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Information and the central dogma

•  DNA as information storage

•  mRNA as information carrier
–  Produced through a process called transcription
–  mRNA is a short-lived copy of the instructions carried in DNA (instances where mRNA lives longer in cell while they're bing translated

1

Different RNAs

Messenger RNA - mRNA
Ribosomal RNA - rRNA
Transfer RNA - tRNA
(Translation)
MicroRNA-miRNA (gene expression)

2

Structure of RNA

Single stranded

Secondary structure can form hairpins

Secondary structures in mRNA can control how transcription can end or how translation can start

3

Transcription in Prokaryotes

•  All RNA’s are made by transcription

•  Template - DNA, DNA dependant RNAsynthesis

•  Enzyme - RNA polymerase helps form RNA DNA hybrids

•  Substrates - Ribonucleoside triphosphates that are added in

•  Only one of the two strands are copied into RNA for a given gene

4

What is special about the first base added in on the hybrid?

RNA pol does not require hydroxyl group to add a base. As long as RNA pol is bound to DNA at a specific part of the enzyme, the complimentary RNA base will be incorporated in on the DNA template, and then every base from then on can use that 3' hydroxyl. First base is special, added based on complimentary base pairing

Additionally, keeps its three phosphates

5

Which DNA strand is copied?

Template strand is what RNA is made from. (Antisense). mRNA that is formed has same base or as sense strand except with Us instead of Ts

6

What do 5' and 3' refer to?

Carbon positions on the sugars of the ribonucleotides

Need to go this way because of the hydroxyl on the 3' carbon

7

What kinds of bonds are formed during synthesis of RNA from DNA template?

Phosphodiester bond formed from RNA polymerase after base pairing occurs. Nucleophilic attack on the 3' OH

Hydrogen bonds between complimentary base pairs

8

The importance of base pairs

Patterns of inverse base pairs are different in the major groove

9

The holoenzyme

RNA polymerase associated with a protein called sigma

Active Site - Mg 2+

It has channels for the DNA to go through and for new RNA bases to come in, and mRNA to trail out

10

What are the five subunits of the holoenzyme?

σ factor recognizes the promoter, binds first

α2β2 subunits-constitutes the core polymerase that catalyzes synthesis of RNA

11

Where is the promoter region in prokaryotes?

-35 and -10 nucleotides upstream from the start site

These consensus sequences are conserved

12

What does sigma do?

Recognizes promoter region (usually sigma 70)

13

How did researchers discover what sigma does?

•  When researchers mixed RNA polymerase, sigma and DNA together in cell free cultures (lysed cells) they found that the holoenzyme would bind only to specific sites on the DNA

•  Sigma is a regulatory protein

they didn't include sigma, they didn't get the same results. Sometimes enzyme would associate, but not as strong.

14

UTR

Untranscribed region, upstream of start point

15

Start point

Where the first RNA base is added

16

Pribnow box

-10 box

17

Specificity of sigma factors

Some sigma factors factors that are specific for certain genes, and those genes will only be transcribed when sigma factor is present.

18

Steps in prokaryotic initiation:

•  Formation of the “closed complex”

•  Unwinding of DNA to yield the “open complex”

•  Synthesis of 5-10 phosphodiester bonds

•  Release of sigma factor- once the RNA begins to form it “outcompetes” with sigma factor and it is displaced

19

Formation of "closed complex"

At first there is a loose association between DNA and core enzyme, it is positioned a site where first base will be added (but RNA chains that begin are not located at the proper sites)

Closed complex- when sigma and RNA polymerase enzyme bind to the promoter region.

20

Unwinding of DNA to yield the “opencomplex”

Sigma opens the DNA helix and transcription begins

Small open complex formed. Its reaction is exergonic.

21

Two hypotheses for initiation

One hypothesis is that sigma associates first and then scans for the promoter region.

The second hypothesis is that sigma binds to the promoter and then it brings in the polymerase and then the holoenzyme is formed on the DNA (accepted)

22

The start of RNA

RNA is initiated with the binding of two rNTPs (ribonucleotide triphosphates) and the formation of the first phosphodiester bond.

23

Synthesis of 5-10 phosphodiester bonds

5-10 bases added in, and the the DNA template's affinity for RNA polymerase goes up, and it is no longer associated with sigma as tightly and sigma will fall off.

After about 10 base pairs the holoenzyme will disassociate to a degree because the sigma factor will no longer be associated with the polymerase

24

Zipper

Helps orient the two DNA strands back together

25

Rudder

helps separate the DNA bases as the mRNA is being made, and stabilizes ssDNA

26

Prokaryotic termination

Rho (a protein) dependent or independent

27

Rho independent

The termination site is heavy in As, which forms Us on 3' end of mRNA

A stem loop (hairpin) is formed that is a palindromic sequence

Only 8 or 10 nucleotides in the RNA DNA hybrid, and Au base pairs (RNA DNA hybrid) weakly associated and the hair pin is stable

hair pin is in the exit channel in the polymerase which stalls the polymerase and causes it to disassociate.

28

What are the factors in disassociation at the t site in prokaryotes?

Stability of hairpin
Where it ends up in the polymerase (exit channel)
Weakness of AU bases

29

Rho dependent termination

Still form hairpin that disrupts polymerase that is stuck in exit channels that stalls polymerase.

During that, rho protein encircles mRNA and spins around it trying to catch up to the polymerase and when the polymerase stalls, rho can catch up. It has helicase activity and it helps to disassociate the DNA RNA hybrid, helps to pull off mRNA.

Rho and pol fall away

30

Similarities in prokaryotic and eukaryotic transcription

•  Both processes go from 5’ to 3’
•  Transcription initiates at a promoter
•  Both utilize RNA polymerases
•  Regulation of transcription initiation is the most common mechanism for control
•  Both involve other transcription activators and repressor proteins that bind to specific DNA sequences and influence transcription rate

31

Differences between prokaryoticand eukaryotic transcription

•  Eukaryotes have 3 different RNA polymerases
•  RNA polymerases have more subunits in euk
•  Prokaryotic promoters are recognized by a subunit of the polymerase. In eukaryotes, the core promoter often contains aTATA box at -30 that is recognized by TBP. TBP recruits the RNA polymerase
•  For prokaryotes “promoter” refers specifically to the RNA polymerase (Holoenzyme) binding site. In eukaryotes, this refers to all of the protein recognition sites
•  In eukaryotes there are many more transcription factors.

32

Eukaryotic Transcription

The initial RNA transcript (primary transcript, HnRNA (hetero nuclear RNA)) is clipped apart and spliced back together!

The mRNA can be as small as 10% of the size of the primary transcript, there are exonsand introns in eukaryotic genes

Lots of regulatory elements

33

Eukaryotic polymerases

•  Pol I - transcribes ribosomal RNA
•  Pol II - transcribes protein coding genes
•  Pol III - transcribes tRNA and other small RNA’s

All of these contain two large subunits and 12-15 smaller subunits

34

How did researchers discoverthe different polymerases?

•  Death cap mushroom and alpha amanitin
•  Different concentrations had different effects.
•  Low Pol II
•  High Pol III

Low concentrations of the poison, pol II activity stops. mRNA can't be made. Cells can't make proteins, some of those proteins detoxify poisons. Also affects pol III, tRNAs.

35

Eukaryotic mRNAs are produced in distinct steps

•  Synthesis of a primary transcript
•  Modification of the 5’ and 3’ ends of the transcript
•  Introns are spliced from the primary transcript
•  Remaining coding sequence is comprised of exons

36

Alternative splicing

all of introns and some exons removed, gene can code for more than one protein

37

RNA pol II transcription unit

Core promoter

Unicellular: Upstream activating sequence

Multicellular: Long range regulatory elements(insulators and enhancers), can be 700-1000 bp away

38

Enhancer elements

They have to be in cis because enhancer elements are on the DNA. Has to be in line with that coding sequence.

Trans regulatory elements can be diffusable- move around in the nucleoplasm Ike RNA pol II.

Enhancers can be anywhere in the sequence

39

Insulators

Euchromatin is transcriptionally active while heterochromatin is not (Some DNA is silenced because it remains tightly bound to the histones)

Insulators keep transcription machinery from being fooled if its surrounded by heterochromatin. There's no way for transcriptionally inactive DNA to inhibit transcription of eukaryotes

Some DNA is silenced because it remains tightly bound to the histones

40

Steps in Initiation for eukaryotes

•  Binding of general transcription factors and RNA polymerase II to promoter
•  Unwinding of DNA
•  Phosphorylation of carboxyl terminus of RNA polymerase II (Post translational modification)
•  Synthesis of first 5-10 phosphodiester bonds
•  Release from promoter and most general transcription factors.

41

Assembly of Active Transcription Complex

–  General Transcription Factors (TFIID, TFIIA, TFIIB) must bind before Pol II. (Accepted)

–  Holoenzyme forms between Pol II and general transcription factors first and then locates promoter.

42

TATA box

–  located at about -30 relative to transcription initiation site
–  Is bound specifically by TFIID
–  Acts similar to a prokaryotic promoter to position RNA polymerase II for transcription initiation.

TATAA

43

Initiator element

–  located at +1
–  bound by specific TAF (TBP associated factors)
–  CpG promoters, ~20-50 CG repeats upstream in the promoterregion. Usually transcribed at a low rate. Cpg islands are GC rich. P is phosphate on sugar phosphate backbone. Sometimes island little upstream of the promoter

44

Transcription factors

TFII (TBP and TAFs), and changes the shape of the DNA a little, signal is sent to other transcription factors

TFIIB (recruits RNAP II-TFIIF) and TFIIA (prevents inhibitor binding) bind to both DNA and TFIID TFIIA doesn't always bind

RNA Pol II- TFIIF binds (TFIIF is a bridge between proteins associated with recognizing the promoter region and RNA pol)

TFIIH binds (helicase activity)

45

TBP (TATA binding protein)

monomeric protein that is highly symmetrical and conserved between species (80% identical between yeast & humans in the conserved domain)

Has 8 beta sheets that line up and fit in the minor groove of DNA(recognizes TA or AT)

Sits like a saddle, and on coils like stirrups on either side of DNA. Causes sharp bend (almost 90 degrees) , change in shape of bps. At has added strain, easier to melt/break

C and N terminus are on the same side

46

How does TBP accomplish the bending?

• High levels of contact between DNA and TBP

• Positively charged lysine and arginine bind with the negative phosphates

• The kink in DNA is produced by phenylalanine residues projecting in between base pairs – further increases contact with DNA (think about TBPs shape

• Strain causes “melting” (what base pairs are at the binding site?)

47

RNA polymerase is modifiedduring transcription initiation

Doesn't get modified until it is associated with the DNA

•  The carboxy terminal domain (CTD) contains 7 aa that arerepeated multiple times.

•  Consensus sequence:
–  Tyr-Ser-Pro-Thr-Ser-Pro-Ser

•  Yeast: 26 repeats

•  Humans: 52 repeats

•  Ser and Tyr modified by phosphorylation

48

TFIIH and carboxy terminal domain kinase activity

Using ATP, phosphorylates carboxy terminus, change in conformation, energizes it and kicks off RNA pol II from the general transcription factors and helps it start elongation

Once RNA pol II is phosphorylated, it can unwind DNA, synthesize RNA, and proofread

49

Everything about phosphorylation

Carboxy terminal domain of II has heptad repeats (the amino acids that occur over and over) different positions on that repeat can be phosphorylated. Tf2h has kinase activity, phosphorylates AA 5 position. Then promoter escape- pol II kicked off, leaves promoter sequence. mRNA is made. Then 2 position is phosphorylated.

Phosphorylation helps initiate transcription and encourage other things that happen. Phosphorylated carboxy terminal domain supports capping enzyme. Supports splicing machinery

50

How does mRNA stay linear

RNA likes to form hairpins and loops but we need to splice out introns. Phosphate groups support protein machinery, allows mRNA not to assume loops and stay linear

51

Assembly of the Pre-Initiation Complex

1  Binding of TFIID (TBP plus TBP associated factors (TAF))

2  Binding of TFIIB (and sometimes TFIIA)

3  Binding of Pol II, TFIIF, TFIIE, TFIIH

4  Phosphorylation of CTD (carboxy terminal domain) ofPol II and unwinding of DNA

5  Formation of first phosphodiester bonds

52

What happens when RNA pol II leaves the promoter region?

Initial recognition proteins are left so new pol II can come. Increased amount of certain protein in cell results if many polymerases bind there

53

Eukaryotic Elongation factors

PTEFb- kinase, helps to phosphorylate c terminus

ELL and SII

54

Eukaryotic termination of transcription

Pol dissassociates from ell and sii, comes back to find another FIID bound to the DNA

FCP1- phosphatase, removes phosphates

Any pol 2 can bind to any promoter region that shows transcriptionally active

55

How do eukaryotic genes vary?

Size and complexity

Due to intron/exon ratio

Human factorVIII gene (blood clotting gene) 200,000 base pairs but barely any exons

Human beta globin gene- 2000 bp, barely any exons

56

RNA processing in eukaryotes

5' cap

3' polyA tail

Splicing

57

5' cap

Eukaryotic mRNA’s have a 5’ cap formed by a 5’-5’ linkage of a 7-methyl GTP residue

Exonucleases in cell, eat from 3' to 5' but can't eat from 5' to 5', so cap resistant to exonucleases.

Also signal for transporting out of the nucleus

58

How can the 5' cap be used against your body?

One Family of viruses can take the cap and put in on the genome, but your own exonucleases can't attack cap

59

Why don't prokaryotes need the cap?

Transcription and translation occur in the same place in prokaryotes. Where in euk, mRNA has to be translated out of the nucleus, cap helps mRNA he carried out of the nucleus. Has to be put on in the nucleus because of the exonucleases in the nucleus

60

How is the cap made?

2'-O-methyl transferase

Guanine-7-Methyl transferase

Guanylyl transferase

61

How was splicing discovered?

•  Early experiments indicated that RNA was synthesized as a longer precursor.

•  This larger class of RNAs was referred to as heteronuclear RNA or hnRNA (many different sizes)

•  The mRNA found associated with polyribosomes was on average much smaller than the hnRNA.

Conclusions:
•  mRNA is derived from hnRNA
•  mRNA’s are smaller in length than hnRNA’s, something must have happened

Other experiments showed:
The size difference between hnRNA and mRNA primarily due to removal of introns.

62

Length of mRNA

•  Are mRNAs as long as the region of DNA encoding the gene?

•  Use of DNA-RNA hybrids to see the splicing. RNA only base paired to the exons

63

Transcription and processing of the ovalbumin gene

Only 24% of the original transcript was left after splicing

64

Addition of poly A tail

•  Specific signal sites dictate where cleavage proteins (endonucelase) and polyadenylation proteins (polyA polymerase) will attach. The upstream poly(A)signal is a specific sequence of nucleotides AAUAAA.

•  The downstream poly(A) signal is a GU-rich region on the pre-mRNA.

•  After transcription has been completed, the first two cleavage factors attach to the up and downstream poly(A) signal sites.

•  PolyA polymerase associates with the cleavage proteins, cleaves the mRNA and then polyadenylation can occur

PolyA polymerase adds about 200 As to the end, protects 3' end of RNA from exonucleases. Needs to last long enough to go into cytoplasm and associate with ribosome

65

Does cleavage and polyadenylation always occur efficiently?

Needs two polyA signals

Upstream polyA signal: AAUAAA, can only mutate to AUUAAA, or polyadenylation is eliminated. Poly a tail wouldn't be added on. Polya polymerase recognizes this sequence

66

Function of 5’ cap and Poly-A

•  Translation initiation

•  Transport out of nucleus

•  Stability
–  Prokaryotes mRNA’s degraded from 5’-3’ –  In eukaryotes mRNA’s are degraded from 3’-5’

•  Poly A tail can be used to purify mRNA

67

Wha sims the spliceosome made of?

SnRNPs (small nuclear ribonucleoprotein particles)- complex of RNA and proteins

Each snRNA is associated with 10 or more proteins

68

What are all the snRNPs called?

Rna components are Uridine rich RNAs, so they are Refferee to as U1, U2, U4, and U5 and U6 (U3 helps splice ribosomal RNA)

69

2 transesterification steps

•  2 Transesterification steps
–  Branch-5’ splice site (formation of lariat)
–  5’ splice site-3’splice site
–  Release of intron as lariat

70

How does spliceosome know where to go?

•  First step is the recognition of the junction between the exons and introns.

•  Recognition involves RNA-RNA base pairing assisted by proteins

71

What four conserved sequences are necessary?

–  5’ splice site, 9 bases
–  branch point, UAAC
–  pyrimidine track
–  3’ splice site, 5 bases

72

Formation of the spliceosome

RNA in these proteins recognize the splice site

1- Binding of U1 snRNP to 5’ splice site

2-Binding of U2 to branch point

3- U4/6 binds to U2, and U5 binds closer to 3' end (5' end of exon)

73

Procedure of splicing

Spliceosome cleaves the phosphodiester bond at the 5' end of intron

U1 is released

U4 is released, lariat is formed, and formation of phosphodiester bond between 5' G of intron and conserved A near 3 end of intron

3' site is cleaved and two exons are joined by phosphodiester linkage

74

Transcription and RNA processing are coupled

Phosphorylated CTD interacts with processing enzymes

Change in the function of CTD - think about the length of some mRNA transcripts

Stabilized by carboxy terminus domain, can be multiple spliceosomes at the same time

75

Group one introns

Self splicing

Require G factor

76

Group two introns

Similar to spliceosomal, but no spliceosomes. Self splice, requires bulging A, catalyze own transesterification reaction

77

RNA editing

another way that alternative mRNAs and proteins are made

Change in base can control what's left in and what's taken out

Differences between closely related cells and their Apolipoprotein DNA sequences due to editing

78

How does alternative splicing affect proteins?

Proteins from genes experiencing alternative splicing are very similar except in key regions that might affect ligand binding, location and activity.

Can do different things in different cells, closely related though

79

What is the advantage of having introns that need to be eliminated before the mRNA can be translated?

•  Main rationale used to explain the existence of introns is that it has allowed evolution to proceed at an increased pace.
–  Exons frequently encode different domains of a protein which can be combined via DNA rearrangements to generate new proteins relatively quickly (exon shuffling)

•  Alternative splicing allows a variety of related proteins to be synthesized from a single gene

•  Introns are thought to be a “safe place” for DNA breaks

NHEJ wouldn't be so bad if intron bases were lost, non coding

Mutations can occur where the introns become exonized and code for the protein making it have higher fitness

80

How do mRNAs get out of the nucleus?

exon junction complexes. Where two exons are joined together and a protein stays there in indicates that the exons were spliced together. Transported out of nucleus and into cytoplasm. Many proteins recycled immediately and go back into the nucleus. Some remain to mark the mRNA for a nonsense mutation