Biol 3301 - Genetics Ch13a - Transcription In Prokaryotes St

Gene Expression: Transcription The synthesis of an RNA copy of a segment of DNA

Gene Expression • 1909, Archibald Gerrod - alkaptonuria, a hereditary disease, was caused by the absence of an enzyme that breaks down a specific substrate, alkapton. • The symptoms of an inherited disease reflect a person’s inability to synthesize a particular enzyme

• 1930s, George Beadle and Boris Ephrussieach mutation affecting eye color in Drosophila blocks pigment synthesis at a specific step by preventing production of the enzyme that catalyzes that step. • Neither the chemical reactions nor the enzymes were known at the time.

One Gene – One Enzyme • Beadle and Edward Tatum - metabolism of a bread mold, Neurospora crassa. • They mutated Neurospora with X-rays and screened the survivors for mutants that differed in their nutritional needs. • Their results provided strong evidence for the one gene – one enzyme hypothesis.

Fig. 17.1 Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings

Human Diseases Due To Metabolic Disorders • Phenylketonuria, 1:12,000, chr12, gene for phenylalanine hydroxylase • Albinism, 1:33,000, tyrosinase • Tay-Sachs, 1:3,600, chr15, hexA, Nacetylhexaminidase, lysosomal storage disease, neurological degeneration • Cystic fibrosis, 1:2,000 in Caucasians, 1:17,000 in African-American; 1:90,000 in Asians, chr7, deletion of three nucleotides, ion transport through membranes is impaired

New Central Dogma • • • •

Not all genes encode proteins Not all gene transcripts are translated Reverse transcription can occur There are more than three types of RNA: - mRNA • - rRNA • - tRNA • - snRNA – small nuclear DNA

What is a gene?

A gene is a DNA sequence on a chromosome that  specifies the information that is required by the cell to make a specific gene product.

What is a gene product? The DNA sequence of a gene also specifies when a gene product  should be made, in what cells a gene product should be made and how  much of the gene product should be made DNA (genetic code) What  Where – tissue specific ( turn on/off) When How much ­ 

General Model For A Gene Structure

RNA Synthesis • RNA is synthesized using DNA as a template • Only one strand is read – template strand and nontemplate strand (sense and antisense) • Requirements for transcription: – Ribonucleoside triphosphate NTPs – DNA template – RNA polymerase

• Enzyme RNA polymerase catalyzes the process of transcription • Transcription occur unidirectionally, 5’ to 3’ • Only a segment of a DNA is transcribed

Most RNA when made in the cell is ssRNA. It’s general chemical structure is very similar to that of DNA, except for the 2’-OH and the use of uracil. DNA has a 2’-H in deoxyribose and thymine instead of uracil.

ssRNA is like water in that it will. . . ???

Transcription in E. coli (in prokaryotes) • Enzyme involved in transcription- DNA dependent RNA polymerase • Rna polymerase activity: some similarities, some differences to DNA polymerase activities

Similarities • Uses nucleoside 5'-triphosphates (NTPs) as precursors • Catalyzes phosphodiester bond between NTPs • Uses DNA as template • Base pairing determines correct base • Growth of nucleic acid chain only in 5’ to 3’ direction • Growing strand antiparallel to template strand

Differences: • Uses ribonucleoside 5'-triphosphates instead of deoxyribonucleoside 5'-triphosphates (ATP, GTP, CTP, UTP) • Can initiate the start of a new strand de novo (no primer necessary) • A single strand of RNA is produced (only one strand of DNA used for RNA synthesis: the DNA template strand with complementary base sequence) • Only short stretches of DNA are transcribed • Only one RNA polymerase to make all RNA, i.e., mRNA, rRNA, and tRNA – in prokaryotes.

E. coli RNA polymerase • Very large protein complex, consists of five subunits. • 2 identical alpha subunits and 1 each of beta, beta', and sigma. • The sigma subunit dissociates from the enzyme easily leaves shortly following initiation, critical for recognition of start of gene. • holoenzyme - complete enzyme - all five subunits together but basic polymerization reaction possible without sigma subunit • Core enzyme: 2 alpha, 1 beta and 1 beta'

RNA Polymerase • Core RNA Polymerase _________________ – Binds DNA – Catalyzes RNA synthesis but with no specificity – Cannot recognize promoter sequence

• Holoenzyme RNAP _________________ - σ reduces affinity to nonspecific DNA - Greatly increases affinity to promoter - Aligns RNAP with transcription site (+1)

RNAP Holoenzyme

Organization of control regions • The base where transcription starts is numbered +1 • Bases distal of the +1 base from the coding region are referred to as upstream and are number as: -1, -2, -3 ... • Those bases adjacent to the +1 base close to the coding region are referred to as downstream and are positively numbered. • The regions of DNA that bind RNA polymerase are called promoters. • The regions of DNA that bind regulatory proteins are called operators.

Promoters • E. coli promoters contain conserved DNA sequences (consensus sequences): – TATAAT, 10 nucleotides upstream if initiation site ( - 10 region), Pribnow box – TTGACA, - 35 region – cis-acting elements ( next to or on the same side)

Alignment of multiple promoters

Promoters • A sequence upstream of the start of the RNA coding sequence • Generally are found at -35 and -10 positions • Consensus sequence for -35 region is 5’TTGACA-3’ • Consensus sequence for -10 position is 5’ – TATAAT-3’ • The more similarity to the consensus sequence, the stronger the promoter

Organization Of Promoter Region In LacZ Operon

Promoters • The RNA polymerase core uses the -10 sequence (TATAAT) to bind and orient where to begin transcription. • The RNA polymerase sigma subunit uses the -35 (and -10 to some degree) sequence to bind and stabilize the polymerase complex.

Promoters • Control the efficiency of transcription • Strong promoters – initiation every 1-2 seconds • Weak promoters – initiation every 10-20 minutes • Mutations of the promoter region may reduce or enhance transcription

Promoters • Different sigma subunits can direct the polymerase to specific genes. Synthesis of new or unique sigma subunits can redirect the polymerase to transcribe a new set of genes • The strength of a promoter is related to its identity to the consensus sequence • Not all genes have the -10 and -35 sequence in the promoter.

Enhancers • Positive regulator proteins often interact with the RNA polymerase and stabilize it at poor promoters that do not closely match the consensus sequence • Enhancers can accelerate the conversion of closed complexes into open complexes • Many bacterial responses are controlled by two-component regulatory systems

Transcription in Prokaryotes • Three stages: – initiation – elongation – termination

Initiation of Transcription In Prokaryotes • RNA polymerase form a holoenzyme - core enzyme (subunits two α, one β and one β’ bound by sigma factor σ) • Holoenzyme binds to the promoter in two steps: – Binds loosely to -35 box (closed promoter complex) – Binds more tightly to -10 box, accompanied by untwisting of DNA (open promoter complex)

• Binding to a promoter allow correct orientation of the holoenzyme

How does the polymerase know what direction to go in?

Specific binding of RNA polymerase • RNA polymerase can bind to double-stranded DNA nonspecifically with low affinity ___________________ • The holoenzyme (with sigma subunit) binds specifically to both the -10 and -35 sequence with high affinity ---- resulting in a closed-promoter complex • The holoenzyme unwinds about 15 bases in double-stranded DNA around the initiation site for transcription ---- resulting in an open- promoter complex • Transcription starts by base pairing of two rNTPs that are joined

Initiation of Transcription In Prokaryotes • Sigma (σ) factor – several types – Allow the holoenzyme to recognize TATA box – TATAAT sequence is recognized by sigma factor σ 70 , protein 70,000Da

Initiation • RNA polymerase catalyzes the insertion of the first 5’-ribonucleoside phosphate, complementary to the first nucleotide at the start site • No primer is required • Process continues in 5’ to 3’ direction

Transcription initiation is critical because this is the step at which cells regulate which genes are actually turned on and which genes remain off.

Elongation of Transcription In Prokaryotes • • • •

DNA helix unwinds to form transcription bubble About 10 nt are synthesized σ factor separates from holoenzyme The core enzyme completes the transcription of the gene • As RNA polymerase moves, it untwists DNA ahead and reanneal DNA behind it • Transcription proceeds at a speed of 30-50 nt/sec • RNA polymerase has two proofreading activities

Gene sequences can be encoded on either strand of the double helix.

5’­TTTTTTTTTTTTTTTTTTTT­3’ 3’­AAAAAAAAAAAAAAAAA­5’

DNA double helix

What will the RNA sequence be of the gene encoded on the top strand?

Gene sequences can be encoded on either strand of the double helix.

5’­TTTTTTTTTTTTTTTTTTTT­3’ 3’­AAAAAAAAAAAAAAAAA­5’

DNA double helix

transcription of the gene on the top strand RNA polymerase uses the bottom strand  as a template to make RNA.

5’­UUUUUUUUUUUUUUUU­3’

Gene sequences can be encoded on either strand of the double helix.

5’­TTTTTTTTTTTTTTTTTTTT­3’ 3’­AAAAAAAAAAAAAAAAA­5’

DNA double helix

transcription of the gene on the  bottom strand RNA polymerase uses the top strand as a template to make RNA.

Elongation • Template strand (3’ to 5’ DNA) – RNA strand 5’ to 3’, complementary (sense RNA) • Nontemplate strand, 5’ to 3’ DNA, RNA sequence is identical (antisense RNA)

Elongation: • The core enzyme leaves the promoter site • The core enzyme travels along DNA template • The DNA is unwound in front and is rewound behind so that approximately 17 base pairs are always unwound • rNTPs are added to growing RNA via base pairing and phosphodiester bond formation

Termination: • The core enzyme encounters a termination signal • RNA is released from DNA template and from the enzyme • RNA polymerase dissociates from DNA

Termination • Enzyme reaches a specific nucleotide sequence that acts as a termination signal • Termination signal is about _____________________ in length • The sequence is actually transcribed into RNA • Newly formed transcript folds back on itself forming _____________________ secondary structure

Termination • Sometimes a termination factor rho (a large hexameric protein) facilitates the termination • Enzyme stops adding nucleotides • RNA transcript is released from template • Core polymerase enzyme dissociates

Polycistronic Transcription • Polycistronic genes Unique to prokaryotes • More than one polypeptide is encoded by a single strand of mRNA • Spacers (after stop codons) separate the sequences so that individual polypeptides can be translated as single entities

Polycistronic Transcription

Transcription And Translation In Prokaryotes • Prokaryotic DNA is located in cytoplasm and ribosomes are distributed in the cytoplasm • Allows for simultaneous occurrence of: – Transciption of DNA to mRNA – Translation of that mRNA to proteins