1. TRANSCRIPTION--- SYNTHESIS OF RNA
Dr. Zakira Naureen

2. Previously we have studied
Central dogma of molecular biology
DNA Structure and Replication
Enzymes and factors involved in Replication
Three types of RNA
m RNA; t RNA; r RNA

3. Learning objectives What is gene ? What is Transcription?
Structure of RNA polymerases

4. Genes
A gene is a region of DNA that controls a discrete hereditary characteristic, usually corresponding to a single mRNA which will be translated into a protein.
In eukaryotes, the genes have their coding sequences (exons) interrupted by non-coding sequences (introns).
In humans, genes constitute only about 2-3% of DNA, the rest is “junk” DNA.

5. protein Eukaryotic Gene Structure transcription translation
5’ - Promoter Exon Intron Exon Terminator – 3’
UTR splice splice UTR
transcription
Poly A
translation
protein

6. Prokaryotic Gene Structure
Promoter CDS Terminator
UTR UTR
Genomic DNA
transcription
mRNA
translation
protein

7. Prokaryote gene versus Eukaryote gene

8. What is Transcription ? It is the process of copying DNA to RNA.
It differs from DNA synthesis in that only one strand of DNA, the template strand, is used to make mRNA.
Unlike DNA replication, it does not need a primer to start.
It can involve multiple RNA polymerases.
Transcription is divided into 3 stages
Initiation
Promoter recognition and binding
Initiation of polymerization of NTPs
Elongation
Termination

9. Transcription Tools Template DNA DNA-dependent RNA polymerase
ATP, GTP, CTP, and UTP are required, Mg2+
Transcription factors
ATP

10. The coding strand and the template strand of DNA
The important thing to realize is that the genetic information is carried on only one of the two strands of the DNA. This is known as the coding strand.
The other strand is known as the template strand, and is complementary to the coding strand.
If you took the template strand and built a new DNA strand on it (as happens in DNA replication), you would get an exact copy of the original DNA coding strand formed.
Almost exactly the same thing happens when you make RNA. If you build an RNA strand on the template strand, you will get a copy of the information on the DNA coding strand - but with one important difference.

11. The Code is read from 5’ to 3’

12. The Basics of Transcription

13. DNA Dependent RNA Polymerase
It’s a holoenzyme
Structure and function are fundamentally similar in Prokaryotes and Eukaryotes ( few exceptions)
Bacterial RNA Polymerases are composed of
4 core subunits (2 small + 2 large ) +
Sigma factor (σ)–
determines promoter
specificity
Core + σ = holoenzyme
Binds promoter sequence

14. continued
In eukaryotes the DNA dependent RNA Polymerase has several other binding factors i.e. Transcription factors
More than one polymerases are required to make all different types of RNA unlike bacterial RNA Polymerase that can catalyze formation of all three RNA molecules.
Three polymerases for 3 RNA molecules
RNA pol I rRNA
RNA pol II mRNA
RNA pol III------tRNA

15. PROMOTER
Sigma factors recognize consensus
-10 and -35 sequences

16. continued Promoter determines: Which strand will serve as a template.
Transcription starting point.
Strength of polymerase binding.
Frequency of polymerase binding.

17. Prokaryotic Promoter One type of RNA polymerase.
Pribnow box located at –10 (6-7bp)
–35 sequence located at -35 (6bp)
Sigma factor actually recognizes this and binds here

18. Eukaryote Promoter Goldberg-Hogness or TATA located at –30
Additional regions at –100 and at –200
Possible distant regions acting as enhancers or silencers (even more than 50 kb).

19. Promoter Sequence

20. Promoter Strong promoter resemble the consensus sequence.
Mutations at promoter sites can influence transcription.
Human gene Beta globin

21. Stages of Transcription
Initiation – the RNA polymerase enzyme binds to a promoter site on the DNA and unzips the double helix.
Elongation – free nucleotides bind to their complementary pairs on the template strand of the DNA elongating the RNA chain which is identical to the informational strand of DNA, except that the nucleotide thymine in DNA is replaced by uracil in RNA. The polymerase moves along the DNA in the 3’ to 5’ direction, extending the RNA 5’ to 3’.
Termination – specific sequences in the DNA signal termination of transcription; when one of these is encountered by the polymerase, the RNA transcript is released from the DNA and the double helix can zip up again.

22. Transcription video

23. 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

24. Initiation

25. 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

26. Elongation

27. Chain Elongation (Cont’d)

28. Chain Termination Two types of termination mechanisms:
• intrinsic termination- controlled by specific sequences, termination sites
Termination sites characterized by two inverted repeats

29. Chain Termination (Cont’d)
Other type of termination involves rho () protein
Rho-dependent termination sequences cause hairpin loop to form

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

31. Control by Different  Subunits

32. 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

33. Elements of a Bacterial Promoter

34. 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
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

35. How Does Repression Work
Repressor protein made by lacI gene forms tetramer when it is translated
Repressor protein then binds to operator portion of operon
Operator and promoter together are the control sites

36. Binding Sites On the lac operon
Lac operon is induced when E. coli has lactose as the carbon source
Lac protein synthesis repressed by glucose (catabolite repression)
E. coli recognizes presence of glucose by promoter as it has 2 regions: RNA polymerase binding site, catabolite activator protein (CAP) binding site

37. Binding Sites On lac operon (Cont’d)

38. Catabolite Repression
CAP forms complex with cAMP
Complex binds at CAP site
RNA polymerase binds at available binding site, and transcription occurs

39. Basic Control Mechanisms in Gene Control
Control may be inducible or repressive, and these may be negatively or positively controlled

40. Control of the trp operon
Trp operon codes for a leader sequence (trpL) and five polypeptides
The five proteins make up 4 different enzymes that catalyze the multistep process that converts chorisimate to tryptophan

41. Alternative 2˚ structures Can Form in trp Operon
These structures can form in the leader sequence
Pause structure- binding between regions 1 and 2
Terminator loop- binding between regions 3 and 4
Antiterminator structure- Alternative binding between regions 2 and 3

42. Attenuation in the trp operon
Pause structure forms when ribosome passes over Trp codons when Trp levels are high
Ribosome stalls at the Trp codon when trp levels are low and antiterminator loop forms

43. Transcription in Eukaryotes
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

44. RNA Polymerase II Most studied on the polymerases
Consists of 12 subunits
RPB- RNA Polymerase B

45. How does Pol II Recognize the Correct DNA?
Four elements of the Pol II promoter allow for this phenomenon

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

47. General Transcription Initiation Factors

48. Transcription Order of Events
Less is known about eukaryotes than prokaryotes
The phosphorylated Pol II synthesizes RNA and leaves the promoter region behind
GTFs are left at the promoter or dissociate from Pol II

49. Elongation and Termination
Elongation is controlled by:
pause sites, where RNA Pol will hesitate
anti-termination, which proceeds past the normal termination point
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

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

51. Eukaryotic Gene Regulation
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)
Response elements all bind proteins (transcription factors) that are produced under certain cell conditions

52. Response Elements

53. Activation of transcription Via CREB and CBP
Unphosphorylated CREB does not bind to CREB binding protein, and no transcription occurs
Phosphorylation of CREB causes binding of CREB to CBP
Complex with basal complex (RNA polymerase and GTFs) activates transcription

54. 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

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

56. Zinc Finger Motif
Motif contains 2 cysteines and 2 His 12 amino acids later
Zinc binds to the repeats

57. Basic Region Leucine Zipper Motif
Many transcription factors contain this motif, such as CREB (Biochemical Connections, page 315)
Half of the protein composed of basic region of conserved Lys, Arg, and His
Half contains series of Leu
Leu line up on one side, forming hydrophobic pocket

58. Helical Wheel Structure of Leucine Zipper

59. Transcription Activation Domains
acidic domains- rich in Asp and Glu. Gal4 has domain of 49 amino acids, 11 are acidic
glutamine-rich domains- Seen in several transcription factors. Sp1 has 2 glutamine-rich domains, one with 39 Glu in 143 amino acids
proline-rich domains- Seen in CTF-1 (an activator). It has 84 amino acid domain, of which 19 are Pro

60. 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
the types of processing in prokaryotes can differ greatly from that in eukaryotes, especially for mRNA
Modifications
trimming of leader and trailer sequences
addition of terminal sequences (after transcription)
modification of the structure of specific bases (particularly in tRNA)

61. Posttranscriptional Modification of tRNA Precursor

62. Modification of tRNA
Transfer RNA- the precursor of several tRNAs is can be transcribed as one long polynucleotide sequence
trimming, addition of terminal sequences, and base modification all take place
methylation and substitution of sulfur for oxygen are the two most usual types of base modification

63. Modification of rRNA Ribosomal RNA
processing of rRNA is primarily a matter of methylation and trimming to the proper size
in prokaryotes, 3 rRNAs in one intact ribosome
in Eukaryotes, ribosomes have 80s, 60s, and 40s subunits
base modification in both prokaryotes and eukaryotes is primarily by methylation

64. Modification of mRNA
Includes the capping of the 5’ end with an N-methylated guanine that is bonded to the next residue by a 5’ -> 5’ triphosphate.
Also, 2’-O-methylation of terminal ribose(s)

65. mRNA Modification
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
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

66. Organization of Split Genes in Eukaryotes

67. The Splicing Reaction Exons are separated by intervening intron
When the exons are spliced together,a lariat forms in the intron

68. Ribozymes
The first ribozymes discovered included those that catalyze their own self-splicing
More recently, ribozymes have been discovered that are involved in protein synthesis
Group I ribozymes
require an external guanosine
example: pre-rRNA of the protozoan Tetrahymena (next screen)
Group II ribozymes
display a lariat mechanism similar to mRNA splicing
no requirement for an external nucleotide

69. Self-splicing of pre-rRNA