Synthesis of RNA and its regulation

1. Transcription
R. C. Gupta
Professor and Head
Department of Biochemistry
National Institute of Medical Sciences
Jaipur, India

2. Synthesis of proteins requires DNA and
RNA
Every protein has got a unique amino
acid sequence
Information about amino acid sequence
of all the proteins is present in DNA

3. The information is present in DNA in a
coded form
The unit of information is a gene
DNA contains a number of genes

4. A gene consists of a specific base
sequence encoding a protein
Three consecutive bases in the gene
constitute a codon
The codons are code words for amino
acids

5. The gene is a series of code words for
amino acids
The information present in genes is
used to synthesize proteins
Different types of RNA are required to
synthesize proteins

6. There are three
types of RNA:
Messenger RNA (mRNA)
Transfer RNA (tRNA)
Ribosomal RNA (rRNA)

7. Functions of different types of RNA
Structure Function
mRNA
Single,
uncoiled
strand
Transmits information from
DNA, serves as a template
for protein synthesis
tRNA
Single strand
folded back
upon itself
Brings amino acids to
ribosomes for protein
synthesis
rRNA
Single strand
folded into
globular shape
rRNA and proteins make up
ribosomes
All types of RNA are synthesized from DNA

8. Information flows from DNA to RNA to
proteins
Flow of information from DNA to RNA is
known as transcription
Use of this information to synthesize
proteins is known as translation
Central dogma of molecular biology

9. DNA RNA Protein
Transcription Translation

10. Transcription is synthesis of RNA
The RNA synthesized is the transcript of a
gene
The base sequence of RNA is comple-
mentary to that of the gene
Transcription

11. A gene is made up of two strands
Only one strand acts as a template for
transcription
The base sequence of RNA is comple-
mentary to this strand

12. The template strand of gene is known as
sense (non-coding) strand
The other strand has a complementary
base sequence
This strand is known as anti-sense
(coding) strand
Sense and antisense

13. Base sequence of RNA is complementary
to that of sense strand
Anti-sense (coding) strandRNA
Sense (template/
non-coding) strand
5’
The gene being
transcribed

14. Roger Kornberg
The process of
transcription was
elucidated by
Roger Kornberg

15. RNA is synthesized by RNA polymerase
(RNAP)
RNAP is a DNA-dependent RNA poly-
merase
It polymerizes ribonucleotides to form
RNA

16. The transcription unit is a gene
The genes for proteins are known as
structural genes
RNA polymerase recognizes a certain
base sequence, and binds to it

17. Every structural gene has got a unique
base sequence
One RNAP cannot recognize thousands
of different base sequences
RNAP recognizes some sequences
common to all the structural genes

18. Each structural gene is preceded by
small sequence known as promoter
Promoter is also known as promoter site
or promoter element or promoter region
All the promoters have some common or
consensus sequences

19. RNA polymerase recognizes the common
sequences in the promoters
A single enzyme can thus transcribe
different structural genes

20. Transcription is catalysed by prokaryotic
RNA polymerase (RNAP)
RNAP recognizes the prokaryotic
promoter
Prokaryotic transcription

21. The promoter has two common sequences
upstream of transcription start site
One sequence, 10 bp (base pairs) up-
stream, is TATAAT (called Pribnow box)
A second sequence, 35 bp upstream of
transcription start site, is TTGACA

23. The RNA polymerase of prokaryotes is a
pentamer
It is made up of two a subunits, a b
subunit, a b’ subunit and an w subunit
The pentamer is known as the core
enzyme

25. The core enzyme can synthesize RNA
but it cannot recognize the promoter
It requires a protein, called sigma factor,
to recognize the promoter
Core enzyme combines with sigma factor
to form RNA polymerase holoenzyme
The holoenzyme binds to the promoter

27. The process of RNA synthesis is similar
to primer synthesis
The portion of DNA which is being
transcribed is unwound (by RNAP)
RNA is synthesized in 5’  3’ direction

28. The substrates are ribonucleoside tri-
phosphates (ATP, GTP, CTP and UTP)
The a-phosphate group of new
nucleotide forms an ester bond with 3'
–OH group of the last nucleotide
An inorganic pyrophosphate is split off

29. The phosphate is thus involved in two
ester bonds – with 3’-OH of last nucleo-
tide and with 5’-OH of new nucleotide
Hence, the linkage between the last and
the new nucleotide occurs by 3’, 5’-
phosphodiester bond

31. The base sequence of template DNA
strand governs the base sequence of RNA
Nucleotides are selected according to the
base-pairing rule:
U opposite A
A opposite T
C opposite G
G opposite C

32. The process of transcription can
be divided into three phases:
Initiation phase
Elongation phase
Termination phase

33. RNAP holoenzyme binds to the
promoter and initiates transcription
The ribonucleotides are joined by
phosphodiester bonds
After initiation of transcription, the sigma
factor dissociates
Initiation phase

34. After release of sigma factor, elongation
phase begins
The core enzyme moves downstream
and adds ribonucleotides one by one
The catalytic function is performed by b
and b’ subunits
Elongation phase

36. A protein called rho (r) factor binds to the
termination site
When core enzyme reaches the r factor,
the newly transcribed RNA is released
The core enzyme and r factor are also
released
Termination

38. The basic process of transcription is
similar in prokaryotes and eukaryotes
The eukaryotic transcription machinery is
more complex and more elaborate
Eukaryotic promoters are slightly different
from prokaryotic promoters
Eukaryotic transcription

39. Eukaryotic promoters also have two common
sequences preceding transcription start site
One consensus sequence is 20-30 bp
upstream of transcription start site
Another consensus sequence is 70-80 bp
upstream of the transcription start site

40. The first consensus sequence is ATATAA
(TATA box or Hogness box)
The second consensus sequence is
GGCCAATC (CAAT box)

42. Unlike prokaryotes, eukaryotes have
different RNA polymerases to synthesize
different types of RNA:
RNA polymerase I synthesizes rRNA
RNA polymerase II synthesizes mRNA
RNA polymerase III synthesizes tRNA
(and also 5S rRNA)

43. The eukaryotic RNA polymerases are
bigger and have more subunits
A number of transcription factors are
required to form the basal transcription
apparatus
Sequences other than promoters affect
the transcription process and its rate

44. Eukaryotic genes may be
divided into three classes:
Class I genes (transcribed by RNA
polymerase I)
Class II genes (transcribed by RNA
polymerase II)
Class III genes (transcribed by RNA
polymerase III)

45. Class I genes are located in the nucleolus
They are transcribed to form 28S rRNA,
18S rRNA and 5.8S rRNA
The rRNAs are not translated
Transcription of class I genes

46. rRNAs combine with some proteins to
form ribosomes
Ribosomes are required in large numbers
Hence, class I genes are present in
multiple copies in DNA

47. Class II genes differ from class I and class
III genes
Class I and class III genes are transcribed
but not translated
Class II genes are transcribed as well as
translated
Transcription of class II genes

48. Class II genes are transcribed to form
hnRNA in eukaryotes
hnRNA is processed to form mRNA
mRNA is translated to form a protein

49. TATA box upstream of class II genes is
the site for attachment of RNAP II
The first event is the binding of TATA
binding protein (TBP) to the TATA box
Several other proteins called TBP-
associated factors (TAFs) bind to TBP

50. The complex of TBP and TAFs is called
Transcription Factor IID (TFIID)
Transcription Factor IIB (TFIIB) joins TFIID
TFIIF brings RNAP II, and both attach to
the complex

51. TFIIF acts like the prokaryotic sigma factor
It positions RNAP II at the correct site for
initiation of transcription

52. TFIIA, TFIIE and TFIIH bind to the complex
This completes the basal transcription
apparatus
The apparatus is analogous to RNA
polymerase holoenzyme of prokaryotes

54. TFIIH possesses kinase activity which is
increased by TFIIE
TFIIH phosphorylates some serine and
threonine residues in RNAP II
This makes the enzyme active
Active RNAP II transcribes the gene

55. CAAT box is another consensus sequence
in eukaryotic promoters
This is present upstream of the TATA box
A protein, CAAT-binding transcription
factor (CTF), binds CAAT box

56. By looping of DNA, CAAT box comes
closer to TATA box
CTF also binds TAFs which are part of
TFIID
This binding increases the frequency of
transcription

57. GC box may also be present upstream of
CAAT box or between TATA box and
CAAT box
A protein, Sp1 binds to GC box and
TAFs, and increases the frequency of
transcription

58. Class III genes encode tRNAs and 5S rRNA
They are transcribed by RNA polymerase
III
Class III genes are present in multiple
copies
Transcription of class III genes

59. The promoters of tRNA genes are
intragenic
The promoters are located within the
gene rather than upstream of the gene
A transcription factor, TFIIIA binds to the
promoter
It positions RNA polymerase III at the
correct site to initiate transcription

61. The newly-synthesized RNA is the
primary transcript of the gene
Primary transcript is not usually the final
and functional RNA
It requires some modifications
Post-transcriptional modifications

62. Except prokaryotic mRNA, all RNAs under-
go post-transcriptional modifications
The modifications differ in different types of
RNA

63. rRNA is a structural constituent of
ribosomes
rRNA combines with some polypeptides
to form ribosomes
The eukaryotic 80S ribosome is made up
of a 40S subunit and a 60S subunit
Post-transcriptional processing of rRNA

64. Made up of 18S rRNA
and about 30 different
polypeptides
Made up of 5S rRNA,
5.8S rRNA, 28S rRNA
and about 50 different
polypeptides
40S Subunit
60S Subunit

65. Both eukaryotic and prokaryotic rRNA are
synthesized initially as large precursors
These are cleaved into final rRNAs
Several bases are methylated

66. The primary transcript of rRNA gene in
eukaryotes is a 45S precursor
This is cleaved sequentially to form 28S,
18S and 5.8S rRNAs
5S rRNA is formed as such from class III
genes

68. Prokaryotic ribosome is 70S in size
It is made up of 30S and 50S subunits
The prokaryotic rRNAs are 5S, 16S and
23S
All are formed by cleavage of a large
precursor

69. Primary transcript of class II genes is
heterogeneous nuclear RNA in eukaryotes
Heterogeneous nuclear RNA (hnRNA) is
the precursor of mRNA
It is also known as pre-mRNA
Post-transcriptional processing of hnRNA

70. hnRNA undergoes extensive
modifications
Some modifications are common to all
hnRNAs
Some are unique to each

71. Two modifications common to
all the hnRNAs are:
Addition of 7-methylguanosine
triphosphate cap (7-methyl GTP
cap) at the 5'-end of RNA
Addition of poly-adenylate tail
(poly-A tail) at the 3'-end of RNA

72. The cap at the 5’-end helps the ribosome
in recognizing mRNA
It also prevents breakdown of mRNA by
5’-exonuclease
The tail at the 3’-end also stabilizes mRNA
by preventing the action of 3’-exonuclease
Some mRNAs do not have a poly-A tail
e.g. mRNAs for histones

73. The third modification is deletion of some
nucleotides from hnRNA
The deletion is different in different
hnRNAs

74. Eukaryotic genes contain some coding
and some non-coding sequences
Coding sequences are expressed, and
are known as exons
Non-coding sequences called introns
intervene between the coding sequences

75. After addition of cap and tail, the introns
are removed and the exons joined
This process is known as splicing
The number and size of exons and
introns are different in different genes

76. An example is b-globin gene
This gene encodes the b polypeptide
chain of haemoglobin
It has three exons interrupted by two
introns
Exon 1 Exon 2 Exon 3Intron 1 Intron 2

77. Splice sites are also known as splice
junctions or intron-exon junctions
Splice sites in all hnRNAs have some
common features
Splice sites

78. The intron begins with GU and ends with
AG
In between these two, there is a branch
site having A
There is a pyrimidine-rich tract of nearly 10
nucleotides between branch site and AG

80. During splicing, 2’ –OH group of A at the
branch site forms an ester bond with
phosphate group of G at the 5’-splice site
Exon 1 is released, and its 3’-nucleotide
forms an ester bond with the
5’-nucleotide of exon 2
The intron is released in lariat form

83. Spliceosome
Spliceosome is an assembly made up of:
hnRNA to be spliced
Small nuclear RNAs (snRNAs)
Some proteins
snRNAs are a species of RNA molecules
<300 nucleotides in length

84. The snRNAs combine with the proteins to
form small nuclear ribonucleoprotein
particles (snRNPs or snurps)
snRNPs are U1, U2, U4, U5 and U6
These combine with hnRNA to form a
spliceosome

85. U1 binds to 5’-splice site
U2 binds to branch site
U5 binds to 3’-splice site of hnRNA
U4 and U6 bind to this complex

87. The splicing reaction is catalysed by the
snRNA components of snRNPs
Auto-antibodies against snRNPs are
formed in systemic lupus erythematosus
This results in wide-spread tissue
damage

88. Prokaryotic genes have no introns
Therefore, prokaryotes do not possess
splicing machinery

89. tRNA is synthesized as a precursor in
prokaryotes as well as eukaryotes
The precursor undergoes extensive post-
transcriptional modifications
Post-transcriptional processing of tRNA

90. The modifications in the
precursor include:
Removal of some nucleotides
Addition of –CCA terminus at 3'-end
Formation of pseudouridine from uridine
Methylation of several bases

92. mRNA is synthesized by RNA poly-
merase II in eukaryotes
This enzyme binds to the promoter site
upstream of the structural gene
Besides promoter site, there are a
number of sequences in DNA which
control the rate of transcription
Regulation of transcription

93. Specific protein factors bind to the
regulatory sequences
The protein factors include transcription
factors, CTF, Sp1, CREB (cAMP
response element binding protein) etc
These protein factors facilitate or
increase the rate of transcription

94. Inducers and repressors also bind to the
regulatory sequences
Inducers increase transcription
Repressors decrease transcription
Mutations in promoter site can decrease
the rate of transcription

95. Enhancer elements are sequences located
far away from the gene they influence
They may be upstream or downstream
A regulatory factor binds to the enhancer
element
This increases the transcription of the
gene influenced by the enhancer element

96. Silencer elements are also located at a
distance from the gene they influence
They may be upstream or downstream
They suppress the transcription of the
genes that they influence

97. Transcription is essential for life
Inhibition of transcription prevents protein
synthesis
No organism can survive without proteins
Hence, selective inhibitors of prokaryotic
transcription can be used as antibiotics
Inhibition of transcription

98. Rifampicin inhibits the b subunit of RNA
polymerase
b Subunit is present only in prokaryotes
Rifampicin doesn’t inhibit the corres-
ponding human enzyme
Therefore, it can be used as an antibiotic