Lecture 20- RNA synthesis I

The central dogma

-DNA makes RNA makes proteins

transcription

-the process of making RNA from DNA

-major control point in the expression of genes and the production of proteins

-the generation of an RNA strand from a complementary DNA molecule

-RNA is not a direct copy of DNA, RNA contains ribonucleic acids instead of deoxynucleic acids (has U instead of T)

-tightly controlled

Machinery for RNA transcription

-highly conserved

-more simple in prokaryotes than eukaryotes

RNA polymerase

-the enzyme that catalyzes the synthesis of RNA

-RNA pol from E. coli is most well-studied

-large protein complex of 5 different subunits (ɑ, ω, β, β’, σ)

-σ subunit is also called a σ factor (responsible for where DNA pol sits then falls off)

Template strand

-anti-sense strand

-RNA pol uses the template strand of DNA to synthesize the RNA transcript

-RNA pol reads the template strand in the 3’ to 5’ direction and synthesizes in the 5’ to 3’ direction (causes RNA transcript to be the same sequence, except U replacing T, as the coding/sense strand of DNA)

-because proteins can be generated from mRNA transcribed in this way, the non-template strand is called the coding strand

because the core enzyme of RNA pol lacks specificity

-it can transcribe either DNA strand

-however, only the template strand contains the correct genetic info that can be used for protein synthesis

RNA polymerase holoenzyme

-ɑ₂ωββ’σ

-contains the sigma subunit that recognizes a promoter sequence within the DNA template

promoters

-regions of DNA that have specific sequences and signal a start site for transcription

-generally lie upstream (on the 5’ side of the gene that is going to be transcribed)

-promoter sequences are usually written out (5’ to 3’) based on the coding strand, even though the RNA polymerase binds and transcribes the template strand

promoter elements

-make up a core promoter

-transcription start site (TSS)

-minus 10 region (pribnow box)

-minus 35 region

transcription start site (TSS)

-the first base to be incorporated into the RNA chain and is said to be at position +1

-there is no “0” position on the DNA stand

-promoter in DNA not RNA

-10 region (pribnow box)

-often considered the first promoter element and is typically 10 bases upstream from the TSS

-35 region

-another promoter element that sits approximately 35 bases upstream of the TSS

-also called the -35 element

3 phases of transcription

-initiation

-elongation

-termination

initiation

-begins when the σ subunit guides the RNA polymerase to a core promoter region of DNA where it binds to form a closed complex, here the DNA is still hydrogen bonded to itself

-following binding, the RNA polymerase opens the DNA strand to an open complex, exposing the single stranded segments that can be read by the RNA polymerase catalytic subunit, this ends the initiation phase

elongation

-occurs once the strands have been separated; this is called a transcription bubble that moves down the DNA strand as the DNA is transcribed

-RNA pol catalyzes the formation of phosphodiester bonds between ribonucleotides taken from the environment; after about 10 nucleotides have been added, the σ subunit dissociates and is recycled to another RNA pol core enzyme

termination

-occurs after the fun RNA transcript has been generated

-intrinsic termination or rho-dependent termination

intrinsic termination

-controlled by specific sequences called termination sites, characterized by inverted repeats in the RNA that can form hairpin structures

-these structures cause the RNA polymerase to dissociate from the DNA strand, thus terminating transcription

rho-dependent termination

-involves the rho (ϱ) protein

-the ϱ protein binds the RNA and chases the polymerase until it catches up and facilitates the dissociation of RNA pol, thus terminating transcription

RNA transcription in prokaryotes

-highly regulated

-not all RNAs are produced at the same time and there is a high degree of temporal control over RNA production

four principal ways transcription in prokaryotes is controlled

-alternative σ subunits

-enhancers

-operons

-attenuation

alternative σ subunits

-these different σ factors can guide the RNA polymerase to different genes and are expressed based on the needs of the cell

enhancers

-these regions are far upstream (-60 to -150 bp) of a core promotor and serve as binding sites for transcription factors, which help to recruit RNA polymerase

operons

-regions of DNA group together proteins that are involved in the same biochemical pathway; this helps the cell generate such proteins at the same time

attenuation

-transcription is controlled after its begun by pausing the early release of incomplete RNA sequences; this keeps the cell from producing proteins that are not needed

operons

-a series of genes that are controlled and transcribed as a group

-ensures that proteins that are involved in the same metabolic pathways can be generated at the same time

-typically under control of the same promotor and are not continuously transcribed; under control of a co-inducer

co-inducer

-small molecules that serve to release a repressor protein from DNA and allow a promotor region to be recognized by RNA polymerase

inducible protein

-B-galactosidase: hydrolyzes the glycosidic bond of lactose, generating galactose and glucose

-transcription of B-gal gene takes place only in the presence of allolactose (a metabolite of lactose), which is the actual co-inducer

LacI gene

-continuously produces a protein called the lac repressor

-in the absence of a co-inducer, this protein binds to an operator site on DNA, which sits upstream of the genes that code for B-gal

-when bound to the O site, the lac repressor inhibits RNA polymerase from binding and keeps B-gal from being transcribed

-when lactose is present, a small amount gets converted to allolactose; this metabolite binds to the lac repressor and removes it from the O site, thus inducing expression of B-galactosidase