1. GENE TRANSCRIPTION AND RNA MODIFICATION
CHAPTER 12
GENE TRANSCRIPTION AND RNA MODIFICATION

3. Anatomy of a gene
A typical protein-encoding gene can be divided into the following elements:
Transcriptional control sequences: bind transcription factors that can activate or inhibit transcription
Promoter: recruits RNA polymerase and marks the start of the transcribed region
Transcript: corresponds to the RNA sequence Can be further subdivided into:
5' untranslated region (5'UTR): before open reading frame – often has sequences that control translation
open reading frame (ORF): sequence translated into polypeptide
3' untranslated region (3'UTR): after ORF; sometime has sequences that control translation
Termination sequences: required to terminate transcription

4. Prokaryotic transcriptional regulation
Transcription factor (TF): Any protein that initiates or regulates transcription
In Prokaryotes: promoters are recognised by the basal transcription apparatus
This is the complex of TFs that assemble to recruit RNA polymerase
A protein called σ factor is required for promoter binding
The exchange of different σ factors and the re-targeting of RNA pol to a different promoter is the basis of selective gene expression in prokaryotes
Basal apparatus + RNA pol holoenzyme = Inititation complex

5. Initiation
RNA polymerase binds to promoter & opens helix
B. Polymerisation
De novo synthesis using rNTPs as substrate
Chain elongation in 5’-3’ direction
C. Termination stops at termination signal

6. Stages in the process Initiation: Closed promoter complex
RNA pol binds the promoter.
Open promoter complex
The DNA is unwound (about turn of the helix from the -10 region to +2/+3) to allow one strand to act as template.
Initiation
2 NTPs are joined together:
N1TP + N2TP –> pppN1-p-N2 + PPi
This stage is often thought to be complete after polymerization of 8-9 nucleotides. This is called promoter clearance: the  subunit dissociates and RNA pol leaves the promoter.
Elongation commences

9. Stages in the process Elongation:
The core polymerase continues using the DNA template strand as a template and adds to the growing RNA strand.
Behind the polymerization site is an RNA-DNA duplex that is only ~1 turn of helix (8-9bp); the other DNA strand will reform a DNA duplex with the template strand after that, displacing the RNA.
A total of ~17 nt of the coding strand is displaced by the "transcription bubble."
The transcription bubble will be preceded by positive supercoils and followed by negative supercoils. Both are relieved by topoisomerases.
RNA pol moves at a rate of ~50-90 nt/sec.

10. Stages in the process Termination:
RNA pol is removed from the gene (otherwise the entire genome would be transcribed).
This usually requires a secondary structure in the sequence of the 3'UTR (most often a stem-loop) and
Rho termination factor (-dependent)
a poly-U sequence (-independent)

11. 12.3 TRANSCRIPTION IN EUKARYOTES
Many of the basic features of gene transcription are very similar in bacteria and eukaryotes
However, gene transcription in eukaryotes is more complex
Larger organisms
Cellular complexity
Multicellularity

12. Eukaryotic RNA Polymerases
Nuclear DNA is transcribed by three different RNA polymerases
RNA pol I
Transcribes all rRNA genes (except for the 5S rRNA)
RNA pol II
Transcribes all structural genes
Thus, synthesizes all mRNAs
Transcribes some snRNA genes
RNA pol III
Transcribes all tRNA genes
And the 5S rRNA gene

13. Genes are first transcribed, and then translated.
Transcription
The first step in gene expression is the production of an RNA copy of the DNA sequence encoding the gene, a process called transcription.
To understand the mechanism behind the transcription process, it is useful to focus first on RNA polymerase, the remarkable enzyme responsible for carrying it out.

14. RNA Polymerase RNA polymerase is best understood in bacteria.
Bacterial RNA polymerase is very large and complex, consisting of five subunits:
Two α subunits bind regulatory proteins.
β′ subunit binds the DNA template
β subunit binds RNA nucleoside subunits.
σ subunit recognizes the promoter and initiates synthesis.

15. Only one of the two strands of DNA, called the template strand, is transcribed.
The strand of DNA that is not transcribed is called the coding strand.
The polymerase adds ribonucleotides to the growing 3′ end of an RNA chain.
Bacteria contain only one RNA polymerase enzyme, while eukaryotes have three different RNA polymerases:
RNA polymerase I: synthesizes rRNA in the nucleolus.
RNA polymerase II: synthesizes mRNA.
RNA polymerase III: synthesizes tRNA.

17. Promoter
Transcription starts at RNA polymerase binding sites called promoters on the DNA template strand.
A promoter is a short sequence that is not itself transcribed by the polymerase that binds to it.
Promoters differ widely in efficiency.
Strong promoters cause frequent initiations of transcription, as often as every 2 seconds in some bacteria.
Weak promoters may transcribe only once every 10 minutes.

18. Initiation
The binding of RNA polymerase to the promoter is the first step in gene transcription.
In bacteria, a subunit of RNA polymerase called σ (sigma) recognizes the –10 sequence in the promoter and binds RNA polymerase there.
Importantly, this subunit can detect the –10 sequence without unwinding the DNA double helix.

19. In eukaryotes, the –25 sequence plays a similar role in initiating transcription, as it is the binding site for a key protein factor.
Other eukaryotic factors then bind one after another, assembling a large and complicated transcription complex.
The eukaryotic transcription complex is described in detail in the following chapter. Once bound to the promoter, the RNA polymerase begins to unwind the DNA helix.

21. Elongation Unlike DNA synthesis, a primer is not required.
The region containing the RNA polymerase, DNA, and growing RNA transcript is called the transcription bubble because it contains a locally unwound “bubble” of DNA.

22. The transcription bubble moves down the DNA at a constant rate, about 50 nucleotides per second, leaving the growing RNA strand protruding from the bubble.
After the transcription bubble passes, the now transcribed DNA is rewound as it leaves the bubble.
Unlike DNA polymerase, RNA polymerase has no proofreading capability.
Transcription thus produces many more copying errors than replication.
These mistakes, however, are not transmitted to progeny.
Most genes are transcribed many times, so a few faulty copies are not harmful.

23. Termination
At the end of a gene are “stop” sequences that cause the formation of phosphodiester bonds to cease the RNA polymerase to release the DNA, and the DNA within the transcription bubble to rewind.
The simplest stop signal is a series of GC base-pairs followed by a series of AT base-pairs.
The RNA transcript of this stop region forms a GC hairpin followed by four or more U ribonucleotides.
How does this structure terminate transcription? The hairpin causes the RNA polymerase to pause immediately after the polymerase has synthesized it, placing the polymerase directly over the run of four uracils.

24. The pairing of U with DNA’s A is the weakest of the four hybrid base-pairs and is not strong enough to hold the hybrid strands together during the long pause.
Instead, the RNA strand dissociates from the DNA within the transcription bubble, and transcription stops.
A variety of protein factors aid hairpin loops in terminating transcription of particular genes.

25. 12.4 RNA MODIFICATION
Analysis of bacterial genes in the 1960s and 1970 revealed the following:
The sequence of DNA in the coding strand corresponds to the sequence of nucleotides in the mRNA
This in turn corresponds to the sequence of amino acid in the polypeptide
This is termed the colinearity of gene expression
Analysis of eukaryotic structural genes in the late 1970s revealed that they are not always colinear with their functional mRNAs

26. 12.4 RNA MODIFICATION
Instead, coding sequences, called exons, are interrupted by intervening sequences or introns
Transcription produces the entire gene product
Introns are later removed or excised
Exons are connected together or spliced
This phenomenon is termed RNA splicing
It is a common genetic phenomenon in eukaryotes
Occurs occasionally in bacteria as well

27. RNA MODIFICATION
Aside from splicing, RNA transcripts can be modified in several ways
For example
Trimming of rRNA and tRNA transcripts
5’ Capping and 3’ polyA tailing of mRNA transcripts
See Next Figure….

29. Trimming
Many nonstructural genes are initially transcribed as a large RNA
This large RNA transcript is enzymatically cleaved into smaller functional pieces
Figure shows the processing of mammalian ribosomal RNA

30. Figure 12.14 RNA poly I 45s rRNA Enzymatic cleavage
This processing occurs in the nucleolus
Functional RNAs that are key in ribosome structure
Figure 12.14

31. Splicing Three different splicing mechanisms have been identified
Group I intron splicing
Group II intron splicing
Spliceosome
All three cases involve
Removal of the intron RNA
Linkage of the exon RNA by a phosphodiester bond

32. Splicing among group I and II introns is termed self-splicing
Splicing does not require the aid of enzymes
Instead the RNA itself functions as its own ribozyme
Group I and II differ in the way that the intron is removed and the exons reconnected
Refer to Figure 12.18
Group I and II self-splicing can occur in vitro without the additional proteins
However, in vivo, proteins known as maturases often enhance the rate of splicing

33. Figure 12.18
12-61

34. In eukaryotes, the transcription of structural genes, produces a long transcript known as pre-mRNA
Also as heterogeneous nuclear RNA (hnRNA)
This RNA is altered by splicing and other modifications, before it leaves the nucleus
Splicing in this case requires the aid of a multicomponent structure known as the spliceosome
Figure 12.16

35. Capping
Most mature mRNAs have a 7-methyl guanosine covalently attached at their 5’ end
This event is known as capping
Capping occurs as the pre-mRNA is being synthesized by RNA pol II
Usually when the transcript is only 20 to 25 bases long
As shown in Figure 12.19, capping is a three-step process

36. Capping
The 7-methylguanosine cap structure is recognized by cap-binding proteins
Cap-binding proteins play roles in the
Movement of some RNAs into the cytoplasm
Early stages of translation
Splicing of introns

37. Tailing
Most mature mRNAs have a string of adenine nucleotides at their 3’ ends
This is termed the polyA tail
The polyA tail is not encoded in the gene sequence
It is added enzymatically after the gene is completely transcribed
The attachment of the polyA tail is shown in Figure 12.20

38. Figure 12.20 Consensus sequence in higher eukaryotes
From a few dozen adenines to several hundred
Appears to be important in the stability of mRNA and the translation of the polypeptide
Length varies between species