GTS 251
STUDY
UNIT 3.1
Lecture Outline
• STUDY UNIT 3.1:
TRANSCRIPTION
(1 of 3 lectures)
• STUDY MATERIAL: Pierce 7th Chapter 13 p 379-386
• CHAPTER SUMMARY: Pierce 7th p 397
✓RNA participates in a variety of Cellular
Functions
✓Transcription- the synthesis of an RNA
molecule from a DNA template
Learning outcomes
• Explain how the structure of RNA allows it to participate in a variety
of cellular functions.
• Summarize the differences in structure of RNA and DNA.
• List the different classes of RNA and their functions.
• Describe the major components required for transcription.
• Identify the parts of a typical transcription unit.
• Give the substrate for transcription and how it is used to create a
polyribonucleotide chain.
• Compare RNA polymerases of bacteria and eukaryotes.
13.1 RNA, Consisting of a Single Strand of
Ribonucleotides, Participates in a Variety of Cellular
Functions
•RNA: evidence suggests RNA was the original
genetic material
•Ribozymes: catalytic RNA
•Tetrahymena thermophila
•Enzymes replaced enzymatic function
•DNA took over as the primary carrier of genetic
information
TABLE 13.1
The structures of DNA and RNA compared
Characteristic
DNA
RNA
Composed of nucleotides
Yes
Yes
Type of sugar
Deoxyribose
Ribose
Presence of 2'–OH group
No
Yes
Bases
A, G, C, T
A, G, C, U
Nucleotides joined by
phosphodiester bonds
Double of single stranded
Yes
Yes
Usually double
Usually single
Secondary structure
Double helix
Many types
Stability
Stable
Easily degraded
13.1 RNA, Consisting of a Single Strand of
Ribonucleotides, Participates in a Variety of Cellular
Functions
TABLE 13.2
Locations and functions of different classes of RNA molecules
Class or RNA
Cell Type
Location of Function in
Eukaryotic Cells*
Function
Ribosomal RNA (rRNA) Prokaryotic
and
eukaryotic
Cytoplasm
Structural and functional
components of the
ribosome
Messenger RNA
(mRNA)
Prokaryotic
and
eukaryotic
Nucleus and
cytoplasm
Carries genetic code for
proteins
Transfer RNA (tRNA)
Prokaryotic
and
eukaryotic
Cytoplasm
Helps incorporate amino
acids into polypeptide
chain
*All eukaryotic RNAs are synthesized in the nucleus.
TABLE 13.2
Locations and functions of different classes of RNA molecules
Class or RNA
Cell Type
Location of Function
in Eukaryotic Cells* Function
Small nuclear RNA (snRNA)
Eukaryotic
Nucleus
Processing of pre-mRNA
Small nucleolar RNA
(snoRNA)
Eukaryotic
Nucleus
Processing and assembly of
rRNA
MicroRNA (miRNA)
Eukaryotic
Nucleus and
cytoplasm
Inhibits translation of mRNA
Small interfering RNA
(siRNA)
Eukaryotic
Nucleus and
cytoplasm
Triggers degradation of other
RNA molecules
Piwi-interacting RNA
(piRNA)
Eukaryotic
Nucleus and
cytoplasm
Suppresses the transcription of
transposable elements in
reproductive cells
CRISPR RNA (crRNA)
Prokaryotic
—
Assists destruction of foreign
DNA
Long noncoding RNA
(lncRNA)
Eukaryotic
Nucleus and
cytoplasm
Eukaryotic Nucleus and
cytoplasm Variety of functions
13.2 Transcription Is the Synthesis of
an RNA Molecule from a DNA Template
Transcription requires 3 major components
1. The DNA template
2. The raw materials rNTPs
3. The transcription apparatus
• Proteins needed to catalyse the synthesis of
RNA
The Template
bot
top
bot
Template
Template
Template
top
The substrate for Transcription
Ribonucleoside triphosphates—rNTPs added to the 3OH group of the growing RNA
RNAn + rNTP
RNAn+1 +PPi
The Transcription Apparatus
RNA polymerase carries out most of the steps in transcription but its action is
enhanced by a number of accessory proteins
The sigma factor guides the core RNA pol to the promoter
consensus sequence i.e. critical for the binding of the molecule
3-D model of RNA-pol in blue, the
DNA in purple and mRNA in red.
TABLE 13.3
Eukaryotic RNA polymerases
Type
Present in
Transcribes
RNA polymerase I
All eukaryotes
Large rRNAs
RNA polymerase II
All eukaryotes
Pre-mRNA, some snRNAs,
snoRNAs, some miRNAs
RNA polymerase III
All eukaryotes
tRNAs, small rRNAs, some
snRNAs, some miRNAs
RNA polymerase IV
Plants
Some siRNAs
RNA polymerase V
Plants
RNA molecules taking part in
heterochromatin formation
Summary
✓RNA participates in a variety of Cellular
Functions
✓An early RNA world
✓The structure of RNA
✓Classes of RNA
✓Transcription- the synthesis of an RNA
molecule from a DNA template
✓The Template
✓The Substrate for transcription
✓The Transcription Apparatus
GTS 251
STUDY
UNIT 3.2
Lecture Outline
• STUDY UNIT 3.2:
TRANSCRIPTION
• STUDY MATERIAL: Pierce 7th Chapter 13 p 387-392
• CHAPTER SUMMARY: Pierce 7th p 397-398
✓Bacterial Transcription
✓Initiation
✓Elongation
✓Termination
(2 of 3 lectures)
Learning outcomes
• Outline the process of bacterial transcription.
• Describe the three major stages of transcription.
• Describe consensus sequences found in a typical bacterial
promoter.
• Explain the differences in rho-dependent and rhoindependent termination.
• Summarize the general principles of bacteria transcription.
13.3 Bacterial Transcription Consists of
Initiation, Elongation, and Termination
Initiation
• Bacterial promoters:
Consensus sequences: sequences that possess considerable similarity.
• –10 consensus: 10 bp upstream of the start site
• Pribnow box:
–5 TATAAT 3
–3 ATATTA 5
• –35 consensus sequence: TTGACA
C
Initiation
• Initial RNA synthesis: no primer is required.
• The location of the consensus sequence determines
the position of the start site.
• Comprises all the steps necessary to begin RNA synth
1.
2.
3.
4.
Promoter recognition
Formation of the transcription bubble
Creation of the first bonds between rNTPs
Escape of the transcription apparatus from the
promoter
Elongation
• RNA elongation is carried out by the action of RNA polymerase.
• At the end of initiation, RNA polymerase changes its conformation
and thereafter is no longer able to bind to the consensus
sequences in the promoter.
• This change lets the polymerase escape from the promoter and
begin transcribing downstream.
Elongation
• As RNA pol moves downstream it progressively unwinds
the DNA at the leading edge of the transcription bubble;
joining nucleotides to the growing RNA molecule
according to the sequence of the template.
• RNA pol then rewinds the DNA at the trailing (upstream)
edge of the bubble.
• Transcription is slower than replication. In bacteria
40nt/s are added.
The Transcription Bubble:
• Short stretch of unwound DNA where transcription takes place
(18nt).
• RNA is continuously synthesized in the transcription bubble.
• As transcription apparatus moves down the template, it
generates positive supercoiling ahead of the transcription
bubble and negative supercoiling behind the transcription
bubble.
• Topoisomerase enzymes probably act to relieve torsional
stress due to winding and unwinding.
Transcriptional pausing:
• A number of features of RNA or DNA such as: secondary
structures, specific sequences or the presence of nucleosomes
cause the RNA polymerase to pause during the elongation
stage.
• Such pauses are often due to backtracking (when RNA pol
slides backward along the DNA template)
• Backtracking disengages the 3’-OH group of the RNA molecule
from the active site of RNA polymerase and temporarily halts
further RNA synthesis.
• Backtracking is important in transcriptional proof reading.
• Sometimes a pause may be stabilised by sequences in the DNA
that ultimately lead to the termination of transcription.
Accuracy of Transcription:
• Errors occasionally arise in transcription, RNA pol has
proofreading functionality.
• When RNA pol incorporates a nt that does not match the
DNA template, the RNA pol backtracks and cleaves the last 2
nt from the growing RNA; RNA pol will then proceed forward,
transcribing the RNA again.
Termination
• Rho-dependent termination: uses rho factor
• Rho-independent termination: hairpin structure formed by
inverted repeats, followed by a string of uracils
Rho-dependent termination
Rho-dependent termination
Rho-dependent termination
Rhoindependent
termination
Rhoindependent
termination
Summary
✓Bacterial Transcription
✓Initiation (Bacterial promoters,
Consensus seq, Abortive initiation)
✓Elongation (Transciptional bubble,
Pausing, Accuracy)
✓Termination (Rho dependent, Rhoindependent)
GTS 251
STUDY
UNIT 3.3
Lecture Outline
• STUDY UNIT 3.3:
TRANSCRIPTION
• STUDY MATERIAL: Pierce 7th Chapter 13 p 392-396
• CONCEPTS SUMMARY: Pierce 7th p 397
✓Eukaryotic Transcription
✓Initiation
✓Elongation
✓Termination
(3 of 3 lectures)
Learning outcomes
• Compare and contrast transcription in bacteria and eukaryotes.
• Describe eukaryotic promoters and how they differ from bacterial
promoters.
• Describe the process of transcription in eukaryotes.
13.4 Eukaryotic Transcription Is Similar to Bacterial
Transcription but Has Some Important Differences
• Transcription and nucleosome structure
• Chromatin modification before transcription
• Promoters
• Basal transcription apparatus
• Transcriptional activator proteins
• RNA polymerase II – mRNA synthesis
• Core promoter TATA box TATAAAA, –25 to
–30 bp, bound by transcription factors
Nucleosome
structure
• Acetyl transferases add
acetyl groups to the
amino acids
Promoters
• In eukaryotic cells, promoter recognition is carried out by accessory proteins
that bind to the promoter and then recruit a specific RNA polymerase.
• One class of accessory proteins comprises general transcription factors. GTFs
• GTFs + RNA pol form the basal transcription apparatus
• Basal transcription apparatus is a group of proteins that assemble near the
start site and is sufficient to initiate minimal levels of transcription (basal rate)
• Another class of accessory proteins consists of transcriptional activator
proteins, which bind to specific DNA sequences and bring about higher levels
of transcription, this is done by stimulating the assembly of the basal
transcription apparatus.
Initiation
• Transcription in eukaryotes is initiated through the assembly of the
transcription machinery on the promoter
• Basal transcription apparatus consists of RNA pol II, GTFs and a mediator.
• GTFs = TFIIA,B,D,E,F,H
• TFII stands for transcription factor for RNA pol II
Elongation
• After about 30bp of RNA have been synthesised , the RNA
pol II leaves the promoter and begins elongation.
• Many of the general TFs are left behind at the promoter
• The RNA pol maintains a transcription bubble during
elongation.
• The molecular structure of eukaryotic RNA polymerase II
and how it functions during elongation have been revealed
through the work of Roger Kornberg and his colleagues.
Termination
RNA pol I:
• Requires termination factor similar to the rho factor used in
bacterial genes.
• Termination factor for RNA pol I binds to a DNA sequence
downstream of terminator.
Termination
RNA pol III:
• Ends transcription after transcribing a terminator sequence
that produces a string of Uracil in the RNA.
• Secondary structures such as hairpins, often occur upstream
of the string of uracil and are necessary for termination, this is
similar to rho-independent termination in bacteria.
Termination RNA pol II
Concepts covered in SU3.3
✓Eukaryotic Transcription
✓Initiation (Different promoter consensus
seq)
✓Elongation
✓Termination (RNA pol II –Rat 1)