ProkarTranscription

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• Transcription in
• Prokaryotes

M.Prasad Naidu MSc Medical Biochemistry, Ph.D,.
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• Gene expression begins with transcription
• RNA copy of a gene made by an RNA
  polymerase
• Prokaryotic RNA polymerases are assemblies of
  several different proteins

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Bacterial Gene Structure of signals
Gene 2
Gene 1

• Bacterial genomes have simple gene structure
• - Promoter
• -35 sequence (T82T84G78A65C54A45) 15-20 bp
• -10 sequence (T80A95T45A60A50T96) 5-9 bp
  (Pribnow Box)
• - Start of transcription initiation start
  Purine90 (sometimes its the A in CAT)
• - Translation binding site (Shine-Dalgarno) 10 bp
  upstream of AUG (AGGAGG)
• - One or more Open Reading Frame
• start-codon (unless sequence is partial)
• until next in-frame stop codon on that strand ..
• Separated by intercistronic sequences.
• - Termination

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• RNA polymerase must know where the start of a
  gene is in order to copy it
• RNA polymerase has weak interactions with the DNA
  unless it encounters a promoter
• A promoter is a specific sequence of nucleotides
  that indicate the start site for RNA synthesis

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General Steps of Transcription

• Initiation
• Binding of RNA polymerase to double stranded DNA
• Development of closed promoter complex
• Development of open promoter complex
• Start of transcription by adding the first two
  ribonucleotides.
• Elongation
• - Formation of transcription bubble or
  Transcription
• elongation complex.
• Progression of the complex gradually in the
  3 direction to elongate the
  initiated RNA chain.
• Rapid process up to 40 nucleotides per second.
• On the same gene there are several RNA strands
  being transcribed in a staggered fashion.
• Termination
• Terminator sequences signal stop of transcription.

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Initiation
Sigma dissociates
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Irreversible form of Open Complex Formation
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Generation of Abortive Initiation Products
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Transcription Bubble
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RNA Elongation

• Reads template 3 to 5
• Adds nucleotides 5 to 3 (5 phosphate to 3
  hydroxyl)
• Synthesis is the same as the leading strand of DNA

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Polymerization is polar enzyme works by adding
to a free 3 hydroxyl in growing mRNA chain.
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RNA Synthesis

• RNA pol moves nt by nt, unwinds the DNA as it
  goes
• Will stop when it encounters a STOP.
• RNA pol leaves, releasing the RNA strand

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Termination of Transcription

• Factor-independent termination
• Factor-dependent termination
• 3 factors
• Rho (?), Tau (?) and NusA
• Rho best studied

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Termination of transcription

• RNA single stranded nucleic acid
• can form secondary structures
• Rho-dependent termination protein signal
• Rho binds to RNA able to cause RNA RNA
  polymerase to leave DNA
• ? termination
• Rho-independent signal hairpin or stem-
• loop RNA structure forms, followed by
• several uracils
• ? termination

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Terminator Sequences

• In prokaryotes there are two types
• 1. Intrinsic Rho (?) independent terminator
• Contains a G-C rich region followed by six
  or more A-T sequences.
• Causes the formation of a double stranded RNA
  called a hairpin loop.
• Retards the movement of the RNA polymerase along
  the DNA molecule, and causes termination at the
  A-T rich region.

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• 2. Extrinsic Rho-dependent terminator
• Requires a protein factor called Rho (?).
• Rho protein trails the RNA polymerase until it
  reaches a GC rich region, when Rho catches up
  with the polymerase.
• Rho protein pulls off RNA from transcription
  bubble.

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1. Rho-independent terminator site

• RNA transcript at the terminating site is
  self-complementary
• The bases can pair to form a hairpin structure
  with a stem and loop, a structure favored by its
  high G-C content
• The stable hairpin is followed by a sequence of 4
  or more U residues
• The RNA transcript ends within or just after them

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Intrinsic termination site
String of Us
Intrastrand complementary bases
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Mechanism of Rho-independent Termination

• RNA polymerase pauses when it encounters such a
  hairpin formed at the terminator site
• The RNA-DNA hybrid helix produced after the
  hairpin is unstable because of its content of
  rU-dA base pairs, the weakest of the four kinds
  of pairs
• Nascent RNA is pulled off from the DNA template
  and then from the enzyme
• DNA template strand now joins its partner to form
  the DNA duplex

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Control of trp operon by attenuation

• stalled translation allows region 2 to interact
  with region 3
• 3 4 cannot interact

• regions 3 4 interact termination results

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Global control systems in E. coli

• In global control systems many genes, pathways
  regulated simultaneously in response to a
  specific environmental signal
• e.g., regulon collection of genes and/or operons
  controlled by common regulatory protein
• Sporulation in Bacillus another global control
  system

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2. Rho-dependent terminator
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Rho (?) Protein

• Rho is an RNA-dependent ATPase
• Also an RNA-DNA helicase
• It is an hexamer, with a mass of 275 kDa (each
  subunit is of 46 kDa)
• It binds to ssRNA at Rut site a stretch of 72
  nt is bound, 12 per subunit
• It is brought into action by sequence located in
  the nascent RNA
• ATPase activity enables it to move
  unidirectionally along the nascent RNA

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Effect of rho protein on the size of RNA
transcripts
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Rho-dependent

• Rho factor factor mediated termination
• In an ATP-mediated reaction, a rho protein
  complex binds to the mRNA and unwinds RNA from
  the DNA template
• Recognition sites may not have hairpins or U
  tracts tend to be C-rich

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(No Transcript)
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One Transcriptional unit
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Two contiguous genes

• RNA is released so we can make many copies of the
  gene, usually before the first one is done
• Can have multiple RNA polymerase molecules on a
  gene at a time

Termination site
Initiation site
RNA fibrils
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Summary
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Types of mRNA

• In Bacteria
• Monocistronic mRNA
• Polycistronic mRNA
• In Eukaryotes
• Monocistronic mRNA

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Polycistronic mRNA

• Many prokaryotic mRNAs are polycistronic
• Contain sequences specifying the synthesis of
  several proteins
• A polycistronic mRNA molecule possesses a series
  of start and stop codons
• In case it codes for three proteins
• Start, Protein1, Stop Start, Protein2,
  Stop
• Start, Protein3, Stop
• Abou 5-20 bases may be present between one stop
  codon and the next start codon. These are called
  Spacers.
• The segment of RNA corresponding to a DNA cistron
  is called a Reading frame

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Polycistronic vs Monocistronic mRNA
spacers
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Processing of pre-rRNA transcripts in E. coli