Lecture 2.1: Transcription

Question 1

What is a Gene?

Answer 1

A gene is a unit of hereditary information/ section of DNA that codes for a specific mRNA or protien

Question 2

Exon & Intron

Answer 2

Exon: A coding section of DNA
Intron: A non-coding section of DNA

Question 3

Transcription Steps (3)

Answer 3

Initiation: Binding of RNA polymerase to promoter region
Elongation: RNA polymerase synthesises mRNA/ RNA transcript
Termination: RNA polymerase reaches termination site, detaches from DNA and then RNA transcript is released

Question 4

Transcription in Prokaryotic Cells (Initiation)

Answer 4

Starts with binding of RNA polymerase to promoter region in DNA

Prokaryotic promoters are characterised by 2 sets of sequences located 10 and 35 base pairs upstream of the transcription start site

Question 5

Prokaryotic RNA Polymerase: structure and what it does during Initiation

Answer 5

It is a holoenzyme consisting of 6 subunits

The role of the sigma part of the RNA polymerase is to bind specifically to the -35 and -10 sequences leading to the transcription

The polymerase then unwinds the DNA around the initiation site, forming an open promoter complex transcription is initiated by polymerisation of free NTs

Question 6

Transcription in Prokaryotic Cells (Elongation)

Answer 6

The sigma subunit of RNA polymerase then dissociates from the core polymerase which migrates along the DNA and elongates the growing RNA chain

Question 7

Transcription in Prokaryotic Cells (Termination)

Answer 7

RNA synthesis continues until the polymerase encounters a termination signal, then transcription stops, the RNA is released from the polymerase, and the enzyme dissociates from its DNA template

The most common type of termination signal consists of a symmetrical inverted repeat of a GC-rich sequence followed by approximately seven A residues, that forms a stable stem-loop structure in the RNA, terminating transcription

Question 8

Types of RNA molecules: Name 5

Answer 8

rRNA, mRNA, tRNA, snRNA, siRNA, miRNA, snoRNA, piRNA, IncRNA

Question 9

PART 1

Transcription in Eukaryotic cells (Initiation): RNA polymerase II

Answer 9

RNA Polymerase II requires transcription factors (TFs) to initiate transcription

General TFs are involved in transcription from most polymerase II promoters

Additional, specialised (gene-specific) transcription factors bind to DNA sequences that control expression of individual genes and are responsible for regulating gene expression

Question 10

PART 2

Transcription in Eukaryotic cells (Initiation): RNA polymerase II

[Promotor Region Table]

Answer 10

Typical Eukaryotic promoter region:

BRE G/C G/C G/A C G C C TFIIB
TATA T A T A A/T A A/T TBP
INR C/T C/T A N T/A C/T C/T TFIID
DPE A/G G A/T C G T G TFIID
———————————————————————————-

For most RNA Polymerase II transcription start points, only 2/3 of the sequences are present

Question 11

PART 3

Transcription in Eukaryotic cells (Initiation): RNA polymerase II

Answer 11

TFIID recognises and binds to the TATA box through its TBP subunit, this enables the binding of TFIIB

The rest of the general TFs (TFIIE, TFIIH) and RNA polymerase II then assemble at the promoter

TFIIH contains DNA helicase as one of its subunits

TFIIH also phosphorylates RNA pol II –> conformational change (in CTD) –> RNA pol II released from TFs –> elongations starts

Question 12

PART 4

Transcription in Eukaryotic cells (Initiation): RNA polymerase II

Answer 12

The phosphorylated CTD the binds to additional proteins to facilitate elongation including “chromatin remodelling factors”

These then displace nucleosomes during transcription

Question 13

What type of enzymes mediate phosphorylation?

Answer 13

Kinases

Question 14

PART 5

Transcription in Eukaryotic cells (Initiation): RNA polymerase II

[Additional Protiens]

Answer 14

Refer to SLIDE 21 of Lecture 2.1 of Molecules, Genes and Diseases

Question 15

Transcription in Eukaryotic cells (Elongation): RNA polymerase II

Answer 15

RNA Polymerase elongation rate 6-70nt/sec

Makes sugar-phosphate backbone

Phosphodiester Bonds

Question 16

Transcription in Eukaryotic cells (Termination): RNA polymerase II

Answer 16

Termination less understood in eukaryotes

Involves cleavage of new transcript followed by template-independent polyadenylation at 3’ end

Question 17

Transcription in Eukaryotic cells: RNA polymerase I

Answer 17

RNA polymerase I is devoted solely to the transcription of ribosomal RNA genes

Question 18

Transcription in Eukaryotic cells: RNA polymerase III

Question 19

(pre) RNA processing

Answer 19

5’ end capping with 7-methylguanosine (m7G)- protects nascent mRNA from degradation and assists in ribosome binding

Splicing by spliceosomes- to remove introns

3’ end is modified via polyadenylation (polyA tail)- protects mRNA from degradation, aids in export of mature mRNA into cytoplasm, assists in ribosome binding

Question 20

Why is splicing important?

Answer 20

Transcripts of many eukaryotic genes (95% human ones) are spliced in more than 1 way

Alternative splicing increases diversity of proteins

It enables eukaryotes to increase the coding potential of their genomes

Question 21

What happens to mature RNA?

Answer 21

They are guided through nuclear pore complexes (NPCs)

Aqueous channels in nuclear membrane leading to cytosol

Question 22

Which DNA strand is transcribed?

Answer 22

The antisense strand is the template for mRNA

As sense strand is the coding strand that contains the true nucleotide sequence of the gene the mRNA needs