Biotech 14: RNA Structure, synthesis, processing, and transciption

where does transcription take place?

where does translation take place?

transcription = DNA—>RNA = nucleus

translation = RNA—> Protein = cytoplasm

can genes be expressed more than once?

YES if you need more than just one protein the gene can be expressed how ever many times you need that protein

what is the only difference between ribose and 2-doxyribose?

the second carbon has an OH and H in ribose and 2 Hs in the 2-deoxyribose

the first carbon is the one attached to the nitrogen base (in ribose the one next to it

the second carbon is between the phosphodiester bond and phosphate (ribose has OH and H deoxyribose has 2H)

Can RNA be both single and double stranded?

NO RNA IS SINGLE STRANDED

what are the three nucleotides that are the same for DNA and RNA

where do they differ?

same for all 3

• Adenine

• Guanine

• Cytosine

differ:

DNA = thymine RNA = uracil

what is mRNA and what does it do?

messenger RNA codes for proteins

What is transcription?

Hydrogen bonds temporarily formed between DNA and mRNA

these bonds are later released as RNA separates from DNA (single stranded)

what are some similarities between transcription and DNA replication

what are some differences?

Similar: DNA unwinding and complementary base pairing

Difference: Hydrogen bonds are not permanent between complementary base pairs and RNA is much shorter as the entire strand of DNA does not get translated all at once (RNA = few thousand DNA = 250 million)

each transcribed segment of DNA is a ________________ ___________ which encodes for one/many genes?

transcription unit = ONE gene

RNA is complimentary to the ___________ strand and identical to the ___________ stand (except U instead of T)

RNA complementary to template (3 to 5)

RNA identical to coding (5 to 3)

what is found upstream from the transcription start site and coding sequence

what is found downstream?

upstream-= regulatory elements = enhancer + promoter

downstream = enhancer

which enzyme catalyzes transcription ?

RNA polymerase

Can RNA polymerase start transcription without a primer?

In which direction does the new nucleotide chain grow?

are there more mistakes in DNA replication or transcription?

YES

5 to 3

transcription

how many polymerases are there in eukaryotic cells?

3

which polymerase is used to create mRNA?

RNA Polymerase II

Rifadin (rifampicin) is a antibiotic. What does it do? What can it treat?

inhibits bacterial RNA polymerase so bacterial proteins cannot be synthesized leading to death

example: tuberculosis

why are alpha—amanitin known as the death cap mushroom?

they inhibit RNA polymerase 2 which is VITAL to make proteins we need to live

Dactinomycin (acitinomycin D) is chemotherapy drug which disrupts RNA polymerase 1. What effect does this have on cancer cells and why is it fatal to normal cells?

RNA polymerase 1 creates rRNA which helps in translation

loss of proteins in normal cells is detrimental

BASIC steps in transcription

1. initiation

2. elongation

3. termination

4. Processing to RNA

Initiation:

what transcription factor binds to the promotor?

where specifically on the promotor does this transcription factor bind to?

how does the transcription factor bind to the promotor?

what happens once they are bound to each other?

once the transcription factor is bound to the promotor what other proteins bind to it?

• transcription factor = TF2D

• TATA box on promotor

• TBP (TATA-binding protein)

• conformational change

• TF2B, A, F, E, H

what makes up the initiation complex?

General Transcription factors + RNA Polymerase

General transcription factors =

TF2D bound to TATA of promotor with TBP

+ TF2B TF2A TF2F TF2E TF2 H

enhancers are ______- regulatory elements located upstream (_____’) to the promotor

cis 5”

• the binding of __________ ________ to an enhancer can contribute to the efficient binding of the ______ __________ to the promotor

• these _________ __________ usually interact with a __________ complex which allows for an interaction between the ______________ and the components of the ______________ complex

• this complex can also recruit

• transcription activators initiation factor

• transcription activators mediator activators initiation

• chromatin-modifying enzymes that modify histones

what is the point of trnascription activators binding to enhancers and attaching to the TATA box through a mediator??

STRENGTHENS connection between promotor (TATA box) and initiation complex

ELONGATION:

how does elongation start?

where does RNA polymerase begin to synthesize mRNA

what happens during elongation?

does the initiation complex remain intact throughout elongation?

• TF2H uses ATP to break apart the initiation complex and phosphorylates C terminal domain (CTD)—tail of RNA polymerase 2

• RNA polymerase adds the first complementary ribonucleotide to the deoxyribonucleotide at the Transcription Start Site (TSS)

• temporary hydrogen bonds between Deoxyribonucleotides and ribonucleotides

• phosphodiester bond form between ribonucleotides

• NO breaks apart

what causes the release of transcription factors from the initiation complex?

TF2H uses hydrolysis of UPT, ATP, CTP, and GTP to provide enough energy to release initiation complex components including itself

what are the 3 roles of TF2H?

1. pull apart DNA strands at Transcription Start Site (TSS) using ATP hydrolysis

2. phosphorylate serines on c-terminal domain (CTD) of RNA polymerase 2

3. hydrolyze UTP, ATP, GTP, CTP so the initiation complex can be released

why does TF2H phosphorylate the CTD of RNA polymerase?

• provide docking site for NEW PROTEINS TO BIND TO RNA POLYMERASE 2 (elongation factors+ RNA processing and modification of histones)

• ensure that RNA polymerase 2 wont dissociate before reaching the end of the gene

where do elongation factors bind?

phosphorylated c-terminal domain (CTD) of RNA polymerase

made possible by TF2H and ATP hydrolysis

what are some examples of elongation factors and what do they do?

ELf1

Spt4 and 5

make sure RNA polymerase does not terminate before it completes transcription and modify histones

without _________ transcription only occurs at basal level

________ increase the level of transcription

binding of _____________ __________ __________ to ____________ can contribute to the binding of general transcription factors and RNA Polymerase 2 to the promoter

• enhancers

• enhancers

• activating transcription factors to enhancer

what are the two roles of the mediators?

1. bind enhancer to initiation complex

2. position TF2H near the tail of RNA polymerase II so it can phosphorylate it and begin elongatioN!

what are the two functions of activating transcription factors (enhancer binding proteins) ?

1. bind modifier to enhancer to initiation complex

2. recruit chromatin-modifying enzymes that can lead to changes in expression

How does RNA polymerase know when to stop?

once RNA polymerase encodes AAUAAA (TTATTT on DNA) also called polyadenylation (poly-A) signal and GU rich region (CTCTCT on DNA) it will stop

what happens once RNA polymerase reaches the polyadenylation cycle and GU rich regions?

what binds to the AAUAAA region? What binds to the GUGUGU region?

Cleavage and Polyadenylation Specificity Factor (CPSF) = AAUAAA

Cleavage Stimulation Factor (CSTF) = GUGUGU

ONCE BOUND THEY RECRUIT CLEAVAGE FACTORS

once CPSF binds to polyA singal and CSTF binds to GU rich region what happens next?

CPSF and CSTF cleave RNA downstream poly- A signal (left so 5’) but upstream GU rich region (not including Poly-A)

mRNA released from DNA !

is the AAUAAA the only signal that elicits termination of transcription?

NO

after termination what are you left with?

pre-mRNA

what three modifications must be made to turn pre-mRNA to mRNA that can be sent to the cytoplasm? IN ORDER

1. 5’ capping

2. Splicing

3. Polyadenylation (Poly A-tail)

the 3” end is modified by _________
the 5"‘ end is modified by a _______________

3’= poly A- tail

5’ = capping

the 5” cap is a methylated (not expressed) _____________ nucleotide called _________________________ (m7G)

when is the cap added?

what does the cap do?

• methylated guanosine called 7methylguanosine

• soon after transcription begins

• helps PROTECT mRNA from degradation and helps PROMOTES TRANSLATION INITIATION

the 3’ poly (A) tail is made up of 250 __________ nucleotides added to the end of mRNA

when is it added?

what does it do?

adenosine

protect mRNA from exonucleases

facilitate export of mRNA from nucleus into cytoplasm for translation

promote translational efficiency

SEQ: 5’ RNA Capping

1. Phosphatase turns triphosphate at 5” end into diphosphate

2. 2 phosphates are taken away from free GTP leaving GMP (monophosphate)

3. GMP is added to diphosphate through Guanyl transferase

4. methyl transferase adds methyl to guanosine at position 7 forming Cap0

how many methyls have been added to guanosines that have Cap 0,1, 2?

cap0 = 1

cap1 = 2

cap3 = 4

function of 5’ RNA cap

• distinguish RNA made by RNA polymerase 2, no other polymerase produces cap because they don’t have CTD on tail to be phosphorylated by TF2H

• cap binds to CBC (cap binding complex) in the nucleus helps the mRNA be further processed and exported

• translation of mRNA in the cystol

at each side of an intron/ exon junction of the mRNA there is a _____________________ _________ ______________ that is similar in all genes and provides information about where splicing will take place

consensus nucleotide sequence

In the process of _________ removal during splicing a structure called the ______ forms and the ____________ gets spliced out

intron lariat intron

Splicing is performed by complex called the ___________ which consists of _______________________ and ___________

spliceosome

small RNA molecules and proteins

what are the small RNA molecules that make up the spliceosome

U1, U2, U4, U5, U6

SEQ: spliceosome/ RNA molecules action

1. _______ base pairs with the 5’ splice junction

2. and 3. _________ and __________ bind at the branch point and the 3’ splice junction

3. ______ displaces _______ and ________

4. Triple Complex (______/_____+ ____) bind along with _____

5. a loop forms and _____ will displace _______

6. _____/______ protein complex positions the entire complex to bring the branch point close to the ______ splice site

1. U1

2. BPP and U2AF

3. U2 displaces BPP and U2AF

4. U4/U6 +U5 bind along U2

5. U6 U1

6. NTC / NTR (nineteen complexes and nineteen related complexes) 5’

what happens once the NTC/NTR Protein complex positions the entire complex to to bring the branch point (3' end) close to the 5” splice site?

1. GU from ___’ end will interact with A on ____’ end

2. This will eventually lead to a cut in the sugar-phosphate backbone at the ______” end leaving a free OH group at the ___” end of the ___” exon

3. Now the end of the intron which still has the _____ sequence covalently binds to the ____ at the 3” branch point

4. The structure rearranges so that the free _____ group of the 3’ exon can interact with the 5’” end of the 3” exon and the ___________(intron) is released and degraded

5. the two exons are now joined together!

1. 5’ 3”

2. 3’ 5’

3. GU A

4. OH lariat

the presence of ________ and ____ allows for the possibility of alternate splicing, increasing hte diversity of genes that can be produced

introns and exons

splicing of mRNA can lead to differed results including

smooth muscle mRNA

fibroblast mRNA

brain mRNA

the Poly A tail is a string of up to ______ or more adenine nucleotides added to the ____” end of the mRNA

when is it added?

250 3’

AFTER cleavage (termination) and splicing

the enzyme _____________________( ) catalyzes the addition of adenosine nucleotides to the 3’ end of the sequence

_______________________ bind to the tail and help protect and stabilize it as it lengthens to its final size

Poly- A polymerase (PAP)

Poly- A binding proteins (PABP)

is the poly-A tail added before or after the AAUAAA sequence?

a little after AAUAAA sequence

______________________ adds about 200 adenosine nucleorides at the 3’ end

what recruits this enzyme to the RNA?

what assembles into the poly-A tail determining its final length?

Poly -A polymerase (PAP)

Cleavage and Polyadenylation Specification Factor (CPSF)

Poly A -binding proteins (PABP)

what helps in

• mRNA stability

• mRNA export from the nucleus

• translation

• regulation of gene expression

poly A tail

what happens to spliced out introns? do they go into the cytoplasm?

they are destroyed in the nucleus

THEY DO NOT GO TO THE CYTOPLASM

what leaves the nucleus? RNA or mRNA?

RNA

Processed mRNA are guided through _______________ ___________ ______________ ( ______) where they leave the nucleus and enter the cytoplasm

nuclear pore complexes (NPC)

how big is the Nuclear Pore Complex? What is needed for mRNA to squeeze through? what receptors are involved?

pores are SMALL

energy (ATP) needed to actively transport mRNA though pores

NUCLEAR TRANSPORT RECEPTORS