Transcription

Learning Objectives:

  • List and describe the main types of RNA

  • Describe transcription, including steps in transcription and how the polymerase functions

  • Define the coding, template, noncoding, nontemplate, and strands

  • List subunits of prokaryotic RNA polymerase and describe their purposes

  • List and compare eukaryotic and prokaryotic promoter elements

  • Describe the function of sigma factor, and how sigma factors are used to control transcription.

  • Describe RNA proofreading mechanisms

  • Describe termination in prokaryotes. Compare intrinsic and rho-dependent termination

  • Describe TFIID and TFIIB in eukaryotes. Explain how they help initiate transcription

Transcription is copying the information in DNA into RNA:

  • Makes it useful to make proteins

    • Central dogma

    • Most RNA is not translated

      • Based on volume, the vast majority is not.

      • most not made into new proteins

  • Three types of RNA:

    • rRNA: Ribosomal RNA, makes up the RNA, and carries out the chemical reactions in the ribosome

      • Run the ribosome in electrophoresis, get two big bands, which is rRNA; the rest is very small

    • mRNA: Messenger RNA, stuff that gets transcribed into RNA, goes to ribosomes and makes the proteins. Messenger between the DNA and the ribosome

      • Most regulated and the most changing

    • tRNA: Transfer RNA, the smartest molecule; speaks both nucleic acid and amino acid

      • An adaptor molecule in between mRNA and the protein

  • The most prominent way of regulating a particular gene pathway is through transcription

    • regulate whether it gets transcribed and still even then weather it gets translated, and if protein is made, whether or not it works

    • Want gene, turn on RNA pol, to make the gene

    • Based on energy, and regulates on the transcriptional level to save energy on the other levels

    • Clotting factors different, becuase we could be dead without it, so it gets cleaved.

    • Energy efficent or fast

All comes back to DNA

How transcription normally works, transcritpion bubbble

RNA:

  • Is a polymer, like DNA

    • Different from DNA because it has the extra OH group on the 2’ carbon

    • Single-stranded, doesn’t have a backup copy of itself

      • very short-lived and can just make another copy of itself

      • it can fold itself up, base pairs within the molecule

      • more reactive than and more unstable than DNA, so can do more chemical reactions

      • Different RNA’s have different purposes

        • Think that there’s mRNA, tRNA, and rRNA all with different jobs, DNA has the same job across the board

  • THINK: Active, transient, reactive, and alays doing stuff

Strand names:

  • Each strand being copied and strand not being copied has a name and a nonname

  • Sequence of RNA is going to be the same as the coding strand

    • ALWAYS 5’-3’

    • Code in DNA is the same as in the RNA

    • Also, the nontemplate strand becuase it’s not the templat

  • Template strand: what is actually physically being coipied by the RNA

    • Also, known as the noncoding strand

RNA is made by RNA polymerase:

  • RNA polymerase basically does the same thing as DNA polymerase

  • The chemical reaction that forms DNA is the same that forms RNA

    • Attacks 3’ hydroxyl group

  • RNA uses uracil instead of thymine

    • everywhere where there is a T in DNA there’s a U in RNA

    • Because RNA is constantly being made and destroyed if the C becomes a U, if it happens a couple of times it’s not a big of a deal

  • RNA polymerase doesn’t need a primer

Prokaryotic RNA polymerase:

  • β, β’ subunits: where the RNA catalysis happens, where nucleotides come together

  • ω subunit helps with assembly

  • α subunits: Help with assembly and also regulatory, bind to other proteins

    • regulatory and structural

What it looks like in prokaryotes

Eukaryotic RNA polyemerase is similar to prokaryotic

is bigger

Cycle of transcription:

Initiation:

  • The start of transcription

  • Starts at the closed complex

    • Have a promoter and RNA polymerase stuck together

      • Promotor: sequence of DNA that RNA polymerase recognizes

  • Then become an open complex

    • RNA polymerase goanna take that DNA

    • and open it up a little bit

    • forming the transcription bubble

Promoter:

  •  DNA sequence that is recognized by RNA pol. 2

  • Mark’s beginning of transcription

  • NOT LIKE A START CODON

  • Can be different promoters

    • Could have a strong promoter for genes you want on all the time

    • could have weak promoters that aren’t bound to the polymerase often for genes you don’t want on all the time

      • So less RNA is being made

    • A better promoter is recognized by the RNA, the more transcription that can occur

  • Position and orientation dependent

    • The way the RNA polymerase faces determines which direction the RNA polymerase goes

  • Sometimes, different combinations of DNA sequences that make up the promoter

Bacterial promoter:

  • The core promoter has a -10 box and a -35 box

    • That many nucleotides upstream from where transcription starts

    • bacterial promoter is 10 and 35 nucleotides upstream from the start of RNA transcription

    • 25 nucleotides apart because that’s the amount of DNA it takes to go all the way around

  • +1 site where transcription starts

    • goes from -1 to +1 where transcription starts

  • UP element: not necessary for transcription, but an A-T-rich sequence of DNA that helps transcription begin,

    • Helps RNA pol. to bind the promotors

  • Have a -35 box, a -10 box, and an UP element will make a pretty strong promotor, and will get lots of RNA

Consensus Sequences:

  • If you took all the promoters in all bacterial sequences and went, what’s the most common nucleotide at -10, -11, or -13, that’s the consensus sequences

    • Standard sequences for the promoters

    • -10: TATAAT

    • -35: TTGACA

  • If the standard sequence is then, RNA polymerase will be able to recognize, and it will be a strong promoter

    • So closer to the exact sequence, the better it will be at transcription

    • Somehow modify something to cause transcription to stop or start

    • Most promoters don’t function unless you do something to make it work.

Sigma factor:

  • Is the intermediary factor that the RNA polymerase binds to

  • Recognizes and binds to the promoter

  • Sigma 70 default factor

  • If in another state, it can change to where it would recognize a different -10 and -30 promotor box, to where it can become resistant to whatever is trying to kill it

  • Need both regular and heat-shock promoters, then you have two promoters, one for each

  • Use if you want to change your entire transctopional system

  • Go from one set of genes to another to get another sigma factor

Structure-function of RNA polymerase (elongation):

  • Making the RNA

  • Have an entry channel where DNA polymerase takes in DNA, or RNA pol. is moving along DNA in that direction

  • Pin pulls DNA apart and holds apart

  • RNA exist channel is where RNA that is being synthesized exits

  • The rNTP entry channel is where the nucleotides enter in.

  • Once made 6-8 nucleotides will hold RNA pol on DNA, so it can keep going

Kinetic Proofreading:

  • Depends on speed/rate of reaction

Nucleolytic proofreading:

  •    RNA polymerase is zipping along when all of a sudden it makes a mistake, and has a internal nuclease

    • cut out a whole chunk of nucleotides that were made and repairs the mispair

  • Lysing, cutting the chain

Termination:

  • End of transcription

  • RNA pol. needs to know when/where to stop

  • Need to have a stop signal

  • 2 ways:

    • intrinsic termination

      • hairpin: folds up on itself

    • and Rho-dependent termination

      • Protein inside bacterial cells

      • Jumps on when there is a rut

      • races after RNA Pol. and then once it reaches a hairpin, Rho is able to catch up, and it will kick RNA pol. off the DNA

    • Both use a hairpin structure

Eukaryotic Transcription:

  • Three different polymerases:

    • Polymerase 1: rRNA

      • Mostly makes rRNA, very busy

    • Polymerase 2: mRNA

      • transcribes messenger RNA, most regulated out of the bunch

    • Polymerase 3: tRNA

      • transcribes tRNA, decodes mRNA

  • All are regulated through:

    • Basal and specific transcription factors:

      • Transcription factor: a protein that binds to DNA and affects transcription

        • Different for each gene, different combinations

      • Basal transcription factor: required for all transcription

        • EX: TF2D

  • promoters are specific fo each polymerase

Eukaryotic Promoters (pol II):

  • Where RNA pol. binds and starts in prokaryotes

  • 4 possible promoter elements in Eukaryotes:

    • The TATA box

    • B response element (BRE

    • Downstream promoter elements

    • or the initiator (INR)

  • Have to have at least 2 of the four for it to successfully start transcription

    • Have to have the initiator and the TATA box for it to work

    • Mostly get bound to general transcription factors in prokaryotes

Eukaryotic basal transcription factors (pol II):

  • TF2D is a complex of differnt proteins so it can bind to different things such as the TATA box or the initator

    • So why need two to allow the factor to bind and do its thing

  • Once binds recruits TF2B which then binds to TBP, and positions the polymerase