1. Authored by Peter J. Russell
CHAPTER 5
Gene Expression: Transcription

2. Gene Expression – an Overview
All life processes depend on the production of gene products present in the cells and tissues
Synthesis of RNA using a DNA template is termed transcription
Only one of the DNA strands (template strand) is transcribed, employing RNA polymerase
Information flow from DNA  RNA  Protein is called the Central Dogma of Molecular Biology

3. There are four major types of RNA molecules:
Messenger RNA (mRNA) encodes the amino acid sequence of a polypeptide
Transfer RNA (tRNA) shuttles amino acids to ribosomes during the translation of mRNA (protein synthesis)
Ribosomal RNA (rRNA) combines with proteins to form a ribosome, the catalyst for translation
Small nuclear RNA (snRNA) combines with proteins to form complexes used in eukaryotic RNA processing

4. Transcription Process
Transcription is controlled by regulatory elements associated with each gene
The unwinding of DNA is accomplished with the help of RNA polymerase in prokaryotes and by a combination of RNA polymerase and other proteins in eukaryotes
Transcription proceeds in the 5’  3’ direction and is catalyzed by RNA pol
RNA polymerization is similar to DNA synthesis, with some notable exceptions…
DNA replication takes place only during S phase; transcription of RNA occurs throughout the cell cycle
Precursors are NTP’s (not dNTP’s)
No primer is needed to initiate synthesis
Uracil is inserted instead of thymine
Animation: RNA Biosyntheis

5. Summary: RNA Transcription v. DNA replication
Make what from what?
DNA from DNA
RNA from DNA
# template strands 2 1
Primer needed?
Yes No
Proofreading?
Precursors
dNTPs (GATC)
NTPs (GAUC)
Main enzyme
DNA Polymerase
RNA Polymerase
Bi or uni?
Bi-directional; growing bubble
Uni-directional; moving bubble

7. Transcription in Prokaryotes
Transcription is divided into 3 steps: initiation, elongation and termination
Whereas elongation is highly conserved, initiation and termination are somewhat different between eukaryotes and prokaryotes (we will discuss prokaryotes here)
A typical prokaryotic gene has three regions:
A Promoter sequence attracts the RNA polymerase to begin transcription
The transcribed sequence is the coding sequence that is transcribed to make RNA
The terminator region specifies where transcription ends

8. Initiation of Transcription in E. coli
The initiation of transcription requires that the RNA polymerase holoenzyme (only one type in bacteria) bind to the Promoter DNA sequence
Promoters are found at -35 and -10 bp upstream from the +1 start site of transcription; their general consensus sequences are:
-35 bp consenus sequence is 5’-TTGACA-3’
-10 bp consensus sequence is 5’-TATAAT-3’ (Pribnow box)
Promoters often deviate from their consensus (in a moment…)
The RNA polymerase holoenzyme is made up of a core enzyme (2 a, 1 b and 1 b’ polypeptides) bound to a s (sigma) factor
The s factor confers promoter-specific binding (DNA sequence) to the RNA polymerase, otherwise RNA polymerase would bind to non-specific sites throughout the DNA
E. coli has more than 1 sigma factor, which can be produced in response to changing conditions, thereby regulating which promoter is recognized and which gene RNA pol will transcribe
The most common factor is s70, which recognizes the exact consensus sequences at -35 and -10 bp
Activators and Repressors are regulatory proteins that bind near the promoter to further ‘tweek’ expression (in Chapter 17)

9. The RNA polymerase holoenzyme binds to the promoter in two steps involving the s factor
First, it loosely binds to the -35 bp consensus sequence of ds DNA (closed promoter complex)
Second, it binds tightly to the -10 bp consensus sequence, untwisting the DNA at the site, beginning transcription (open promoter complex)
Figure 5.4 Action of E. coli RNA polymerase in the initiation and elongation stages of transcription. (a) In initiation, the RNA polymerase holoenzyme first binds loosely to the promoter at the -35 region. (b) As initiation continues, RNA polymerase binds more tightly to the promoter at the -10 region, accompanied by a local untwisting of about 17 bp around that region. At this point, the RNA polymerase is correctly oriented to begin transcription at +1(c) After eight to nine nucleotides have been polymerized, the sigma factor dissociates from the core enzyme. (d) As the RNA polymerase elongates the new RNA chain, the enzyme untwists the DNA ahead of it; as the double helix re-forms behind the enzyme, the RNA is displaced away from the DNA.

10. Elongation of an RNA Chain
As transcription proceeds, the s factor dissociates from the core enzyme after about 8 nucleotides are added (note the compaction of RNA pol)
As the RNA polymerase core enzyme continues to add NTPs to the growing RNA strand, the DNA helix reforms, displacing the RNA transcript
The transcription bubble moves downstream across the gene at about nucleotides per second

11. Eine kleine aufschreiben (a little note)…
RNA polymerase has proofreading activity similar to that of DNA polymerase
The enzyme moves back one or more nucleotides, excises the errant RNA and then resumes synthesis in the forward direction (termed 3’  5’ exonuclease activity)

12. Termination in Prokaryotes
Terminator sequences are used to end transcription
In prokaryotes there are two types of terminator sequences and two mechanisms involved in termination:
Rho-independent (r-independent or type I terminators)
Rho-dependent (r-dependent or type II terminators)

13. Rho-independent termination
Rho-independent (r-independent) or type I terminators (DNA) consist of an inverted C/G rich repeat sequence that is about 20 bp upstream of the transcription termination point (A rich)
RNA pol transcribes the terminator sequence, which is part of the initial RNA-coding sequence of the gene
Because of the inverted repeat arrangement (twofold symmetry), the RNA forms a hairpin loop, casing RNA pol to slow its catalysis of synthesis. The string of U nucleotides downstream of the hairpin destabilizes the DNA-RNA hybrid, facilitating the release of RNA transcript and RNA polymerase

14. Rho-Dependent Termination
Rho-dependent (r-dependent) or type II terminators are C-rich, G-poor sequences that have no hairpin structure and require the rho factor protein for termination
Rho binds to the C-rich terminator sequence in the RNA transcript (which is upstream of the termination site) and moves along the transcript until encountering the stalled RNA polymerase (which is downstream of Rho)
It then acts as a helicase, destabilizing the RNA-DNA hybrid at the termination region, thereby terminating transcription
This process is dependent on ATP hydrolysis

15. Transcription in Eukaryotes
Prokaryotes contain only one RNA polymerase, which transcribes all types of RNA for the cell
RNA polymerase is multi-subunit protein in prokaryotes and eukaryotes
Eukaryotes have three different polymerases, each transcribing a different class of RNA:
RNA polymerase I, located in the nucleolus, transcribes the three major rRNAs (28S, 18S, and 5.8S).
RNA polymerase II, located in the nucleoplasm, transcribes mRNAs and some snRNAs.
RNA polymerase III, located in the nucleoplasm, transcribes the tRNAs, 5S rRNA and and additional snRNAs
R,M,T – 1,2,3…

16. Transcription of mRNA by RNA Polymerase II
Transcription begins at the promoter; there are two types of promoter elements in eukaryotes:
Core promoter elements are located near the transcription start site and specify the origin of transcription. Examples include…
The initiator element (Inr) spans the transcription start site (+1)
The TATA box (Goldberg-Hogness box) aids in local DNA denaturation. Its full sequence is TATAAAA (-30)
Proximal promoter elements are required for high levels of transcription. They are further upstream from the start site (at positions between -50 and -200)
The CAAT box is located at about -75
The GC box is located at about -90 (consensus sequence 5’-GGGCGG-3’ )

17. Activators are recognized by proximal promoter elements and determine the efficiency of transcriptional initiation
Housekeeping genes (used in all cells for basic function) have common proximal promoter elements that are recognized by activator proteins found in all cells
Genes expressed only in some cell types or at particular times have proximal promoter elements recognized by activator proteins found only in specific cell types or times

18. Enhancers are another cis-acting element required for the maximal transcription of a gene
Enhancers are usually far upstream (1000’s of bp) of the transcription initiation site but may also be downstream
Enhancers contain short sequence elements, some similar to promoter sequences
Activators bind to these sequences and other protein complexes form, bringing the enhancer complex close to the promoter, thereby increasing transcription

19. Transcription Initiation in Eukaryotes
In eukaryotes, RNA polymerases cannot bind to the promoter on its own; it needs the binding of general transcription factors (GTF’s) on the core promoter
TFIID is a multisubunit protein composed of the TATA-binding protein (TBP) and TBP-associated factors (TAFs). TFIID binds to the TATA box forming the initial committed complex
Sequentially, other factors are recruited, as is RNA polymerase II, to progress through the minimal and complete transcription initiation complexes
TFIIH acts like a helicase…
Figure 5.7 Assembly of the transcription initiation machinery. First, TFIID binds to the TATA box to form the initial committed complex. The multisubunit TFIID has one subunit called the TATA-binding protein (TBP), which recognizes the TATA box sequence, and a number of other proteins called TBP-associated factors (TAFs). In vitro, the TFIID–TATA box complex acts as a binding site for the sequential addition of other transcription factors. Initially, TFIIA and then TFIIB bind, followed by RNA polymerase II and TFIIF, to produce the minimal transcription initiation complex. (RNA polymerase II, like all eukaryotic RNA polymerases, cannot directly recognize and bind to promoter elements.) Next, TFIIE and TFIIH bind to produce the complete transcription initiation complex, also called the preinitiation complex (PIC). TFIIH’s helicase-like activity now unwinds the promoter DNA, and transcription is ready to begin.

20. Structure and Production of Eukaryotic mRNA
Mature mRNA has a 5’ untranslated region (called the leader sequence), a protein coding sequence and a 3’ untranslated region (called the trailer sequence)
Eukaryotes and prokaryotes produce mRNA differently
Animation: mRNA Production in Eukaryotes

21. In prokaryotes, polycistronic transcription and translation are coupled processes taking place in the cytoplasm (there are no organelles in prokaryotes)

22. In eukaryotes, monocistronic transcription and processing of precursor mRNA takes place in the nucleus, producing mature mRNA that is exported to the cytoplasm.
The processes of transcription, processing and export are coupled (continuous)
Processing includes:
The addition of a 5’ cap
The addition of a 3’ tail
The removal of introns.

23. 5’ Methyl Guanosine Cap Modification
The newly made 5’ end of the mRNA is modified by a 5’ cap. A capping enzyme adds a guanine, usually 7-methyl guanosine (m7G), to the 5’ end using a 5’-to-5’ linkage. Sugars of the two adjacent nucleotides are also methylated
Functionally, the cap:
Protects the mRNA from degradation
Facilitates ribosome binding to mRNA during the initiation of translation
Figure 5.10 Cap structure at the end of a eukaryotic mRNA. The cap results from the addition of a guanine nucleotide and two methyl groups.

24. 3’ poly-A Tail Modification
~200 adenine nucleotides are added to the 3’ end of mRNA in 2 steps…
Transcription continues through the poly(A) consensus sequence (AAUAAA). CPSF (cleavage and polyadenylation specificity factor) CstF (cleavage stimulation factor), and CFI and CFII (cleavage factor proteins) bind and cleave the mRNA
Poly-A polymerase (PAP) then adds the poly-A tail on mRNA, to which poly-A binding proteins (PABII) attach
RNA pol II stops transcription by an unclear mechanism (5’3’ exonuclease on mRNA?)
Functionally, the tail:
Protects the mRNA from degradation
Facilitates export of the mRNA from the nucleus
Figure 5.11 Schematic diagram of the end formation of mRNA and the addition of the poly(A) tail to that end in mammals. In eukaryotes, the formation of the end of an mRNA is produced by cleavage of the lengthening RNA chain. CPSF binds to the AAUAAA signal, and CstF binds to a GU-rich or U-rich sequence (GU/U) downstream of the poly(A) site. CPSF and CstF also bind to each other, producing a loop in the RNA. CFI and CFII bind to the RNA and cleave it. Poly(A) polymerase then adds the poly(A) tail to which poly(A) binding proteins attach.

25. Introns and splicing in Eukaryotic pre-mRNA
Eukaryotic pre-mRNAs have intervening (intron) sequences between the expressed (exon) sequences. Spliceosomes remove the introns during RNA processing
Spliceosomes are small nuclear ribonucleoprotein particles (snRNPs are RNA and protein) associated with pre-mRNA
Animation: RNA splicing

26. Spliceosome model summarized...
The 5’ end of an intron is typically GU; the 3’ end is typically AG. Near the end of the intron is an A nucleotide located within the branch-point sequence, which, in mammals, is YNCURAY, where Y=pyrimidine, N=any base, R=purine, A=adenine. In yeast, the sequence is UACUAAC. (The italicized A in each sequence is where the 5’ end of the intron bonds).
With the aid of snRNPs, intron removal begins with a cleavage at the first exon–intron junction. The G at the released 5’ of the intron folds back and forms an unusual 2’-5’ bond with the A of the branch-point sequence. This reaction produces a lariat-shaped intermediate. Cleavage at the 3’ intron–exon junction and ligation of the two exons completes the removal of the intron.
Figure 5.13 Model for intron removal by the spliceosome. At the end 5’ of an intron is the sequence GU, and at the 3’ end is the sequence AG. Near the end of the intron is an A nucleotide located within the branch-point sequence, which, in mammals, is YNCURAY, where Y=pyrimidine, N=any base, R=purine, A=adenine, and, in yeast, is UACUAAC. (The italic A in each sequence is where the 5’ end of the intron bonds). With the aid of snRNPs, intron removal begins with a cleavage at the first exon–intron junction. The G at the released 5’ of the intron folds back and forms an unusual 2’-5’ bond with the A of the branch-point sequence. This reaction produces a lariat-shaped intermediate. Cleavage at the 3’ intron–exon junction and ligation of the two exons completes the removal of the intron.

27. so why have introns, you ask…?
There can be mixing and matching of similar introns between 2 different genes, thereby increasing beneficial crossover events, creating novel genes
Alternate splicing of introns takes place in different tissues (cells), producing different gene products and proteins which possess optimal functions in that tissue (cells)
Thus, 1 gene > many polypeptides

28. Alternative Splicing: Many Transcripts from One Gene

29. Self-splicing Introns
Most rRNA genes nave no introns
Tetrahymena has an intron interrupting its 28S rRNA sequence
The intron self-splices by folding into a secondary structure that catalyzes its own excision (no proteins are involved in Group I excision!)
Such RNAs with enzyme-like activity are called ribozymes (not classically an enzyme since the catalyst is not in its original form)
Group I introns are also found in nuclear rRNA, mitrochondrial mRNA and tRNA in phages
Group II introns use a different molecular mechanism for self-splicing
Lead to “RNA world” hypothesis of the origin of life
Figure 5.14 Self-splicing reaction for the group I intron in Tetrahymena pre-rRNA. Cleavage at the splice junction takes place first, accompanied by the addition of a G nucleotide to the end of the intron. Cleavage at the splice junction releases the intron; the two exons are spliced. The intron now circularizes by the nucleotide bonding to an internal nucleotide, producing a lariat-shaped molecule. Cleavage of the lariat molecule produces a circular RNA molecule and a linear molecule.

30. RNA editing
RNA editing adds or deletes nucleotides from a pre-mRNA, or chemically alters the bases, resulting in an mRNA with bases that don’t match its DNA coding sequence.
In Trypanosome brucei, the cytochrome oxidase subunit III gene (from mitochondrial DNA) does not match its mRNA. Uracil residues have been added and removed, and over 50% of the mature mRNA consists of posttranscriptionally added Uracil.

32. Transcription of Other Genes
Genes for rRNA, tRNA, and snRNA
Ribosomal RNA and Ribosomes:
Ribosomes are sites for protein synthesis.
Ribosomes are made up of 2 unequal subunits.
Prokaryotic ribosomes are 70S in size: 50s and 30s
Eukaryotic ribosomes are 80s in size: 60s and 40s
Transcription of rRNA genes
DNA regions that encode rRNA are called ribosomal DNA (rDNA) or rRNA transcription units

33. Ribosome made up of 2 subunits
Figure 5.18 rRNA genes and rRNA production in E. coli. (a) General organization of an E. coli rRNA transcription unit (an rrn region). (b) The scheme for the synthesis and processing of a precursor rRNA (p30S) to the mature 16S, 23S, and 5S rRNAs of E. coli. (The tRNAs have been omitted from this depiction of the processing scheme.)
Transcription of rRNA genes

34. Transcription of tRNA
All tRNAs can be shown in a cloverleaf structure, with complementary base pairing between regions to form four stems and loops (Figure 5.20). Loop II contains the anticodon used to recognize mRNA codons during translation. Folded tRNAs resemble an upside-down “L.”
All tRNAs have CCA (added post-transcriptionally) at their 3’ ends
Figure 5.20 Transfer RNA. (c) Photograph of a space-filling molecular model of yeast phenylalanine tRNA. The CCA end of the molecule to which the amino acid attaches is at the upper right, and the anticodon loop is at the bottom.