1. Chapter 11 Transcription and RNA Processing
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2. Chapter Outline Transfer of Genetic Information: The Central Dogma
The Process of Gene Expression
Transcription in Prokaryotes
Transcription and RNA Processing in Eukaryotes
Interrupted Genes in Eukaryotes: Exons and Introns
Removal of Intron Sequences by RNA Splicing
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3. Transfer of Genetic Information:
The Central Dogma

4. Transfer of Genetic Information:The Central Dogma
The central dogma of biology is that information stored in DNA is transferred to RNA molecules during transcription and to proteins during translation.
DNA RNA proteins
Genotyping Phenotyping
RNA DNA/RNA proteins
virus
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5. The Central Dogma Transcription (U-A bp) --one strand of DNA to one
complementary strand of RNA
--Uracil replaces Thymine
(U-A bp)
--primary transcripts
messenger RNA (mRNA)
-----chemical modification
(splice=remove introns)
(spliceosomes)
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6. The Central Dogma Translation --RNA nucleotide into amino acids
UUU--Phe ( F) phenylalanine(*)
--genetics code (codons)
--Ribosomes (ribonucleoproteins)
(*) sense codon
Non sense codon=stop codon
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7. Transcription and Translation in Prokaryotes
The primary transcript is equivalent to the mRNA molecule.
The mRNA codons on the mRNA are translated into an amino acid sequence by the ribosomes.
Retroviruses
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8. Transcription and Translation in Eukaryotes
The primary transcript (pre- mRNA) is a precursor to the mRNA.
The pre-mRNA is modified at both ends, and introns are removed to produce the mRNA.
After processing, the mRNA is exported to the cytoplasm for translation by ribosomes. 3
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9. Types of RNA Molecules
Messenger RNAs (mRNAs)—intermediates that carry genetic information from DNA to the ribosomes.
Transfer RNAs (tRNAs)—adaptors between amino acids and the codons in mRNA.
Ribosomal RNAs (rRNAs)—structural and catalytic components of ribosomes.
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10. Types of RNA Molecules
Small nuclear RNAs (snRNAs)—s tructural components of spliceosomes.
Micro RNAs (miRNAs)—short single-stranded RNAs (20 to 22 bp) that block expression of complementary mRNAs.
RNAi is similar to miRNA (RNA interference, double strand RNA, plant) siRNA (small interference)
piRNA (Piwi-interacting RNA) is a small non-coding RNA molecules
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11. The Central Dogma
The central dogma of molecular biology is that genetic information flows
-----from DNA to DNA during chromosome replication,
----from DNA to RNA during transcription,
----from RNA to protein during translation.
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12. Transcription involves the synthesis of a single strand RNA transcript complementary to one strand of DNA of a gene.
Translation is the conversion of information stored in the sequence of nucleotides in the RNA transcript into the sequence of amino acids in the polypeptide gene product, according to the specifications of the genetic code.
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13. © John Wiley & Sons, Inc.

14. The Process of Gene Expression

15. The Process of Gene Expression
For non-viral proteins…
Information stored in the nucleotide sequences of genes is translated into the amino acid sequences of proteins through unstable intermediaries called messenger (m)RNAs.
Synthesis of viral proteins… in infected bacteria involved an unstable RNA molecule synthesized from the viral DNA.
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16. RNA synthesis 1-Single strand of DNA (template strand)
2-ribonuleoside triphosphate (NTP)
3-no pre-existing primers (de novo)
template strand=transcribed
Protein
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17. --3’ hydroxyl group of the RNA strand
Nucleophilic attacks
--3’ hydroxyl group of the RNA strand
--nucleotidyl phosphorus on the
nucleoside triphosphate
“nucleotide,
nucleoside monophosphate”
“RNA polymerase”
“Transcriptional factors”
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18. The Transcription Bubble
Prokaryotes:
--RNA polymerase binds specific
nucleotide sequences (promoter regions) plus transcriptional factors
--Single RNA polymerase
--DNA unwinding (AT regions)
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19. The Transcription Bubble
Eukaryotes:
--several RNA polymerases
--no direct recognition binding
--transcriptional factors
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20. General Features of RNA Synthesis
Similar to DNA Synthesis except
The precursors are ribonucleoside triphosphates.
Only one strand of DNA is used as a template.
RNA chains can be initiated de novo (no primer required).
The RNA molecule will be complementary to the DNA template (antisense) strand and identical (except that uridine replaces thymidine) to the DNA non-template (sense) strand.
RNA synthesis is catalyzed by RNA polymerases and proceeds in the 5’3’ direction.
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21. In eukaryotes, genes are present in the nucleus, whereas polypeptides are synthesized in the cytoplasm.
Messenger RNA molecules function as non-stable intermediaries that carry genetic information from DNA to the ribosomes, where proteins are synthesized.
RNA synthesis, catalyzed by RNA polymerases, is similar to DNA synthesis in many respects.
RNA synthesis occurs within a localized region of strand separation (Transcription Bubble), and only one strand of DNA functions as a template for RNA synthesis.
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22. RNA synthesis, catalyzed by RNA polymerases, is similar to DNA synthesis in many respects.
Prokaryotic:
OriC (245 bp)
AT-rich region (replication bubble)
Eukaryotic:
ARS (Autonomously Replicating Sequences)
AT-rich region 11 bp
T O T
cDNA
5’ ’
5’ ’ 22

23. 23

24. Transcription in Prokaryotes
---The first step in gene expression
---Transfers the genetic information stored in DNA (genes) into messenger (m)RNA molecules that
---Carry the information to the sites of protein synthesis in the cytoplasm.
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25. Stages of Transcription
--DNA dependent RNA polymerase
--5’ to 3’ direction
--Walk (literally) on the DNA
--Upstream and downstream
regions
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26. E. Coli RNA Polymerase Tetrameric core: 2 ’ Holoenzyme: 2 ’ 
(480,000 Daltons; bp~650 Daltons)
Functions of the subunits:
: assembly of the tetrameric core
: ribonucleoside triphosphate binding site
’: DNA template binding region
(sigma factor): initiation of transcription (*)
(*) in vivo
In vitro: RNA polymerase works…just fine on both DNA strands

27. Initiation of RNA Chains
Binding of RNA polymerase holoenzyme to a promoter region in DNA ( promoter region)
Localized unwinding of the two strands of DNA by RNA polymerase to provide a single-stranded template
Formation of phosphodiester bonds between the first few ribonucleotides in the nascent RNA chain
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28. A Typical E. coli Promoter
..,-2,-1,+1,+2,..
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29. Numbering of a Transcription Unit
The transcript initiation site is +1 (A/T).
Bases preceding the initiation site are given minus (–) prefixes and are referred to as upstream sequences.
Bases following the initiation site are given plus (+) prefixes and are referred to as downstream sequences.
Consensus sequences: highly conserved
Recognition sequences: Sigma factor (
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30. Elongation Sigma factor needs to be released
---Re- and Un-winding activities
-- Walk (literally) on the DNA
5’ to 3’
--growing RNA chain
RNA polymerase binds both
DNA template and growing RNA chain
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31. Termination Signals in E. coli
Rho-dependent terminators—require a protein factor ()
Rho-independent terminators—do not require 
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32. Rho-independent terminators—do not require  intrinsic termination)
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33. RNA transcription stops --when the newly synthesized RNA
molecule forms a G-C-rich hairpin loop
followed by a run of As
--Create a mechanical stress
--Pulls the poly-U transcript out of the
active site of the RNA polymerase
--A-U has very weak interaction
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34. Termination Signals in E. coli
Rho-dependent terminators (non-intrinsic) — require a protein factor () and rut site
Rut proteins bind specific RNA sequences (>>Cs and <<<Gs)
Not hairpins or other secondary Structures
Rho utilization (rut)
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37. Coupled Transcription and Translation in E. coli
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39. stop (aka "nonsense") codon

40. Rho factor binds to specific regions in naked RNA

41. RNA synthesis occurs in three stages: (1) initiation, (2) elongation, and (3) termination.
RNA polymerases—the enzymes that catalyze transcription—are complex multimeric proteins.
The covalent extension of RNA chains occurs within locally unwound segments of DNA.
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42. Chain elongation stops when RNA polymerase encounters a transcription-termination signal.
Transcription, translocation, and degradation of mRNA molecules often occur simultaneously in prokaryotes.
RNAase
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43. Transcription and RNA Processing in Eukaryotes
Five different enzymes catalyze transcription in eukaryotes, and the resulting RNA transcripts undergo three important modifications, including the excision of noncoding sequences called introns.
The nucleotide sequenced of some RNA transcripts are modified post-transcriptionally by RNA editing.
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44. Modifications to Eukaryotic pre-mRNAs
A 7-Methyl guanosine cap is added to the 5’ end of the primary transcript by a 5’-5’ phosphate linkage.
( stability and protection)
A poly(A) tail (a nucleotide polyadenosine tract, As) is added to the 3’ end of the transcript. The 3’ end is generated by cleavage rather than by termination. (stability and protection)
When present, intron sequences are spliced out of the transcript. (stability)
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45. © John Wiley & Sons, Inc.

46. Eukaryotes Have Five RNA Polymerases
RNA polymerase II Nucleus miRNA
Pre-mRNA~Heterogeneous nuclear RNA (hnRNA)
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47. A Typical RNA Polymerase II Promoter (mRNA)
Promoter: short sequence of conserved elements (seq. of DNA)
located upstream from the transcript starting point.
--~200 bp ( DNA linear)
--~10 Kdp ( DNA bending)
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48. Initiation by RNA Polymerase II
Transcriptional factors (proteins)
--Help/modulate/assist
Basal transcriptional factors
--bind close to the transcript starting point
Other factors (enhancers and silencers)
-TFIID
--TATA-biding proteins (TBP)
-TFIIA
-TFIIB
Where is the enzyme?
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49. -TFIIF (together with the RNA Pol-II)
--enzymatic activity (DNA-unwinding)
-TFIIE
--binds downstream regions
-TFIIH (helicase activity)
-TFIIJ
Helicase activity:
it separates two annealed nucleic acid strands
RNA Pol-II DNA-unwinding activity:
RNA Polymerase bends and wraps around DNA.
TFIIF alters (nonspecific) DNA binding by RNA polymerase II,
resulting in substantial DNA unwinding but not DNA strand separation.
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50. When RNA polymerase II binds to
-TFIIH (helicase activity and kinase activity)
When RNA polymerase II binds to
the complex, it initiates transcription.
Phosphorylation of the CTD is
required for elongation to begin.
CTD: carboxy-terminal domain

51. Some subunits are common to all three RNA polymerases.
All eukaryotic RNA polymerases have ∼12 subunits and are aggregates of >500 kD. (nucleotide pair~0.660 kD)
Some subunits are common to all three RNA polymerases.
The largest subunit in RNA polymerase II has a CTD (carboxy-terminal domain) consisting of multiple repeats of a heptamer.
-Typical RNA polymerase isolated from yeast (S. cerevisiase) ( and  subunits)
-  subunits: CTD – carboxy-terminal domain, which consists in multiple repeats of 7 amino acids, unique and important of regulation (tyrosine (Try, Y), serine (Ser, S) and threonine (Thr, T) residues)
-Some subunits are common to all three polymerases.
Figure 24.2

52. RNA Polymerase I Has a Bipartite Promoter
The RNA polymerase I promoter consists of:
--a core promoter
--an upstream control element (UPE)
RNA Pol I transcribes rRNA genes.
Core promoter: -45 to +20 seq.,
G-C-rich and A-T-rich (Inr-initiator) regions,
Binding factors - protein complexes formed by TFIs and TBP-(TATA binding protein)

53. RNA Polymerase III Uses Both Downstream and Upstream Promoters
RNA polymerase III has (3) types of promoters.
-RNA Pol III transcribes tRNA
-Core promoters (boxes)
-Transcriptional Factors(TF) III: general and specifics
*proximal sequence element *
Figure 24.7

54. RNA Chain elongation
--Model
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55. The 7-Methyl Guanosine (7-MG) Cap
Histones:?
FACT (facilitates chromatin transcriptional)
Energy
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56. RNA Chain termination Pol-II vs Pol I and III The 3’ Poly(A) Tail
Termination signal: specific DNA seq.
-1000 to 2000 nucleotides
--AAUAAA seq.
--GU-rich seq.
--poly(A) polymerase
Endonuclease
Pol-II vs Pol I and III
-Terminator proteins (Rho-indep. Terminator)
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57. RNA Editing
Usually the genetic information is not altered in the mRNA intermediary.
Sometimes RNA editing changes the information content of genes by
Inserting or deleting uridine monophosphate residues.
Changing the structures of individual bases
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58. Editing of Apoplipoprotein-B mRNA
(Amino groups)
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59. Eukaryotic gene transcripts usually undergo three major modifications:
Three to five different RNA polymerases are present in eukaryotes, and each polymerase transcribes a distinct set of genes.
Eukaryotic gene transcripts usually undergo three major modifications:
the addition of 7-methyl guanosine caps to 5’ termini,
The addition of poly(A) tails to 3’ ends,
The information content of some eukaryotic transcripts is altered by RNA editing, which changes the nucleotide sequences of transcripts prior to their translation.
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60. Interrupted Genes in Eukaryotes: Exons and Introns
Most eukaryotic genes contain noncoding sequences called introns that interrupt the coding sequences, or exons.
The introns are excised from the RNA transcripts prior to their transport to the cytoplasm.
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61. Hybridization: annealing
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62. R-Loop Evidence of an Intron in the Mouse -Globin Gene
mRNA
Missing in actions
Pre-mRNA
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63. Introns
Introns (or intervening sequences) are noncoding sequences located between coding sequences.
Introns are removed from the pre-mRNA and are not present in the mRNA.
Introns are variable in size and may be very large ( 50 bp to bp).
Exons (both coding and noncoding sequences) are composed of the sequences that remain in the mature mRNA after splicing.
The biological significance of introns is still open to debate.
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64. Removal of Intron Sequences by RNA Splicing
The noncoding introns are excised from gene transcripts by several different mechanisms.
Eukaryotes
No prokaryotes (excepts a few a prokaryotes virus and others)
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65. Excision of Intron Sequences
Conserved seq. for mRNA
Exon-GT…AG-exon
intron
99%
Ribonucleoproteins:
Spliceosomes (1981)
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66. Splicing Removal of introns must be very precise.
Conserved sequences for removal of the introns of nuclear mRNA genes are minimal.
Dinucleotide sequences at the 5’ and 3’ ends of introns.
Exon-GU…AG-exon
TACTAAC box (branch site with A) about 30 nucleotides upstream from the 3’ splice site.
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67. Spliceosomes: snRNA plus ~40 proteins 1%: CG…AG AT…AC
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68. Nuclear splicing involves trans-esterification
GU…UACUAAC….AG
“Branch site”
Does splicing require energy?
Does splicing ever occur in DNA?
Figure 26.7

70. tRNA A splicing endonuclease makes two cuts at the end of the intron.
A splicing ligase joins the two ends of the tRNA to produce the mature tRNA.
Specificity resides in the three-dimensional (secundary) structure of the tRNA precursor, not in the nucleotide sequence.
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71. rRNA (Autocatalytic Splicing)
G-3’-OH:
absolute requirement
“Co-factor”
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72. Noncoding intron sequences are excised from RNA transcripts in the nucleus prior to the transport to the cytoplasm.
Introns in tRNA precursors are removed by the concerted action of a splicing endonuclease and ligase, whereas introns in some rRNA precursors are spliced out autocatalytically—with no catalytic protein involved.
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73. The introns in nuclear pre-mRNAs are excised on complex ribonucleoprotein structures called spliceosomes.
The intron excision process must be precise, with accuracy to the nucleotide level, to ensure that codons in exons distal to introns are read correctly during translation.
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