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Transcription
R. C. Gupta
Professor and Head
Department of Biochemistry
National Institute of Medical Sciences
Jaipur, Ind...
Synthesis of proteins requires DNA and
RNA
Every protein has got a unique amino
acid sequence
Information about amino acid...
The information is present in DNA in a
coded form
The unit of information is a gene
DNA contains a number of genes
A gene consists of a specific base
sequence encoding a protein
Three consecutive bases in the gene
constitute a codon
The ...
The gene is a series of code words for
amino acids
The information present in genes is
used to synthesize proteins
Differe...
There are three
types of RNA:
Messenger RNA (mRNA)
Transfer RNA (tRNA)
Ribosomal RNA (rRNA)
Functions of different types of RNA
Structure Function
mRNA
Single,
uncoiled
strand
Transmits information from
DNA, serves...
Information flows from DNA to RNA to
proteins
Flow of information from DNA to RNA is
known as transcription
Use of this in...
DNA RNA Protein
Transcription Translation
Transcription is synthesis of RNA
The RNA synthesized is the transcript of a
gene
The base sequence of RNA is comple-
ment...
A gene is made up of two strands
Only one strand acts as a template for
transcription
The base sequence of RNA is comple-
...
The template strand of gene is known as
sense (non-coding) strand
The other strand has a complementary
base sequence
This ...
Base sequence of RNA is complementary
to that of sense strand
Anti-sense (coding) strandRNA
Sense (template/
non-coding) s...
Roger Kornberg
The process of
transcription was
elucidated by
Roger Kornberg
RNA is synthesized by RNA polymerase
(RNAP)
RNAP is a DNA-dependent RNA poly-
merase
It polymerizes ribonucleotides to for...
The transcription unit is a gene
The genes for proteins are known as
structural genes
RNA polymerase recognizes a certain
...
Every structural gene has got a unique
base sequence
One RNAP cannot recognize thousands
of different base sequences
RNAP ...
Each structural gene is preceded by
small sequence known as promoter
Promoter is also known as promoter site
or promoter e...
RNA polymerase recognizes the common
sequences in the promoters
A single enzyme can thus transcribe
different structural g...
Transcription is catalysed by prokaryotic
RNA polymerase (RNAP)
RNAP recognizes the prokaryotic
promoter
Prokaryotic trans...
The promoter has two common sequences
upstream of transcription start site
One sequence, 10 bp (base pairs) up-
stream, is...
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 subuni...
The core enzyme can synthesize RNA
but it cannot recognize the promoter
It requires a protein, called sigma factor,
to rec...
The process of RNA synthesis is similar
to primer synthesis
The portion of DNA which is being
transcribed is unwound (by R...
The substrates are ribonucleoside tri-
phosphates (ATP, GTP, CTP and UTP)
The a-phosphate group of new
nucleotide forms an...
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 base sequence of template DNA
strand governs the base sequence of RNA
Nucleotides are selected according to the
base-p...
The process of transcription can
be divided into three phases:
Initiation phase
Elongation phase
Termination phase
RNAP holoenzyme binds to the
promoter and initiates transcription
The ribonucleotides are joined by
phosphodiester bonds
A...
After release of sigma factor, elongation
phase begins
The core enzyme moves downstream
and adds ribonucleotides one by on...
A protein called rho (r) factor binds to the
termination site
When core enzyme reaches the r factor,
the newly transcribed...
The basic process of transcription is
similar in prokaryotes and eukaryotes
The eukaryotic transcription machinery is
more...
Eukaryotic promoters also have two common
sequences preceding transcription start site
One consensus sequence is 20-30 bp
...
The first consensus sequence is ATATAA
(TATA box or Hogness box)
The second consensus sequence is
GGCCAATC (CAAT box)
Unlike prokaryotes, eukaryotes have
different RNA polymerases to synthesize
different types of RNA:
RNA polymerase I synth...
The eukaryotic RNA polymerases are
bigger and have more subunits
A number of transcription factors are
required to form th...
Eukaryotic genes may be
divided into three classes:
Class I genes (transcribed by RNA
polymerase I)
Class II genes (transc...
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...
rRNAs combine with some proteins to
form ribosomes
Ribosomes are required in large numbers
Hence, class I genes are presen...
Class II genes differ from class I and class
III genes
Class I and class III genes are transcribed
but not translated
Clas...
Class II genes are transcribed to form
hnRNA in eukaryotes
hnRNA is processed to form mRNA
mRNA is translated to form a pr...
TATA box upstream of class II genes is
the site for attachment of RNAP II
The first event is the binding of TATA
binding p...
The complex of TBP and TAFs is called
Transcription Factor IID (TFIID)
Transcription Factor IIB (TFIIB) joins TFIID
TFIIF ...
TFIIF acts like the prokaryotic sigma factor
It positions RNAP II at the correct site for
initiation of transcription
TFIIA, TFIIE and TFIIH bind to the complex
This completes the basal transcription
apparatus
The apparatus is analogous to ...
TFIIH possesses kinase activity which is
increased by TFIIE
TFIIH phosphorylates some serine and
threonine residues in RNA...
CAAT box is another consensus sequence
in eukaryotic promoters
This is present upstream of the TATA box
A protein, CAAT-bi...
By looping of DNA, CAAT box comes
closer to TATA box
CTF also binds TAFs which are part of
TFIID
This binding increases th...
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, ...
Class III genes encode tRNAs and 5S rRNA
They are transcribed by RNA polymerase
III
Class III genes are present in multipl...
The promoters of tRNA genes are
intragenic
The promoters are located within the
gene rather than upstream of the gene
A tr...
The newly-synthesized RNA is the
primary transcript of the gene
Primary transcript is not usually the final
and functional...
Except prokaryotic mRNA, all RNAs under-
go post-transcriptional modifications
The modifications differ in different types...
rRNA is a structural constituent of
ribosomes
rRNA combines with some polypeptides
to form ribosomes
The eukaryotic 80S ri...
Made up of 18S rRNA
and about 30 different
polypeptides
Made up of 5S rRNA,
5.8S rRNA, 28S rRNA
and about 50 different
pol...
Both eukaryotic and prokaryotic rRNA are
synthesized initially as large precursors
These are cleaved into final rRNAs
Seve...
The primary transcript of rRNA gene in
eukaryotes is a 45S precursor
This is cleaved sequentially to form 28S,
18S and 5.8...
Prokaryotic ribosome is 70S in size
It is made up of 30S and 50S subunits
The prokaryotic rRNAs are 5S, 16S and
23S
All ar...
Primary transcript of class II genes is
heterogeneous nuclear RNA in eukaryotes
Heterogeneous nuclear RNA (hnRNA) is
the p...
hnRNA undergoes extensive
modifications
Some modifications are common to all
hnRNAs
Some are unique to each
Two modifications common to
all the hnRNAs are:
Addition of 7-methylguanosine
triphosphate cap (7-methyl GTP
cap) at the 5...
The cap at the 5’-end helps the ribosome
in recognizing mRNA
It also prevents breakdown of mRNA by
5’-exonuclease
The tail...
The third modification is deletion of some
nucleotides from hnRNA
The deletion is different in different
hnRNAs
Eukaryotic genes contain some coding
and some non-coding sequences
Coding sequences are expressed, and
are known as exons
...
After addition of cap and tail, the introns
are removed and the exons joined
This process is known as splicing
The number ...
An example is b-globin gene
This gene encodes the b polypeptide
chain of haemoglobin
It has three exons interrupted by two...
Splice sites are also known as splice
junctions or intron-exon junctions
Splice sites in all hnRNAs have some
common featu...
The intron begins with GU and ends with
AG
In between these two, there is a branch
site having A
There is a pyrimidine-ric...
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
...
Spliceosome
Spliceosome is an assembly made up of:
hnRNA to be spliced
Small nuclear RNAs (snRNAs)
Some proteins
snRNAs ar...
The snRNAs combine with the proteins to
form small nuclear ribonucleoprotein
particles (snRNPs or snurps)
snRNPs are U1, U...
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
The splicing reaction is catalysed by the
snRNA components of snRNPs
Auto-antibodies against snRNPs are
formed in systemic...
Prokaryotic genes have no introns
Therefore, prokaryotes do not possess
splicing machinery
tRNA is synthesized as a precursor in
prokaryotes as well as eukaryotes
The precursor undergoes extensive post-
transcript...
The modifications in the
precursor include:
Removal of some nucleotides
Addition of –CCA terminus at 3'-end
Formation of p...
mRNA is synthesized by RNA poly-
merase II in eukaryotes
This enzyme binds to the promoter site
upstream of the structural...
Specific protein factors bind to the
regulatory sequences
The protein factors include transcription
factors, CTF, Sp1, CRE...
Inducers and repressors also bind to the
regulatory sequences
Inducers increase transcription
Repressors decrease transcri...
Enhancer elements are sequences located
far away from the gene they influence
They may be upstream or downstream
A regulat...
Silencer elements are also located at a
distance from the gene they influence
They may be upstream or downstream
They supp...
Transcription is essential for life
Inhibition of transcription prevents protein
synthesis
No organism can survive without...
Rifampicin inhibits the b subunit of RNA
polymerase
b Subunit is present only in prokaryotes
Rifampicin doesn’t inhibit th...
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Synthesis of RNA and its regulation

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  1. 1. Transcription R. C. Gupta Professor and Head Department of Biochemistry National Institute of Medical Sciences Jaipur, India
  2. 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. 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. 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. 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. 6. There are three types of RNA: Messenger RNA (mRNA) Transfer RNA (tRNA) Ribosomal RNA (rRNA)
  7. 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. 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. 9. DNA RNA Protein Transcription Translation
  10. 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. 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. 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. 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. 14. Roger Kornberg The process of transcription was elucidated by Roger Kornberg
  15. 15. RNA is synthesized by RNA polymerase (RNAP) RNAP is a DNA-dependent RNA poly- merase It polymerizes ribonucleotides to form RNA
  16. 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. 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. 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. 19. RNA polymerase recognizes the common sequences in the promoters A single enzyme can thus transcribe different structural genes
  20. 20. Transcription is catalysed by prokaryotic RNA polymerase (RNAP) RNAP recognizes the prokaryotic promoter Prokaryotic transcription
  21. 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
  22. 22. 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
  23. 23. 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
  24. 24. 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
  25. 25. 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
  26. 26. 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
  27. 27. 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
  28. 28. The process of transcription can be divided into three phases: Initiation phase Elongation phase Termination phase
  29. 29. 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
  30. 30. 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
  31. 31. 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
  32. 32. 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
  33. 33. 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
  34. 34. The first consensus sequence is ATATAA (TATA box or Hogness box) The second consensus sequence is GGCCAATC (CAAT box)
  35. 35. 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)
  36. 36. 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
  37. 37. 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)
  38. 38. 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
  39. 39. 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
  40. 40. 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
  41. 41. Class II genes are transcribed to form hnRNA in eukaryotes hnRNA is processed to form mRNA mRNA is translated to form a protein
  42. 42. 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
  43. 43. 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
  44. 44. TFIIF acts like the prokaryotic sigma factor It positions RNAP II at the correct site for initiation of transcription
  45. 45. TFIIA, TFIIE and TFIIH bind to the complex This completes the basal transcription apparatus The apparatus is analogous to RNA polymerase holoenzyme of prokaryotes
  46. 46. 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
  47. 47. 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
  48. 48. 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
  49. 49. 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
  50. 50. 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
  51. 51. 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
  52. 52. 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
  53. 53. Except prokaryotic mRNA, all RNAs under- go post-transcriptional modifications The modifications differ in different types of RNA
  54. 54. 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
  55. 55. 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
  56. 56. Both eukaryotic and prokaryotic rRNA are synthesized initially as large precursors These are cleaved into final rRNAs Several bases are methylated
  57. 57. 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
  58. 58. 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
  59. 59. 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
  60. 60. hnRNA undergoes extensive modifications Some modifications are common to all hnRNAs Some are unique to each
  61. 61. 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
  62. 62. 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
  63. 63. The third modification is deletion of some nucleotides from hnRNA The deletion is different in different hnRNAs
  64. 64. 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
  65. 65. 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
  66. 66. 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
  67. 67. Splice sites are also known as splice junctions or intron-exon junctions Splice sites in all hnRNAs have some common features Splice sites
  68. 68. 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
  69. 69. 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
  70. 70. 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
  71. 71. 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
  72. 72. 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
  73. 73. 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
  74. 74. Prokaryotic genes have no introns Therefore, prokaryotes do not possess splicing machinery
  75. 75. tRNA is synthesized as a precursor in prokaryotes as well as eukaryotes The precursor undergoes extensive post- transcriptional modifications Post-transcriptional processing of tRNA
  76. 76. 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
  77. 77. 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
  78. 78. 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
  79. 79. 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
  80. 80. 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
  81. 81. 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
  82. 82. 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
  83. 83. 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
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Synthesis of RNA and its regulation

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