Transcription

gene expression

how genes produce things; the gene has the “recipe”/useful info, but something has to actually do the work to “bake the cake”

transcription

synthesizing an RNA molecule from a gene’s DNA sequence

nucleic acids

• information molecules

• deoxyribonucleic acid and ribonucleic acid

• polymers of individual nucleotides linked together by phosphodiester bonds

nucleotide sugars

• distinction between DNA and RNA (for us and enzymes)

• ribose has OH (hydroxyl group) on 2’

nitrogenous bases

• chemistry of nucleotides

• pyrimidines (CUT)

• purines (AG), bigger with 2 rings

• thymine and uracil have similar structures, so uracil will bind with adenine

what is RNA a polymer of

ribonucleotides

phosphodiester bond

bond between 5’ phosphate group and 3’ hydroxyl group

RNA polymer chain

• 3’ hydroxyl breaks high energy triphosphate bond

• energy is used to form covalent phosphodiesteer bond (3’ → 5’)

• RNA polymer chain will have 5’ to 3’ direction

central dogma

the process of getting from genes to gene expression with the end goal being to make protein

process of central dogma

gene has information that will be sent down through transcription/lation to later on produce protein

DNA → transcription → mRNA → translation

gene size

• VERY small

• 100,000 genes per chromosome

mRNA

• messenger mRNA

• intermediate messenger on the way to making protein

expression

make product of gene

“players” of transcription

• DNA

• mRNA

• transcription factors

• RNA polymerase

DNA

• double stranded

• gene with info

• ACTG

• has template that is used but unchanged during transcription

mRNA

• messenger RNA

• single stranded

• intermediate between transcription and translation

• ACGU (found in ribonucleotides)

• made complementary to the template strand

transcription factors

• proteins that bind DNA

• finds start of gene and tells a cell when it should make proteins in response to a cell signal or stimulus

• regulatory- tells RNA polymerase where to start transcription

RNA polymerase

• enzyme

• starts at beginning of gene

• 5’ to 3’

• breaks hydrogen bonds, briefly separating d.s. DNA to s.s to get a peek of template sequence to produce mRNA

transcription

making a transcript

template strand

read/used by RNA polymerase in making mRNA

which way are ribonucleotides added

• 5’ to 3’

• complementary and antiparallel to the template strand

who is doing the work in transcription

RNA polymerase

• 5’ → 3’ RNA polymerase activity = individual RNA triphosphates come in and RNA polymerase takes hydroxyl group on 3’ end and breaking 5’ triphosphate on incoming nucleotide to form phosphodiester bond

• unzipping/helicasing d.s. DNA to see template

what is being catalyzed in transcription

phosphodiester bond

where does the energy for transcription come from

triphosphates (bonds) of incoming nucleotides

which DNA strands are antiparallel and complementary to each other

• non template to template

• template to mRNA

how does RNA polymerase know where to start and stop transcription

the termination signal and promoter on the gene

gene

unit of transcription because the start and stop of the gene is where the start and end of transcription is

termination signal

• where transcription stops

• stretch of DNA with specific sequence on both strands (not just template)

promoter

• promotes transcription/where transcription starts

• stretch of DNA with specific sequence on both strands (not just template)

transcription factors

• binds to promoter

• tells polymerase where template is and where the start is so it knows which way to transcribe

can both strands of DNA serve as template strands

yes

• gene 1 can use top template, gene 2 can use bottom

• promoter is specific for gene, telling it where to start

• 50/50

steps of prokaryotic transcription

1) initiation

2) elongation

3) termination

prokaryotic initiation

• gene with promoter and termination

• sigma protein binds to promoter and indicates template, there is a transcription bubble

• RNA polymerase binds to start of the gene because it feels the sigma bonded to the d.s DNA

• sigma bond recognizes -10 sequence and binds to open DNA so RNA polymerase can recognize and then bind

sigma protein

• binds to promoter and indicates template

• many different ones that are specific for specific genes because it will not make proteins all at once

• recognizes specific promoters that need to be turned “on”

consensus sequence

most common nucleotide at each space from each gene

prokaryotic elongation

• RNA polymerization in 5’ to 3’ direction, complementary to template

• individual nucleotides being added

• sigma protein floats away once RNA polymerase binds and starts

• RNA polymerase moves away from the promoter, binding and making DNA complementary to the template

• transcription bubble forms so there is enough space to do transcription, then DNA will come back to being d.s.

prokaryotic termination

RNA polymerase hits termination signals then disassociates and floats away

eukaryotic transcription steps

1) initiation

2) elongation

3) termination

eukaryotic initiation

• start with d.s DNA

• MANY transcription factors bind to promoter

• RNA polymerase recognizes and binds, the TF tells RNA polymerase the template strand and direction so it can start transcription

TATA box

• TBP TF

• TATA-binding protein

• consensus sequence necessary for initiating transcription

eukaryotic elongation

same as prokaryotic

eukaryotic termination

same as prokaryotic

would a promoter region need to be inserted in from of the human insulin gene

yes because we need to include a bacterial promoter since all the machinery that is going to be doing transcription is bacterial

when a sigma factor binds to DNA, what is the nature of the binding

noncovalent bonds because it just interacts (does its job, then floats away)

when RNA polymerase binds sigma factors, what is the nature of the binding

noncovalent bonds because it just interacts (does its job, then disassociates and floats away)

in eukaryotes, when is RNA processed

after transcription

steps that need to occur to primary transcript in eukaryotes so the transcript will be ready for translation

1) 5’ cap added on 5’ end of transcript

2) Poly(A) tail added

3) removal of introns

mature mRNA

• ready to be used for translation

• exons stuck together

where do the steps of preparing the primary transcript in eukaryotes for translation occur

in the nucleus, once it is complete, it is sent to the cytoplasm

5’ cap

• step 1 of getting primary transcript ready for translation in eukaryotes

• modified nucleotide

• stuck on 5’ end of mRNA

• 5’ to 5’ is unique which signals to cell that it is the 5’ end of mRNA

• it also protects the unstable mRNA from degradation

poly(A) tail

• second step in getting primary transcript ready for translation in eukaryotes

• added to 3’ end

• unique (100s of As) and signals to cell to export to cytoplasm

• protects unstable mRNA from degradation because if a few As are lost, the cell will be fine

removal of introns

• 3rd step of getting primary transcript ready for translation in eukaryotes

• noncoding RNA (contains no info) that does not end up in final product of mature

splicing

• removes portions of genes that are unneeded

• in pre-mRNA introns are removed (phosphodiester bond cut), so exons join together for final product

• exons are kept in mRNA

introns

non-coding DNA/RNA; removed

exons

• code for protein and extra RNA

• not all exons and all portions of exons code for proteins though

• kept in mRNA (because not everything kept in mRNA codes for proteins)

alternative splicing

• allows one gene to code for more than one protein

• produces final mRNA

• occurs in nucleus

• one or the other (not keeping all of the exons)

pre-mRNA

spliced to form mRNA

when splicing can you make a transcript that rearranges the order

no (ex: 1, 3, 2)

this can happen though: 1, 3, 4

how many ways can molecules of mRNA be spliced

one way

how many ways can a cell splice

hundreds of ways because transcription can produce hundreds of copies of transcripts from that one gene (keeping certain exons)

• certain cells keep certain exons (ex: muscle cells)

what happens to exons that are not used

their individual ribonucleotides can be recycled

eukaryotes RNA polymerase

• RNA pol I

• RNA pol II

• RNA pol III

what does RNA pol I transcribe

ribosomal RNA

what does RNA pol II transcribe

• genes that code for protein

• any gene that goes that full step of transcription, then translation

what does RNA pol III transcribe

transfer RNA (tRNA)

how does initiation in transcription differ between prokaryotes and eukaryotes

prokaryotes: promoter DNA sequences are recognized by the sigma protein transcription factor

eukaryotes: promoter DNA sequences are recognized by many transcription factors (TATAA)

do prokaryotes have a 5’ cap tail in transcription

no

do eukaryotes have a 5’ cap tail in transcription

yes, for signaling and protection

do prokaryotes have a 3’ poly(A) tail in transcription

no

do eukaryotes have a 3’ poly(A) tail in transcription

yes for signaling and protection

do prokaryotes have splicing in transcription

no

do eukaryotes have splicing in transcription

yes, for signaling and protection

in transcription, how many RNA polymerases do prokaryotes have

one

in transcription, how many RNA polymerases do eukaryotes have

three

• RNA pol I (transcribes ribosomal RNA)

• RNA pol II (codes proteins)

• RNA pol II (transcribes transfer RNA)

can most genes be transcribed from either strand of DNA

no

• must start at promoter and mRNA is made 5’ → 3’ being added to 3’ end

• each gene can only be transcribed from one strand

• different genes can be transcribed from different strands