3.3 transcription - mechanism

Question 1

location of transcription and fate of RNA in bacteria vs eukaryotes

Answer 1

Bacteria
- occur in cytoplasm at the same place and time
- RNA is NOT processed

Eukaryotes
- transcription takes place in the nucleus
- transcript is processed (spliced) and then exported to the cytoplasm
- T and T take place in separate compartements

Question 2

RNA polymerase

Answer 2

• separates DNA strands
• Catalyzes the addition of new rNTPs
• moves in 3’ to 5’ direction
• but!! newly synthesized RNA 5’ - 3’
• threads the newly created RNA through a channel
• does not bind directly to the promoter…
• made up of several protein subunits (quat. structure)

Question 3

RNAP II

Answer 3

one of the tree forms of RNA polymerase in eukaryotes

transcribes genes for mRNA (and a few other RNAs such as small nuclear RNA, miRNA)

Question 4

what happens in transcription?

Answer 4

1. RNA POL binds to a region called a PROMOTER
2. transcription starts at a position called the +1 site/transcription start site (transcribes the first base/nucleitide)
3. transcription stops after the RNA POL passes through a region called the terminator

Question 5

RNA polymerization steps

Answer 5

1. incoming nucleotides are accepted if they correctly base pair with the template DNA
2. the 3’-OH of the growing stand attacks the high energy phosphate bond of the incoming ribonucleotide, providing the energy to drive the reaction
3. the two phosphates of the incoming ribonucleotide are released as pyrophosphate

Question 6

holoenzyme

Answer 6

Sigma factor + RNA POL

Bacteria

moves in the direction from -10 box and +1 site, downstream

orientation of the sigma/RNA POL relative to the -35 and -10 box is important — the RNA complex “know” which direction to move along the strand

ability to bind tightly to the promoter is a level of control

Question 7

general transcription factors bind where

Answer 7

bind to the promoter

Question 8

where do transcriptional activator proteins bind

Answer 8

enhancers

Eukaryotes

Question 9

imitation of transcription at a eukaryotic promoter

Answer 9

TATA binding protein (TBP) and general transcription factors (activators) band to the promoter to recruit the binding of RNA POL

Question 10

Basal transcription complex

Answer 10

minimum complex needed to initiate transcription

TATA protein and General Transcription Factors (and activators) bound to the promoter

Eukaryotes

Question 11

where does transcription start in eukaryotes vs bacteria

Answer 11

bacteria: +1 site (10 bases downstream from -10 box)

eukaryotes: transcription start ~25 bases downstream from the promoter

Question 12

when does transcription stop

Answer 12

after RNA POL passes through the terminator

transcribed but not translated

Question 13

end of transcription in eukaryotes vs bacteria

Answer 13

EUKARYOTE
- termination sequence is transcribed as well
- involves proteins called TERMINATION FACTORS (term not tested)
- binding of these termination factors to the DNA disturbs the RNA POL

BACTERIA
- transcribes through a termination sequence
- a hairpin loop forms on the mRNA, causing the RNA POL to dissociate from the GENE

Question 14

DNA binding proteins

Answer 14

• bind in the major and minor grooves of DNA
• recognize specific sequences of base pairs
• bind by H-bonds and other non-covalent interactions interact with the bases
• how strongly a protein binds will determine how long it will stay on the DNA strand and whether transcription and/or translation will proceed

Question 15

how can transcription be regulated

Answer 15

how OFTEN and how TIGHTLY RNA POL (and sigma or other transcription factors) bind to the promoter

binding more often and tightly results in more RNA transcripts made from that gene!!

Question 16

5’ cap

Answer 16

modified base linked by its 5’ carbon to the 5’ end of the primary transcript by a bridge composed of 3 phosphates

basically a nucleotide added in reverse

Essential for translation because this is how the ribosome recognizes mRNA!!

Question 17

3 chemical modifications of Eukaryotic Primary RNA transcript

Answer 17

1. addition of 5’ cap
2. polyadenylation (forming poly (A) tail)
3. splicing of introns and exons

Question 18

Exons

Answer 18

expressed regions
code for amino acids
sometimes removed

Question 19

introns

Answer 19

intervening sequences
do not code for everything
always removed

Question 20

splicing

Answer 20

cutting out introns

Question 21

alternative splicing

Answer 21

• primary transcripts from the same gene can be spliced in different ways to yield different mRNA
• Allows the same transcript to be processed in diverse ways
• when cells also splice out some of the EXONS
• leads to 2 cells producing 2 proteins with similar function but varying number of AA found in the protein

Question 22

5’ and 3’ UTRs untranslated regions

Answer 22

mRNAs contain regions before the start codon and after the stop codon

Question 23

alternative splicing

Answer 23

different splicing pattern

creates splice variants

means that more than one type of protein can be made from a single gene coding for an mRNA

Question 24

DNA packaging as transcriptional control

Answer 24

BACTERIA
- promoters are unpackaged
- accessible to RNA polymerase
- default state is always on

EUKARYOTES
- DNA is packaged tightly
- must be unpackaged for the RNA polymerase to access the DNA and start transcription

Question 25

Primary transcript

Answer 25

RNA transcript that comes off the template DNA strand and contains the complement of every base that was transcribed from the DNA template

○ Includes the information needed to direct the ribosome to produce the protein corresponding to the gene

Question 26

mRNA

Answer 26

RNA molecules that combines with the ribosome to direct protein synthesis, carries genetic message from DNA to ribosome

Question 27

Primary transcripts for protein-coding genes in prokaryotes features not shared with those in eukaryotes:

Answer 27

primary transcripts are translated immediately
In prokaryotes, the primary transcript IS the mRNA
often contain genetic info for the synthesis of 2 or more different proteins

Question 28

Polycistronic mRNA

Answer 28

molecules of mRNA that code for multiple proteins

Question 29

RNA processing

Answer 29

chemical modification of the primary transcripts, converts it into the finished mRNA, which can then be translated by the ribosome

Question 30

steps of RNA processing

Answer 30

1. 5’ end of the primary transcript is modified by the addition of 5’ cap
2. Polyadenylation – addition of a string of ~250 consecutive A-bearing ribonucleotides to the 3’ end, forming poly(A) tail
3. Removal of non-coding introns

Question 31

• Ribosomal RNA (rRNA)

Answer 31

○ Essential in translation
○ Genes and transcripts for rRNA are concentrated in the nucleolus
§ Non-membrane bound spherical structure observed within the nucleus

Question 32

• Transfer RNA (tRNA)

Answer 32

○ Carries individual amino acids for use in translation

Question 33

most abundant transcripts in mammalian cells

Answer 33

ribosomal RNA and transfer RNA

Needed in large amounts to synthesize the proteins enconded in mRNA

Question 34

transcription bubble

Answer 34

where transcription takes place, strands of DNA duplex are separated

Question 35

RNA-DNA duplex

Answer 35

the growing end of the RNA transcript is paired with the template strand

Question 36

General process of polymerization of RNA

Answer 36

incoming ribonucleoside triphosphate is accepted by RNA polymeras only if it undergoes proper base pairing with the base in the template DNA strangd

• RNA polymerase orients the 3’ end of the growing strand so that the oxygen in the hydroxyl group can attack the innermost phosphate of the triphosphate of the incoming ribonucleoside, competing for the covalent bond

When cleaved, provides the energy to drive the reaction that creates the phosphodiester bond attaching the incoming nucleotide to the 3’ end of the growing chain