ncRNAs (ie ribosomal RNA, tRNA and spliceosome snRNAs)
• Also includes regulatory ncRNAs:

1. microRNA - control the translation of perhaps most genes
2. siRNA / RNAi – viral defence, experimental tool
3. piRNA – fascinating, clearly important for germ cell production
4. long ncRNA:- Xist – important for X chromosome inactvation
   H19 – involved in imprinting of the IGF-1 gene plus many more anti-sense RNAs with potential for imprinting and gene regulation

X-inactivation (also called lyonization)

• The process by which one of the two copies of the X chromosome present in female cells is inactivated
• The inactive X chromosome is silenced by its being packaged in such a way that it has a transcriptionally inactive structure called heterochromatin
• X-inactivation prevents female cells from having twice as many X chromosome genes as males cells, which only possess a single copy of the X chromosome
• The choice of which X chromosome to inactivate is random. Once an X chromosome is inactivated it will remain inactive throughout the lifetime of the cell and it’s derivatives
• long ncRNA:- Xist controls X chromosome inactivation

Question 8

Explain the mechanisms underlying gene regulation through RNA interference, explain what micro RNAs (miRNAs) are and how they act in gene regulation

Answer 8

The function of regulatory RNA (both microRNA and siRNA) is to control what mRNA is translated via RNA interference

• RNA silencing is used by plants as a defence mechanism against virus infection.
• RNA interference (RNAi):

1. A strand of RNA complimentary to mature mRNA is made (ANTISENSE RNA)
2. Hydrogen bonds form between the two
3. The mRNA is now trapped in the dsRNA and cannot be translated
4. The difference between microRNA and siRNA:
5. siRNA is made exogenously and cuts the target mRNA at one point
6. microRNA is made endogenously and cuts the target mRNA at multiple points

Modulating Translation: siRNA Mechanism (image)

1. Dicer; Rnase III-like endonuclease activity. Digests dsRNA into 21-25bp fragments –> Creates 3’ overhangs
2. Ago proteins attach to overhangs –> siRNA-guided, endonuclease activity acts to remove one of the siRNA strands, known as the “passenger strand”.
3. Multiprotein, RNA-induced silencing complexes, or “RISC” complexes formed.
   These recognize and cleave target mRNA molecules and silences them

Dicer is an essential gene: Lethality in early embryonic stages, embryonic stem cells unable to differentiate, depletion of multipotent stem cells.

Genomically encoded siRNAs: “Micro RNAs”

• Experimental evidence: Caenorhabditis elegans needs to decrease its levels of Lin-14 (a transcription factor) after a certain point during the larvae stage
• Lin-4 inversely correlated with Lin-14
• Lin-4 was found to code for microRNA that silenced the mRNA that would have produced Lin-14
• microRNA (abbreviated to miRNA) is synthesised in nucleus
• Hairpin structure roughly 18-26 nucleotides in length, and play key role in gene regulation
• It is synthesised with a 3’ overhang over the 5’ end of 2 nucleotides

miRNA transcription and processing

1. Transcribed by RNA polymerase II or III into a long primary pri-miRNA transcript with a stem-loop structure of up to several kilobases in length.
2. pri-miRNA processed by the RNase III endonuclease, Drosha, in a complex with DiGeorge syndrome critical region 8 (DGCR8). Produces 60 – 80 nucleotide precursor miRNA (pre-miRNA).
3. The pre-miRNA has a 5’ phosphate and two nucleotide overhang at the 3’ end. This is transported from the nucleus to the cytoplasm by the export receptor, Exportin-5, using Ran-GTP.

• DGCR8 interacts with the pri-miRNAs, through ssRNA regions
• 33 bp stem assists Drosha to cleave the substrate
• Drosha cleaves the pri-miRNA at approximately two helical turns into the stem from the junction of the loop of the miRNA hairpin

miRNA binding sites

• miRNAs inhibit target mRNAs through base pairing with incomplete complementarity
• The “Seed” region - the most important region of the miRNA for targeting. Lies between nucleotide positions 2 to 8 from the 5’ end, and is often flanked by adenosines
• Mismatches, predominantly in the centre forms a bulge, often followed by a less stringent degree of complementarity in the 3’ region
• Each miRNA can target several different mRNA’s, leading to co-ordinated gene regulation
• The vast majority of known miRNA target sites lie within 3’UTRs. The position and number of binding sites can influence repression efficiency.

​