Nucleic acids 3/4 -Gene Organisation & Transcription

Question 1

Describe what is meant by “Transcription”

Answer 1

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

Question 2

What protiens are involved with transcription (2)

Answer 2

• Gene transcription is carried out by enzymes called “RNA Polymerases”
• regulatory proteins called “Transcription Factors”

Question 3

List the major functional classes of RNA

Answer 3

Functional classes of RNA
 Transfer
 Ribosomal
 Messenger

Question 4

Name the three Eukaryotic RNA Polymerases

Answer 4

 RNA Polymerase I -Transcribes rRNA genes
 RNA Polymerase II - Transcribes genes encoding proteins into mRNA
 RNA Polymerase III- Transcribes tRNA and 5S RNA genes

Question 5

Describe what is meant by a “Transcription factor”

Answer 5

The rate/level of transcription from a given gene is regulated by the activity of DNA binding proteins, or
Transcription Factors.

These can be transcriptional activators, or transcriptional repressors,

Question 6

Describe the process involved with the basal transcription complex

Answer 6

Step 1
TF IID binds to TATA
• Partially unwinds the DNA helix, widening the minor grove to allow extensive contact with bases within the
DNA.
• This unwinding is asymmetric with respect to the TBP-TATA complex,thereby assuring transcription is
unidirectional

Step 2
• TFII A and TFII B bind
• TFII B is particularly important, as it is able to bind to TFII D and RNA Polymerase II

Step 3
• RNA polymerase binds to TFII B (with TF IIF bound to itself)

Step 4
• involve the binding of TFII E,TFII H and TFII J to RNA polymerase
• TFII H promotes further unwinding of the DNA helix to facilitate RNA synthesis by RNA Polymerase II.

Question 7

Factors affecting Transcription Factor Expression

Answer 7

Cell lineage

Transcription factor expression is altered by signals outside the cell (e.g. Hormones, Growth Factors, Mechanical Stress)

Mutated transcription factors have been implicated in several human genetic disorders.

Abnormal transcription factor expression is found in several human factors.

Question 8

Gene Regulation in Human Disease

Answer 8

• 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

Question 9

Distinguish “sense” and “anti-sense” DNA template strands.

Answer 9

Sense strand: The sense DNA strand will read in the same direction as the RNA Strand formed. This will not be complementary to the RNA single strand, but rather will have the same sequence (with U replacing T)

Antisense strand: the DNA which the RNA is built up from, i.e. is complementary to the synthesised RNA, and will be read in the opposite direction

Question 10

What is the basal transcription complex

Answer 10

The Basal Transcription Complex produces a low level of transcription in the absence of other transcription factors.

The Basal Transcription Complex allows RNA Polymerase II to be phosphorylated and then engage in transcription.

Question 11

What does TF IID consist of

Answer 11

consists of TATA Binding Protein (TBP) and TBP Accessory Factors (TAFs).

Question 12

What is RNA Processing

Answer 12

Turning pre-mRNA -> mRNA

• The initial RNA produced from a gene is known as a “primary transcript” or “Pre- mRNA” or “heterogenous
nuclear RNA” ( hn RNA)

• This needs to be processed to make mRNA before translation
• This occurs in the nucleus, and then the mRNA is exported to the cytoplasm for translation

Question 13

The anatomy of gene

Answer 13

• The promoter lies at the 5’ end
• The sequence information contained in the final mRNA is encoded “discontinuously” in the DNA of the gene
• Segments of the gene which contain sequences that form part of the final RNA are called “exons”
• “Introns” are sequences in the gene which are transcribed but are edited out of the final mRNA

Question 14

What is the sequence of events in mRNA splicing/processing (6)

Answer 14

1. snRNP U1 binds to splice donor sequence
2. snRNP U2, U4, U5 and U6 bind
3. The end G of the intron bends round and an ‘A residue’ in the intron acts as a branchpoint in an intermediate step in splicing
4. Phosphodiester bonds forms between G and A residue
5. The phosphodiester bond beween end G of intron and the exon breaks so intron is removed as LARIAT structure
6. Adjacent exons are ligated together

Question 15

Define the function of the “Spliceosome”

Answer 15

The spliceosome is the complex formed by the binding or the small ribonuclear proteins with pre-mRNA during
mRNA processing that coordinates the splicing of pre-mRNA to form mature mRNA.

Question 16

Describe the Cap added to the 5’ end of the mRNA in Post-transcriptional modification of mRNA. (3)

Answer 16

Cap is formed by hydrolysis of terminal triphosphate of mRNA to a diphosphate.

This then reacts with a phosphate of GTP to form 5’-5’phosphate linkage.

Cap is further modified by Methylation at the N7 position in the purine ring to form 7-Methylguanylate cap.

Question 17

What does the CAP do?

Answer 17

CAP ACTS TO PROTECT mRNA AT 5’ END AND GREATLY ENHANCES THE TRANSLATION OF mRNA.

Question 18

Addition of Poly-A tail

Answer 18

• Known as Polyadenylation
• Added one base at a time
• added 11-30 bases downstream (3’ end) of the sequence AAUAAA, which is found in all mRNA’s
• artificially engineered mRNA without the poly-A tail are more unstable and degenerate rapidly

Question 19

describe polio

Answer 19

Interferes with recognition of cap during translation

Highly infectious disease

Virus can invade nervous system and cause total paralysis within hours

Mainly affects children under the age of three

Virus enters the body through the mouth, multiplies in the intestines.

Initial symptoms include fever, fatigue, headache, vomiting, stiffness in the neck and pain in limbs.

5-10% die because muscles involved in breathing become immobilised.

Question 20

describe how mutations in splice sites feature in human disease.

Answer 20

A mutation is a heritable change in the sequence of a gene.
• ~ 13% of cases feature mutations in gene promoters
• ~ 6 % feature mutations of the polyadenylation sequence
• ~ 1.2 % feature mutations of RNA capping
• ~ 33% feature mutations in splice donor / acceptor sequences
• the remaining 47% of cases involve mutations that alter the structure / function of the protein being
produced e.g. in cancer

Question 21

Describe thalassemia

Answer 21

• Example of mutations in splice sites in human disease
• very common in the Mediterranean, SE Asia, China
• Several types of b-thalassemia feature splice site mutations in b-Globin gene

Question 22

What is the junction between exon and intron called?

Answer 22

SPLICE DONOR SITE

Question 23

What is the junction between intron and exon ?

Answer 23

SPLICE ACCEPTOR SITE

Question 24

What do introns and exons end with ?

Answer 24

AG

Question 25

What type of protein does RNA processing use?

Answer 25

small Ribonuclear Proteins (snRNPs).

Question 26

What are the features of Thalassemia (3)

Answer 26

Iron Overload (Hemosiderosis) - elevated absorption of Iron due to chronic anaemia.

Iron Overload results in: Hepatic fibrosis and cirrhosis, darkening of skin, cardiomyopathy.

Extramedullary hematopoiesis

Hepatomegaly/Hepatosplenomegaly

Question 27

What is the gene promoter

Answer 27

The DNA sequence at which the transcription complex assembles is the gene promoter.