Transcription and RNA Processing

Question 1

coding strand

Answer 1

same sequence as the mRNA

Question 2

template strand

Answer 2

3’ to 5’ strand of DNA

Question 3

basic structure of genes

Answer 3

promoter region upstream of start site

start site of transcription

teminator sequence that stops transcription

genes can be overlapping or coded in the opposite direction

Question 4

prokaryotic RNA polymerase

Answer 4

synthesizes all RNA in bacteria except for the primers used in DNA replication

five subunit core enzyme, one that binds template, one that has polymerase activity, and two copies of another necessary for complex formation, role of fifth is unknown

Question 5

sigma factor

Answer 5

used in prokaryotes, associates with the core RNA polymerase enzyme to form the holoenzyme, recognizes the promoter

Question 6

Name the three eukaryotic RNA polymerases and describe what they do.

Answer 6

Pol I - ribosomal RNA genes

Pol II - mRNA genes, most snRNA, snoRNAs, and miRNAs

Pol III - tRNAs, 5S rRNA, and some snRNA and snoRNA genes

polyA polymerase and poly ADP-ribose polymerase - two non-template dependent RNA polymerases responsible for synthesizing RNA polymers

Question 7

prokaryotic gene transcription

Answer 7

promoters 35bp stream of start stie

second site at -10 called the Pribnow box or the TATA box

sequences is where the helix is unwinding, forms a transcription bubble

topoisomerase relieves supercoiling, short RNA molecules made by polymerase until RNA longer than 10 nucleotides is made

polymerase can extend the transcript

termination sequence encodes hairpin structure RNA

Question 8

Describe the RNA Pol I promoter and how it promotes transcription.

Answer 8

contains the upstream control element (UCE) which binds the upstream binding factor (UBF)

UBF binds UCE and CPE and recruits SL1 which recruits RNA polymerase I

Question 9

rRNA transcription

Answer 9

greatest mass of RNA in cells is rRNA transcribed by RNAP I

genes linked in tandem with region inbetween containing terminator of one gene and promoter for the next

promoters for rRNA genes are immediately 5’ to the start of transcription and extend into the beginning of the gene

ribosomes assembled with protein structures and processed before export into the cytoplasm where the two subunits of the ribosome are put together

assembly of the 5S subunit happens in the cytoplasm

Question 10

RNA Pol III promoters

Answer 10

transcribe tRNA genes and recognize DNA sequences near start site, but the actual promoter lies downstream of the start site, split into two segments

Question 11

processing of tRNAs

Answer 11

synthesized as longer molecules that are trimmed at the ends to form matrue tRNA

uracil residues at the 3’ end are removed and replaced by CCA

some tRNAs also have a small intron in the anticodon loop tha tis removed

other extensive modifications include dihydrouracil, speudocuracil and other methylated bases

Question 12

structure of an RNA Pol II gene

Answer 12

promoters of some RNAP II genes have a TATA or Hognes Box ~25bp upstream of the start site

constitutively expressed genes usually do not have a TATA box here, replaced by a GC-rich sequence

CAAT boxes often found ~70-80 bps upstream of the start site thatare recognition binding sites for transcription factors

also have start sites, splice sites, stop signals and polyA addition signal

Question 13

transcription factors

Answer 13

two main categories, general and specific

general TFs bind to the core promoter and are the minimal requirement for recruiting the polymerase and initiating transcription

specific TFs bind within and outside the core promoter and moedulate the frequency of initiation

Question 14

general TF complex

Answer 14

TFIID binds TATA box, TFIIB binds TFIID, TFIIF recuits the polymerase, and TFIIH acts as a helicase and protein kinase to allow for the promoter to clear

Question 15

enhancers

Answer 15

can be upstream or downstream of the start site, can also be thousands of bps away in any orientation

bind specific TFs that bend or loop DNA to interact with other TFs and the promoter

can increase or decrease transcription

Question 16

Describe the transcription rpocess after initiation.

Answer 16

elongation proceeds past stop codon and through 3’ UTR

first a guanine cap is added, then splicing, then polyadenylation

enzyme binds polyadenylation signal, claves mRNA 10-20 nucleotides downstream and then adds ~200 nucleotide polyA tail in a non-templated reaction

all the necessary protein complexes attached to the CTD

Question 17

RNA splicing

Answer 17

as the lengthening RNA transcript is released from the RNA-DNA duplex, it immediately associates with RNA binding proteins to form a ribonucleoprotein complex

snRNPs facilitate the splicing activity, complex of U1 to U6 excluding U3 is called the spliceosome

Question 18

processing of rRNA

Answer 18

synthesized in a long rRNA precursor containing the 28S, 18S, and 5.8S rRNAs

eukaryotic 5SrRNA is synthesized separately by RNAP III

pre-rRNA is claved by endoribonucleases, ends of individual rRNAs trimmed and some of the bases modified

ribosomal proteins previously synthesized bind to rRNAs and ribosome assembly occurs

rRNA modifications and assembly are assisted by snoRNAs

Question 19

processing of tRNA

Answer 19

synthesized as longer molecules

trimmed at their ends to form mature tRNA

uracil residues at the 3’ end are removed and replaced by CCA

some tRNAs have a small intron in the anticodon loop that is removed, extensive base modification

Question 20

processing of mRNA

Answer 20

collection of all RNAP II transcripts in the nucleus called heterogenous nuclear RNA (assembled with proteins into hnRNPs)

includes transcripts that will become mRNa and transcripts that will be long non-coding RNAs

5’ capping first, 7-methylguanosine cap is added to the 5’ end

introns are removed

polyA tail is added, 40-200 nucleotide long polyA tail is added and interacts with binding proteins to help stabilize the tail against degradation by nucleases

Question 21

snRNAs

Answer 21

U1-U12, functions in splicing of mRNA, histone mRNA transcription termination

Question 22

snoRNA

Answer 22

rRNA processing, modification of bases in rRNA

Question 23

SMN complex

Answer 23

important protein complex for the assembly of snRNPs

Question 24

miRNA processing

Answer 24

transcribed by RNA pol II, capped at 5’ end and polyadenylated and called primary miRNA

microprocessor complex associates and cuts out pre-miRNA from long precursor

modification by RNA editing include deamination of adenosines to inosines

transported to cytoplasm, cleaved by Dicer to generate mature RNA duplex of 22 nucleotides

one strand of the miRNA duplex is incorporated in to an RNA-induced silencing complex (RISC)