Unit II- Transcription and mRNA processing

Question 1

Central dogma of molecular biology

Answer 1

• genetic information flows from DNA to RNA to protein
• exceptions: some viruses use RNA as a source of genetic information as well as a template for replication (Polio virus)
• HIV converts its RNA into DNA to integrate into the host genome
• some RNAs work as ribozymes or carry out functions without being translated into protein

Question 2

Genes transcribed according to needs of cell

Answer 2

• many identical copies of RNA can be made from the same gene, and each RNA molecule can direct the synthesis of many proteins
• this allows amplification in the number of proteins
• each gene can be transcribed and translated with a different efficiency, allowing copious amounts of some proteins while only minute amounts of others as dictated by their functions
• a cell can change the expression of each of its genes according to the needs of the moment

Question 3

Function of different RNAs

Answer 3

mRNA- code for prooteins
rRNAs-form the core of the ribosome and catalyze protein synthesis
miRNAs-regulate gene expression
tRNAs-serve as adaptors between mRNA and amino acids during protein synthesis
Other small RNAs- used in RNA splicing, telomere maintenance, and many other processes

Question 4

Differences between RNA and DNA

Answer 4

• ribose rather than deoxyribose
• uracil (U) rather than thymine (T)
• RNA can remain single stranded
• RNA can fold up or base pair with other nucleic acids *DNA or RNA
• the structure formed by RNA-RNA hybrids can have catalyic activity (ribozymes) or serve a regulatory function (miRNAs)

Question 5

RNA polymerase

Answer 5

• transcription begins with the unwinding of a small portion of the DNA double helix to expose the bases on each strand
• nucleotide sequence of the RNA chain is determined by complementary base-pairing of incoming ribonucleotides according the sequence of the DNA template
• transcription from 5’ to 3’
• NTPs serve as substrates for RNA synthesis and provide the energy for the reaction via their high energy phosphate bonds
• the enzymes that carry out transcription are RNA polymerases which catalyze the formation of phosphodiester bonds that link nucleotides together and form the sugar-phosphate backbone of the RNA chain
• the RNA sequence is the same as the non-template DNA strand except there is U

Question 6

Promoter and Terminator sequences

Answer 6

• start sequences are located upstream DNA adjacent to the 5’ end of the gene, called promoter
• stop signals, called terminators are sequences on the 3’ end, terminators are transcripted

Question 7

3 parts of transcription

Answer 7

Initiation- where RNA polymerase binds to the promoter and begins RNA synthesis. The main point at which cells regulate which proteins are produced and at what rate

Elongation- where RNA synthesis proceeds down the gene uninterrupted

Termination- where RNA polymerase recognize the terminator sequence and falls off the template strand

Question 8

Transcription start/stop sites and coding region of gene

Answer 8

• the promoter is not transcribed
• there is a sequence prior to the first AUG initiation codon that is present in the RNA transcript but is not translated called the 5’ untranslated region
• there is a sequence present in the RNA transcript that is after the final termination code of the protein sequence called the 3’ untranslated region (3’ UTR)
• the terminator is transcribed into RNA (but may be trimmed away during processing)

Question 9

Watson/Crick strand

Answer 9

• genes can be encoded on either strand
• watson is the top strand
• bottom is the Crick strand

Question 10

RNA polymerases vs. DNA polymerases

Answer 10

Similarites:
-synthesis of RNA via phosphodiester bond formation is always 5’ to 3’ using template strand

Differences:

• RNA polymerases use rNTPS as substrates not dNTPs
• uracil rather than thymine base
• transcription involves de novo synthesis, no “primer” is required
• no proof-reading function
• no exonuclease activities
• only one DNA strand serves as a template for a given gene

Question 11

Types of RNA polymerases

Answer 11

RNA Pol I - most rRNA genes
RNA Pol II- protein coding genes, miRNA genes, plus genes for some small RNAs (those in spliceosomes)
RNA Pol III- tRNA genes, 55 rRNA genes, genes for many other small RNAs

Question 12

Problems with RNA polymerases

Answer 12

• bacteria have only one RNA polymerase enzyme that transcribes all types of RNA
• the bacterial RNA polymerase can be specifically targeted by antibiotics like rifampicin- used to treat Myobacterium infections like TB

-poisonous mushrooms contain a toxin, alpha-amanitin that specifically inhibits eukaryotic RNA pol II. Upon ingestion and uptake by liver cells, it binds and inhibits RNA pol II causing cytolysis of hepatocytes. Mortalilty= 15%

Question 13

Steps for RNA pol II transcription in eukaryotes

Answer 13

1) TBP binds TATA box (TBP part of TFIID)
2) Other (basal) TF are recruited, along with RNA polymerase to form pre-initiation complex
3) Pol II must be phosphorylated to leave promoter and start transcribing mRNA

Question 14

mRNA processing in prokaryotes/eukaryotes

Answer 14

• in prokaryotes, mRNA is translated, co-transcriptionally, so no processing needed
• eukaryotes, mRNA is covalently processed and transported out of the nucleus. In the cytoplasm, mRNA can be translated into protein

Question 15

5’ Capping and 3’ polyadenylation

Answer 15

• eukaryotic RNAs are capped (by guanyl and methyl transferases) just after RNA pol II has synthesized the 5’ end of the primary transcript and before it has completed transcribing the whole gene
• the 5’ end is a 5’ to 5’ linage of the 7-methyl G to the RNA
• polyadenylation occurs following cleavage of the primary transcript, and provides a 3’ end tail consisting of ~200 A’s which are non-templated

Question 16

Purpose of mRNA processing

Answer 16

1) transport to the cytoplasm
2) increased stability
3) translational efficiency

Question 17

Splicing

Answer 17

• eukaryotic transcripts are interrupted by noncoding introns
• introns must be removed from the primary transcript or pre-mRNA to produce a mature mRNA suitable for translation by the protein synthesis machinery
• performed in the nucleus co-transciptionally and produces a mature mRNA containing only exons

Question 18

Why are eukaryotic genes interrupted

Answer 18

1) expand the repertoire of gene products via alternative splicing
2) expand the target size of genes, increasing genetic diversity by enhancing the rate of crossing over of homologous recombination
3) provide evolutionary diversity by exon shuffling
4) introns may provide regulatory advantage (splicing is regulated)
5) introns are “selfish DNA” (remnants of transposon invaders)

Question 19

Mechanisms of pre-mRNA splicing (cis acting sequences)

Answer 19

• doesn’t matter the sequence for the introns except for key parts at the end that act for cues of removal
• they are recognized by snRNPs which cleave the RNA at the intro-exon borders and covalently link the exons together

Question 20

The RNA splicing mechanism

Answer 20

• RNA splicing is catalyzed by assembly of snRNPs which bring the ends of the intron together
• after the assembly of the snRNPs a specific adenine nucleotide in the intron sequence attacks the 5’ splice site and cuts the sugar phosphate backbone of the RNA at this point
• the cut 5’ end of the intron becomes covalently linked to the adenine nucleotide, forming a loop, or lariat, in the RNA molecule
• the free 3’ OH end of the first exon sequence then reacts with the beginning of the second exon sequence, cutting the intron at its 3’ end and joining the two exons together

-lariat formation- 2’ to 5’ phosphodiester linkages
U-snRNPS- set of proteins plus a uracil rich RNA

Question 21

Splicing is regulated

Answer 21

• alternate splicing generates different protein isoforms
• depending on cell types in which the gene is being expressed, or the stage of development of the organism
• most human genes are alternatively spliced
• 30,000 genes give rise to 500,000 proteins

Question 22

Hutchinson-Gilford Progeria Syndrome

Answer 22

• error in RNA splicing have profound consequences
• abnormal splice in lamin gene
• defective processing because the enzyme recognition site is deleted which results in lamin A that remains bound to the membrane instead of being released