1. Chapter Eleven Transcription of the Genetic Code: The Biosynthesis of RNA

2. Transcription Overview of Transcription
• synthesized on a DNA template, catalyzed by DNA-dependent RNA polymerase
• ATP, GTP, CTP, and UTP are required, as is Mg2+
• no RNA primer is required
• the DNA base sequence contains signals for initiation and termination of RNA synthesis; the enzyme binds to and moves along the DNA template in the 3’ -> 5’ direction
the RNA chain is synthesized in the 5’ -> 3’ direction
• the DNA template is unchanged

3. Transcription in Prokaryotes
E. coli RNA Polymerase:
molecular weight about 500,000
four different types of subunits: ,  , ’, and s
the core enzyme is 2’
the holoenzyme is 2’s
the role of the s subunit is recognition of the promoter locus; the s subunit is released after transcription begins
of the two DNA strands, the one that serves as the template for RNA synthesis is called the template strand or antisense strand; the other is called the coding (or nontemplate) strand or sense strand
the holoenzyme binds to and transcribes only the template strand

4. The Basics of Transcription

5. Promoter Sequence
Simplest of organisms contain a lot of DNA that is not transcribed
RNA polymerase needs to know which strand is template strand, which part to transcribe, and where first nucleotide of gene to be transcribed is
Promoters-DNA sequence that provide direction for RNA polymerase

6. Promoter Sequence

7. How does RNA polymerase know where to begin transcription?
Polymerase moves along template strand from 3’-5’ and RNA is formed from 5’-3’
Binding site for polymerase is upstream of start of transcription – away from 5’ of coding strand
Promoter sequence is based on coding strand and RNA polymerase is binding to template strand

8. How does RNA polymerase know where to begin transcription?
Promoter are upstream towards 5’ of coding and 3’ template strand
The first base to be incorporated is at position + 1 = transcription start site
All nucleotides upstream from this start site are given –ve numbers
The first promoter element is about 10 bases upstream is – 10 region or pribnow box

9. How does RNA polymerase know where to begin transcription?
After PB bases are variable
Next promoter element is about 35 bases upstream of TSS = -35 region or -35 element
Area from -35 element to TSS = core promoter
Upstream of core element is UP element = enhances binding of RNA polymerase
Region from UP element to TSS = extended promoter
BASE SEQUENCE of promoter is A and T

10. Chain Initiation First phase of transcription is initiation
Initiation begins when RNA polymerase binds to promoter and forms closed complex
After this, DNA unwinds at promoter to form open complex, which is required for chain initiation

11. Initiation and Elongation Transcription

12. Chain Elongation (Cont’d)

13. Chain Elongation
After strands separated, transcription bubble of ~17 bp moves down the DNA sequence to be transcribed
RNA polymerase catalyzes formation of phosphodiester bonds between the incorp. ribonucleotides
Topoisomerases relax supercoils in front of and behind transcription bubble

14. Chain Termination Two types of termination mechanisms:
• intrinsic termination- controlled by specific sequences, termination sites
Termination sites characterized by two inverted repeats
The inverted repeats are sequences of bases that are complementary such that they can loop back on themselves. Dna codes a series of uracils. The A-U base pairs between template strand forming hairpin loop. These are weakly bonded compared with gc pairs and rna dissociates from transcription bubble ending transcription

15. Chain Termination Other type of termination involves rho () protein
Rho-dependent termination sequences cause hairpin loop to form
Rho sequences form hair loop and rho protein will stall polymerase from proceeding

16. Transcription Regulation in Prokaryotes
In prokaryotes, transcription regulated by:
• alternative s factors
enhancers
operons
transcription attenuation

17. Alternative s factors
Viruses and bacteria exert control over which genes are expressed by producing different s-subunits that direct the RNA polymerase to different genes.

18. Enhancers
Certain genes include sequences upstream of extended promoter region
These genes for ribosomal production have 3 upstream sites, Fis sites
Class of DNA sequences that do this are called enhancers
Bound by proteins called transcription factors

19. Elements of a Bacterial Promoter

20. Operon
Operon: a group of operator, promoter, and structural genes that codes for proteins
the control sites, promoter, and operator genes are physically adjacent to the structural gene in the DNA
the regulatory gene can be quite far from the operon
operons are usually not transcribed all the time

21. Example of Operon system
b-Galactosidase, an inducible protein
coded for by a structural gene, lacZ
structural gene lacY codes for lactose permease
structural gene lacA codes for transacetylase
expression of these three structural genes is controlled by the regulatory gene lacI that codes for a repressor

22. Transcription in Eukaryotes is complex
Three RNA polymerases are known - each transcribes a different set of genes and recognizes a different set of promoters:
• RNA Polymerase I- found in the nucleolus and synthesizes precursors of most rRNAs
• RNA Polymerase II- found in the nucleoplasm and synthesizes mRNA precursors
• RNA Polymerase III- found in the nucleoplasm and synthesizes tRNAs, other RNA molecules involved in mRNA processing and protein transport
Prokaryotes have single RNA polymerase responsible for three kinds of prokayotic RNA - m RNA, t RNA and r RNA. THE polymerase switch sigma factors to interact with different promoters but core polymerase stays same. All three eukaryotic RNA are large and consist of 10 or more subunits. Their overall structure differs but have two larger subunits that share homology with ß and ß1 of prokaryotic RNA polymerase. There are no sigma subunits to direct polymerases to promoters.

23. RNA Polymerase II Most studied in the polymerases
Consists of 12 subunits
RPB- RNA Polymerase B
RPB1-RPB12. I, II, III also known as A, B and C

24. How does Pol II Recognize the Correct DNA?
Four elements of the Pol II promoter .

25. Pol II promoters
Variety of upstream elements – activators and silencers
GC box (-40) – Consensus sequence – GGGCGG
CAAT box (extending to – 110) – Consensus sequence - GGCCAATCT

26. Pol II promoters
Second element found at -25 = TATA box has consensus sequence of TATAA (T/A)
Transcription start site at position + 1 surrounded by a sequence called initiator element (Inr)
Inititator and TATA box = core promoter
Fourth element – downstream regulator - rare
ALL four are not present in all eukaryotes. Core promoter is most consistent in most species and genes

27. Initiation of Transcription
Transcription factor -Any protein regulator of transcription that is not a subunit of Pol II
Initiation begins by forming the preinitiation complex - Transcription control is based here
General transcription factors

28. Transcription Order of Events
The phosphorylated Pol II synthesizes RNA
Leaves the promoter region behind
GTFs are left at the promoter or dissociate from Pol II
Less is known about eukaryotes than prokaryotes. Pol II gets dephosphorylated and recycles back

29. Elongation and Termination
Elongation is controlled by:
pause sites - where RNA Pol will hesitate
positive transcription elongation factor (P-TEF) and negative transcription elongation factor (N-TEF)
Termination
begins by stopping RNA Pol; the eukaryotic consensus sequence for termination is AAUAAA
Elongation factors increase the productive form and decrease abortive form

30. Gene Regulation
Enhancers and silencers- regulatory sequences that augment or diminish transcription, respectively
DNA looping brings enhancers into contact with transcription factors and polymerase

31. Response elements
Response elements are enhancers that respond to certain metabolic factors
• heat shock element (HSE)
• glucocorticoid response element (GRE)
• metal response element (MRE)
• cyclic-AMP response element (CRE)

32. Structural Motifs in DNA-Binding Proteins
Most proteins that activate or inhibit RNA Pol II have two functional domains:
DNA-binding domain
transcription-activation domain
DNA-Binding domains have domains that are either:
• Helix-Turn-Helix (HTH)
• Zinc fingers
• Basic-region leucine zipper
Interact through hydrogen bonding, electrostatic attractions and hydrophobic interactions

33. Helix-Turn-Helix Motif
Hydrogen bonding between amino acids and DNA

34. Zinc Finger Motif
Motif contains 2 cysteines and 2 His – after every 12 amino acids
Zinc binds to the repeats

35. Basic Region Leucine Zipper Motif
Many transcription factors contain this motif - CREB
Half of the protein composed of basic region of conserved Lys, Arg, and His
Half contains series of Leu

36. Post Transcriptional RNA Modification
tRNA, rRNA, and mRNA are all modified after transcription to give the functional form
the initial size of the RNA transcript is greater than the final size because of the leader sequences at the 5’ end and the trailer sequences at the 3’ end
Modifications
trimming of leader and trailer sequences
addition of terminal sequences (after transcription)
modification of specific bases (particularly in tRNA)
the types of processing in prokaryotes can differ greatly from that in eukaryotes, especially for mRNA

37. Modification of tRNA Transfer RNA-
trimming, addition of terminal sequences, and base modification - take place
methylation and substitution of sulfur for oxygen are the two most usual types of base modification

38. Modification of rRNA Ribosomal RNA
processing of rRNA - methylation and trimming to the proper size

39. Modification of mRNA
Capping of the 5’ end with an N-methylated guanine
A polyadenylate “tail” that is usually nucleotides long, is added to the 3’ end before the mRNA leaves the nucleus
This tail protects the mRNA from nucleases and phosphatases

40. Organization of Split Genes in Eukaryotes

41. Modification of mRNA
Eukaryote genes frequently contain intervening base sequences that do not appear in the final mRNA of that gene product
Expressed DNA sequences are called exons
Intervening DNA sequences that are not expressed are called introns
These genes are often referred to as split genes

42. The Splicing Reaction Exons are separated by intervening intron
When the exons are spliced together - lariat forms in the intron

43. Ribozymes
The first ribozymes discovered included those that catalyze their own self-splicing
ribozymes have been discovered that are involved in protein synthesis
Group I and II

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