Transcription Flashcards
1
Q
Define transcription
A
The process by which cellular DNA directs the synthesis of a complementary strand of RNA which reflects the genetic information encoded within the DNA
2
Q
Three types of RNA
A
Messenger RNA (mRNA) Ribosomal RNA (rRNA) Transfer RNA (tRNA)
3
Q
How is transcription carried out?
A
The transcription of DNA to RNA is carried out by RNA polymerases
4
Q
Similarities between RNA and DNA polymerase
A
LIKE DNA polymerase, RNA polymerase:
- Uses nucleoside 5’-triphosphates as precursors
- Catalyses phosphodiester bond formation between NTPs
- Uses DNA as template
- Base-pairing interaction determine corrects base
- 5’ → 3’ growth of new nucleic acid chain
- Growing strand is the reverse complement to template
5
Q
Differences between RNA and DNA polymerase
A
UNLIKE DNA polymerase, RNA polymerase:
- Uses RIBOnucleoside 5’-triphosphates (ATP, GTP, CTP, UTP)
- Can initiate de novo synthesis of a new strand (no primer needed)
- A single strand of RNA is produced (only one strand of DNA is used at a time for RNA synthesis)
- Only short stretches of DNA are transcribed
6
Q
Transcription in Bacteria vs Eukaryotes
A
- All cells contain RNA polymerase
- In bacteria, one RNA polymerase synthesises all of the cell’s RNA
(except for the short RNA primers used in DNA replication) - In eukaryotic cells, there are around five different RNA polymerases. Each synthesises a different class of RNA
- The process of transcription in bacterial cells is less complex
7
Q
Structure of E. coli RNA Pol
A
E. coli RNA polymerase, a large 449 kD protein complex, consists of:
- A core tetramer of subunits (involved in DNA template-binding and phosphodiester bond formation)
- A sigma subunit that only associates with the core enzyme for its critical role in transcription initiation
8
Q
Four Main Steps of Transcription
A
- Binding of RNA polymerase to the specific site where a gene starts (called the promoter)
- Initiation of transcription at the start site
- Elongation in the 5’ → 3’ direction
- Termination of transcription
9
Q
Start of Transcription
A
- RNA synthesis is initiated at specific sites on the DNA
- The DNA strand that serves as the template is known as the antisense or non-coding strand
- The other DNA strand is known as the sense or coding strand and has the same sequences as the transcribed RNA (except for the replacement of U with T)
10
Q
Step 1 - RNA Pol Binds to Promotors
A
- RNA polymerase binds to transcription initiation sites known as promoters
- Specific promoters are recognised by the corresponding sigma factor
- E. coli promoters are approximately 40 bp in length and are located on the 5’ side of the transcription start site
11
Q
Step 2 - Promotor Recognition and Transcription Initiation
A
- The complete RNA polymerase holoenzyme (core tetramer plus sigma) binds to non-promoter DNA LOOSELY, allowing the enzyme to move rapidly along the DNA in search of the sigma subunit’s corresponding promoter
- At the promoter, the core enzyme binds TIGHTLY and the sigma factor dissociates from the core RNA polymerase which carries on with elongation in the 5’ → 3’ direction
- Note that different sigma factors recognise different promoters
12
Q
Step 3 - Elongation: RNA Chain Growth
A
- As the polymerase advances, the DNA unwinds ahead of the RNA’s growing 3’ end and rewinds behind it
- In the region being transcribed, the DNA double helix is always unwound by ~ 17 bp, forming a “transcription bubble”
13
Q
Step 4 - Termination of Transcription
A
- In many E. coli genes, the transcription termination signal is an inverted repeat of GC-rich sequence followed by 4 or more adenines
- The transcribed RNA forms a self-complementary stem-loop “hairpin” structure that is terminated by several U residues
- Weak hydrogen bonding at site of transcription results in RNA being released from DNA template and from the enzyme
14
Q
Transcription in Eukaryotes
A
- The fundamental principles of transcription are similar in bacteria and eukaryotes
- However, eukaryotic transcription is distinguished by having multiple RNA polymerases and complicated control sequences
- The eukaryotic transcription machinery is also far more complex
15
Q
Eukaryotes have several RNA Polymerases
A
Eukaryotic nuclei contain three distinct types of RNA polymerase:
- RNA Polymerase I
- Located in nucleoli (dark straining nuclear bodies where ribosomes are assembled)
- Synthesise most rRNA precursors - RNA Polymerase II
- Located in nucleoplasm
- Synthesises mRNA precursors - RNA Polymerase III
- Located in nucleoplasm
- Synthesises 5s rRNA precursors, tRNAs and others
Note that eukaryotic cells also contain separate mitochondrial and chloroplast RNA polymerases
16
Q
Different polymerases recognise different types of promotors
A
- In eukaryotes the RNA polymerase complex does not include a removable sigma factor
- Instead, a number of accessory proteins (e.g. transcription factors) identify promoters and recruit RNA polymerase to the transcription site
- Eukaryotic promoters are more complex and diverse than bacterial promoters
- The three main eukaryotic RNA polymerases recognise different types of promoters
17
Q
RNA Pol II
A
RNA Polymerase II synthesises mRNA precursors for both constitutively and non-constitutively expressed genes
- Constitutively transcribed structural genes are expressed in all tissues
- Selectively transcribed structural genes are expressed in fewer cell types
18
Q
Sequence/Promotor Elements in Typical Eukaryotic Gene
A
TATA box (TATAAAA)
- Located approximately 25-30 bp upstream of the +1 start site
- Determined the exact start site (not in all promoters)
- Binds the TATA binding protein (TBP) which is a subunit of TFIID
GC box (CCGCCC) - Binds Sp1 (specificity factor 1)
CAAT box (GGCCAATCT) - Binds CTF (CAAT box transcription factor)
19
Q
Other Regulatory Elements
A
- Other regulatory elements of RNA polymerase II act to determine the frequency or efficiency of transcription
- Can be located many bp UPSTREAM or DOWNSTREAM of start site or even within genes
- Such transcription elements include:
- Enhancers (increase transcription rate)
- Silencers (decrease transcription rate)
- Response Elements (target sequences for signalling molecules)
- Regulatory proteins (activators or repressors) may bind to these elements
20
Q
Post-Transcriptional RNA Processing
A
- Eukaryotes lack precise transcription termination sites
- This is largely due to the fact that most primary transcripts undergo various post-transcriptional modification processes in order to acquire biological activity
- The three major classes of RNA are altered in different ways:
- mRNA undergoes 5’ capping, 3’ polyadenylation and splicing events
- rRNA undergoes endonucleolytic cleaving, methylation and splicing
- tRNA undergoes nucleotide removal, addition and modification
21
Q
mRNA Processing
A
- 5’ capping
- 3’ Polyadenylation
- Splicing
22
Q
mRNA Processing - 5’ Capping
A
- Eukaryotic mRNAs have a cap structure consisting of 7-methylguanosine residue joined to the transcripts initial 5’ nucleotide via a 5’ - 5’ triphosphate bridge
- Capping involves several enzymatic reactions
- The cap is added to the growing transcript when it is ~ 30 nucleotides long
- The cap identifies the eukaryotic translation start site
- The cap also protects the polynucleotide from degradation by 5’ exonucleases
23
Q
mRNA Processing - 3’ Polyadenylation
A
- Mature eukaryotic mRNAs have well defined 3’ ends terminating in poly(A) tails of ~250 nucleotides
- The poly(A) tail is generated from ATP through the action of poly(A) polymerase
- Multiple copies of a poly(A) binding protein associate with the tail and help protect the 3’ end of the mRNA transcript from 3’ exonucleases
- The poly(A) tail may also serves as a handle for the proteins that deliver mRNA to ribosomes
24
Q
mRNA Processing - Splicing
A
- A major difference between the structural genes of eukaryotes and bacteria is that the coding sequences of most eukaryotic genes are interspersed with unexpressed regions
- The primary transcripts are also called pre-mRNAs or heterogeneous nuclear RNAs (hnRNAs)
- Pre-mRNAs are processed by the excision of non-expressed intervening sequences (introns), following which the flanking expressed sequences (exons are spliced (joined together))
