1 - RNA Transcription

Question 1

Major Diff Between RNA and DNA

Answer 1

DNA RNA

Function is genetic info genetic expression

Sugar is 2-deoxyribose ribose

Pyrimidine bases are cytosine cytosine and uracil

and thymine

Usually double stranded single stranded

Size can exceed 1 mil base pairs 60-20,000 bp

No enzymatic function ribozyme

Conformation is restrained by Greater flexibility

being double stranded

• Ribozyme = RNA that is an enzyme ; can also catalyze its own replication also found in our own body
• Example of single stranded RNA: in case of viral infection, signals for interferons.

Question 2

Major Diff Between RNA and DNA

Answer 2

DNA RNA

Found in nucleus nucleus and cytoplasm

Deoxyribose sugar is not Ribose sugar is more

very reactive. Stable in alkaline reactive. Not stable in

conditions. Small grooves alkaline conditions. Has

where nucleases attach. larger grooves.

Chromosomal DNA is Less sensitive to UV rays

primarily B form. Sensitive

to UV rays.

Question 3

Types of RNA in Eukaryotes

Answer 3

• Messenger RNA conveys genetic information from the DNA to the protein. Makes up about 5% (subsequently small percentage) of the total RNA in the cell
• Ribosomal RNA …18S constitutes part of the small subunit of the ribosome
• 28S,5.8S, 5s constitute part of the large subunit of the ribosome ( we do not need to know the numbers or percentage)
• Make up approximately 80% of the total RNA in the cell
• Transfer RNA …Serves as adaptor molecule in protein translation
• Makes up about 15% of the total RNA in a cell
• There are 32 different types of tRNA in most eukaryotic cells
• Contain no more than 93 nucleotides (very small)
• Small nucleolar RNA are found in the nucleolus, where they are involved in:
  • Synthesis of ribosomes
  • Chemical modifications of other RNA molecules
  • Alternative splicing
  • Synthesis of telomeres
• MicroRNA: contains about 20 nucleotides, are involved in regulating the levels of expression of certain genes
• XIST RNA: large molecules of RNA responsible for inactivation of X chromosome in females
  
  • Two X’s exist early in development but one of them is inactivated forever; considered to be a backup system where having two X’s increase the likelihood of at least one being a viable/”healthy” gene.
• Small nuclear RNA….Are involved in processing of other RNA’s (e.g., are found in splicesomes)

Question 4

Transcription

Answer 4

Question 5

Prokaryotic and eukaryotic RNA’s

Answer 5

• Just know they have different sizes, shapes, and functions
• Ribosomes also consist of different proteins on top of rRNA’s

Question 6

Ribosomes

Answer 6

• Cells can have many ribosomes, which is the reason why rRNA can account for 80% of the total RNA in a cell (It’s been reported that a very active mammalian cell can synthesize almost over 1000 rRNA molecules each second, each of which associates with 150 different snoRNA’s , which subsequently combine with 80 different proteins to create the ribosomes; especially greater for more active cells).
• The genes for 28S, 18S, and 5.8S rRNA are usually
  repeated in tandem clusters; they are transcribed by RNA Polymerase I as a single 45S rRNA precursor, which is subsequently processed (chopped off) to produce the 3 rRNA mature transcripts; only rRNA’s go through this NOT mRNA’s

Question 7

Eukaryotic rRNA and its
posttranscriptional processing

Answer 7

Question 8

tRNA

Answer 8

• has double stranded character
• tRNA has a cloverleaf structure (in 3D sense) with a significant amount of tertiary structure composed of stems and loops
• Many of the nucleotides are modified (Modified nucleotides can include ribothymidine, pseudouridine, dihydrouridine, etc.)
• Eukaryotic tRNA genes are transcribed by RNA
  Polymerase III, and then undergo additional processing, which may includes the following: cleavage of the additional 5’ sequence, intron splicing, addition of the CCA sequence at the 3’end, and base modification.
  
  • No need to know the RNA pol # except for Pol II, which is associated with mRNA.
• Both mRNA and tRNA undergo significant post translational processing.

Question 9

Post-transcriptional
Modification of RNA

Answer 9

• Ribosomal and Transfer RNA both go through extensive post-transcriptional processing
• Pos-transcriptional processing of tRNA:
• A 16 nucleotide sequence at the 5’ end is cleaved by RNAse P in pre-tRNA
• A 14-nucleotide intron in the anticodon loop is removed by splicing in pre-tRNA
• Many bases are converted to characteristic modififed bases
• Uracils at the 3’ end are replaced by the CCA sequence found in mature tRNAs

Question 10

Eukaryotic mRNA

Answer 10

• Only very specific portion of Eukaryotic mRNA actually codes for protein i.e. the Coding Region
• Eukaryotic mRNA contains:
  
  • A cap (a methylated guanosine triphosphate attached through a 5’ to 5’ linkage to a the hydroxyl on the ribose at the 5’ end of the mRNA)
    
    • It doesn’t look like anything else and very unique thus cannot be processed by nucleases; protection from degradation
    • NOT something coded by DNA
• Poly (A) tail ( up to 200 adenine molecules at the 3’ terminus of the mRNA)
  
  • Simply added on; NOT something that originated from DNA
• Both the cap and the poly A tail serve to stabilize the mRNA against exonucleolytic degradation and participate in polypeptide chain initiation; play a role in translation and stabilization
• The cap is added immediately after synthesis, followed by the addition of the poly A tail.
• Neither the cap or tail are coded for in the DNA, protects it so nucleases won’t attack it

Question 11

Base pairing between DNA and RNA

Answer 11

• Antiparallel, complementary base paring between DNA and RNA

Question 12

RNA Synthesis

Answer 12

• RNA polymerase can initiate the synthesis of the RNA chain without the need for a primer.
• The essential precursors for the synthesis of the chain are CTP, UTP, GTP, and ATP
• The DNA template is copied in the 3’ to 5’ direction
• The RNA chain is synthesized in the 5’ to 3’ direction
• Local unwinding of the DNA occurs after binding of the RNA polymerase; the template strand (vs. Non-template) is subsequently transcribed; RNA will ook very much alike non-template strand of DNA

Question 13

Prokaryotic RNA polymerase

Answer 13

• The prokaryotic RNA polymerase consists of the core enzyme plus a dissociable sigma factor. The sigma factor recognizes transcriptional start sites, and the core enzyme polymerizes nucleotides into RNA. (Except for the short RNA primers associated with DNA synthesis), there is only one species of prokaryotic RNA Polymerase.
• Omega imparts specificity on what to transcribe and where to the polymerase should bind and work.

Question 14

Prokaryotic promoter region

Answer 14

• Polymerase will recognize and bound upstream of +1 in the DNA strand.
  
  • There is no “zero”th bp; transcription starts at +1
  • Anything upstream of +1 is given Negative numbers
• No need to memorize the specific bp sequence but know their names and whereabouts
  
  • Pribnow box from -7 to -12 (around -10)
  • -35 sequence from -32 to -37 (around -35 hence the name)
• Sigma recognizes the -35 sequence and Pribnow box.
• If the consensus sequence are close enough, they are fine, doesn’t have to be 100% perfect
• However, there is a possibility of going much slower and lesser quality translation.
• The prokaryotic promoter serves as the binding site for the RNA polymerase.
• The prokaryotic promoter consists minimally of the -35 site ( consensus sequence is TTGACA) and the -10 site ( Pribnow box).
• The nucleotides in the Pribnow box dissociate (Pribnow box consensus sequence is TATAAT), allowing the RNA Polymerase to separate the DNA double helix and to initiate transcription.
• The less the specific recognition sequences resemble the consensus sequence, the slower the transcription.

Question 15

Local unwinding of DNA caused by RNA polymerase

Answer 15

• Either strand of DNA can serve as the template.
• RNA Polymerase unwinds and rewinds the DNA, adding 5’ nucleoside triphosphates to the RNA’s 3’ hydroxyl.
• The template stand is read 3’ to 5’, and the direction of synthesis is 5’ to 3’.
• RNA Polymerase does possess some proofreading ability and has an error rate of about 1 in 10,000, while DNA Polymerase III has an error rate of about 1 in 10,000,000. (Please discuss.)
  
  • It doesn’t matter that much. it’s one RNA molecule out of many many.
  • If DNA get mutated, thats a big deal, however.
• Actinomycin D and Mitomycin intercalate between two G-C base pairs in the DNA and inhibit the RNA Polymerase thusly acting as antibiotic
• Rifampicin binds to the beta subunit of bacterial RNA Polymerase, and subsequently prevents the formation of the first phosphodiester bond.
  
  • Used for treatment of tuberculosis; does NOT work on eukaryotes.
• Figure -
  
  • Start with unwinding of the stands going from 5’ –>3’
  • Once RNA is made, old RNA will start to dissociated from DNA and just dangle around

Question 16

Termination of transcription

Answer 16

• Rho-independent termination of transcription
• Top: DNA template sequence generates a self-complementary sequence in the RNA
• Bottom: Hairpin structure formed by the RNA (N represents a non-complementary base)
  
  • Signals termination
• Termination can be either associated with the rho factor or be independent of this factor.
• The rho –independent termination occurs as a result of the RNA’s forming a hairpin loop when it transcribes a palindromic sequence ( “A man, a plan, a canal, Panama“).

Question 17

Rifampicin

Answer 17

• Rifampicin does NOT bind eukaryotic RNA Polymerase.
• Does NOT affect eukaryotes
• Prevent prokaryotic RNA polymerase activity.

Question 18

Histone

Answer 18

• Acetylation/deacetylation of a lysine residue in a histone
  
  • Acetylation removes the positive charge; not going to be able to bind to DNA thus leading to more transcription as DNA strands are more available.
• HAT is histone acetyltranssferase;
• HDAC is histone deacetylase
• Exposure to BPA lead to hyper-acetylation leading to autism in child

Question 19

Chromatin

Answer 19

• Euchromatin is the more relaxed form of chromatin, allowing a greater degree of transcription. Heterochromatin is the more condensed form, with a lesser amount of transcription, e.g. female barr body; inactivated X chromosome
• The acetylation of the lysine residues on histones by histone acetyltransferase (a) reduces the positive charge on the lysine and (b) decreases the interaction of the histone with the DNA (c) relaxing the chromatin.
• The acetyl group is removed by histone deacetylase, condensing the chromatin.
• This ongoing process is an integral part of histone remodeling.

Question 20

Trans acting factors

Answer 20

• Trans acting factors
• There are three eukaryotic RNA polymerases:
  
  • RNA Polymerase I…transcribes the 28S, 18S, and 5.8S rRNA genes in the nucleolus
  • RNA Polymerase II…transcribes mRNA genes,snoRNA, snRNA genes (the only one we have to know)
  • RNA Polymerase III…transcribes tRNA genes and 5S rRNA gene

Question 21

Transcription factors

Answer 21

• There also are general transcription factors that bind to promoters. They include the following; TFIIA, TFIIB,…THIIH, also, TBP (TATA binding protein) and TBF’s (TBP Associated Factors). Usage of specific transcription factors depends on the specific gene and the specific situation.
• These factors are characterized as having structural motifs as leucine zippers, zinc fingers, helix-loop-helix, bromodomains, etc.
  
  • Transcription factor work as combinatorial fashion; work together to induce specific transcription
  • Promotor region is cis, whereas protein or other factors that bind to them are trans.
  • Transcription fcators for prokaryotes = sigma factor (the only one)
  • Eukaryotes are a lot more complicated and lot more varieties
• Cis acting factors are nucleotide sequences on the DNA, as opposed to trans acting (protein) factors that interact with these cis factors.
  
  • cis-Regulatory Elements (CREs) are regions of non-coding DNA which regulate the transcription of nearby genes (wiki)
• The eukaryotic promoters contain the following conserved sequences:
  
  • TATA (Hogness) box, found at -25
  • CAAT box, found at -70
  • GC boxes, found between -40 and -110
  • These regions of the eukaryotic promoter bind the trans acting factors.
• Figure: Eukaryotic gene promoter consensus sequence

Question 22

Transacting factors

Answer 22

• The information can arise from outside of the cell and be transmitted through signaling pathways.
• An example would be JAK-STAT ( Janus Kinase Signal Transducer and Activator of Transcription) [we don’t have to know this]. The cellular receptor is activated by a signal from different cytokines ( interferon, interleukin, etc.). Ultimately, the STAT protein translocates into the cell nucleus, binds to the promoter, and initiates the transcription of specific genes.
• Enhancers (or silencers) function in the stimulation or the inhibition of transcription
• They can be located either (a) 5’ or 3’ to the transcription start site, (b) close to or far away from the transcription start site (up to 50,000 base pairs away), (c) on either strand of the DNA.
• These enhancers contain response elements ( Hormone response element, Glucocorticoid response element, Cyclic AMP response element, etc.) that interact with specific trans acting factors.
• Transacting factors are activated by physiological change/message from somewhere in the body ; involve some genes to be transcribed, cascade of events, eventually leading to TF’s binding to DNA
• The effect of these enhancers/silencers is achieved by the looping out of the DNA between the enhancer and the promoter. This allows for contact between the transcription factors bound to the enhancer with those bound to the promoter.
• Figure:
  
  • A: Assembly of eukaryotic initiation complex
  • B: Enhancer stimulation of RNA polymerase II
  • Transcription factosrs bind promotoer and amplify transcription
  • Enhancers, after being binded to transcription factors, will affect TF’s that are very far downstream, which then either lead to enhancing or silencing effect.

Question 23

Possible locations of enhancer sequences

Answer 23

• Possible locations of enhancer sequences

Question 24

Posttranscriptional Modification of RNA

Answer 24

• Messenger RNA precursor molecules are called heterogeneous nuclear RNA (hnRNA)–>immature mRNA, which is sent to ribosomes
  
  • 5’ “capping”: to allow translation beggins
  • Poly-A tail: to help stabilize the mRNAs and facilitate their exit from the nucleous

Question 25

Eukaryotic rRNA and its posttranscriptional processing

Answer 25

* Eukaryotic rRNA and its posttranscriptional processing

Question 26

tRNA

Answer 26

Question 27

RNA splicing

Answer 27

* RNA splicing is the removal of intervening sequences (introns) from the hnRNA (heterogeous nuclear RNA) and the subsequent bringing together the neighboring exons into the correct alignment for translation. * Has to be done perfectly, otherwise induce frameshift mutation * Cut out the introns and bind the neighboring exons * Introns in eukaryotes are very long * Introns are cut out in the mRNA, NOT in the DNA itself. * Splicing is carried out by uracil-rich small nuclear RNA’s bound to proteins (snRNP’s, small nuclear ribonucleoproteins). These “snurps” base pair with the consensus sequence at both ends of the intron (GU….AG) to form the spliceosome. The intron loops out (lariat) and is excised. * These transacting factors (TF's) are snRNP's binding to proteins in the ends of the introns and cut'em out * Consensus sequences at the each end * Introns are thought to comprise 21% of the human genome, while exons make up only a little over 1%. * About 15% of all human genetic disorders are caused by mutations associated with improper splicing. * Exons sometimes code for protein domains, both structural and functional. It is believed that exon shuffling has contributed to the assembly of new genes in the course of evolution. * Alternative splicing allows a single transcript to be processed in difference ways, giving us a highly increased number of different isoforms

Question 28

Posttranscriptional Modification of RNA –messenger RNA

Answer 28

* Removal of introns * Role of small nuclear RNAs: facilitate the splicing of exon segments by forming base pairs with the consensus sequences at each end of the intron * Mechanism of excision of introns: the GU and AG sequences at the branch site are invariant * Alternative splicing of mRNA molecules * No need to know the specifics of the mechanisms

Question 29

Alternative splicing patterns

Answer 29

* Calcitonin was the first example of study of alternative splicing * Most of the genes in humans that express proteins have some kind of alternative splicing; 90% of them do * Genes have multiple variants in terms of alternative splicing * One gene can lead to multiple kinds of proteins * D-scam 38,000 varients in this single gene