Biol 202 Notes

Transcription

The Central Dogma
RNA Polymerase Ribosome

DNA transcription replication

RNA translation Protein

One gene encodes one protein? (Beadle and Tatum, 1941)
• • • Cellular machinery is largely made up of proteins. Proteins are made up of chains or sequences of amino acids. The sequence of these amino acids are "encoded" in the cell's DNA. DNA is transcribed into RNA, which is then translated into protein. When one gene is mutated, one protein is affected (usually disabled). But, the analysis of human genome (and many “higher” eukaryotes) suggests that in general, multiple proteins arise from each gene in humans as a result of alternative splicing (will discuss in later lecture).

• •

Differences Between RNA and DNA
• RNA contains ribose, rather than deoxyribose (difference of a single oxygen). • RNA uses uracil rather than thymidine as a base; Uracil still base pairs with adenine. • RNA is generally single stranded, rather than double stranded and thus is capable of folding into complex three-dimensional shapes.

Differences between RNA and DNA building blocks
5’

P
4’ 3’ 2’ 1’

P

Phosphate linkage here

(RNA)

Phosphate linkage here

(DNA)

• Like DNA, RNA has POLARITY (5’ and 3’) • Unlike DNA, RNA is usually single-stranded and flexible

5ʹ′ and 3ʹ′ refer to the carbon positions on the sugar ring of RNA or DNA

5’ 4’ 1’ 3’ 2’

This hydroxyl group is required for RNA polymerization

RNA contains a different base → Uracil
O H 3C C C H
5 6 1

O H
3 2

C
4

H C C
5 6

C
4 3 2 1

H N C O

N C O H

N H

N H

thymine (T)

uracil (U)

RNAs can fold into unique structures

tRNAs are highly specialized RNAs that function during translation

tRNA secondary structure

tRNA tertiary structure

RNA secondary structures can be very complex

16S Ribosomal RNA secondary structure

Two kinds of RNA
• Messenger RNA (= mRNA; translated into proteins). • Structural (functional) RNAs – not translated. – includes: • ribosomal RNA (rRNA, which is an integral component of ribosomes) • transfer RNA (tRNAs which are involved in translation) • small nuclear RNAs (snRNAs, involved in RNA splicing) • cytoplasmic RNAs (scRNAs involved in protein trafficking) • microRNA, siRNAs, piRNAs (regulate the expression of genes)

Transcription
• Catalyzed by RNA polymerase – a complex, multisubunit enzyme that catalyzes the formation of the phosphodiester bonds that link together the nucleotides in an RNA chain

Gene Structure
“upstream” “downstream”

Transcription Transcription start site Translation stop site

5’(CAP)

AUG

TAA

(AAAA) 3’UTR

5’UTR Translation start site

Note: 5’ CAP, poly A tail and intron only present in eukaryotes UTR = untranslated region.

Gene can be transcribed from either strand of DNA

RNA is always synthesized 5’ to 3’
(coding strand)

Therefore, the template strand is read 3’ to 5’

Transcription
5’ GATCTGACTGACATAGACATAGAT 3’ coding (= non-template) strand 3’ CTAGACTGACTGTATCTGTATCTA 5’ template strand 5’ GAUCUGACUGACAUAGACAUAGAU 3’ mRNA

E. coli promoters contain characteristic DNA sequences that associate with sigma factors and RNA Polymerase holoenzyme

Pribnow box

Mutations in -10 and -35 sequences affect transcription

DNA complexed with RNA polymerase

In both prokaryotes and eukaryotes, transcription proceeds in stages called Initiation, Elongation, and Termination Sigma factor

Prokaryotic Transcriptional Initiation

Comparison of Regulation of Gene Expression Between Prokaryotes and Eukaryotes
Prokaryotes
• Control of transcription through specific DNA-binding proteins • Role played by chromatin structure • Coordination achieved by operons • Differential splicing • Attenuation • Differential polyadenylation • Differential transport from nucleus to cytoplasm • Differential rates of translation

Eukaryotes Yes Yes No Yes No Yes Yes Yes

Yes No Yes No Yes No No Yes

Transcription initiation in eukaryotes

Figure 8-12 step 1

Transcription initiation in eukaryotes

Figure 8-12 step 2

Transcription initiation in eukaryotes

Figure 8-12 step 3

Transcription initiation in eukaryotes

Figure 8-12 step 4

Cotranscriptional processing of RNA

Figure 8-13 step 1

Cotranscriptional processing of RNA

Figure 8-13 step 2

Cotranscriptional processing of RNA

Figure 8-13 step 3

Eukaryotic mRNAs are spliced

Exon1

Intron

Exon2

Pre-mRNA

RNA splicing mRNA

Self-splicing introns

Structure of a group II self-splicing intron

Alternative Sigma Factors

Transcription proceeds in stages called Initiation, Elongation, and Termination
Sigma factor

Prokaryotic Transcriptional Initiation

E. coli promoters contain characteristic DNA sequences that associate with sigma factors and RNA Polymerase holoenzyme

Pribnow box

Different Sigma factors allow RNA polymerase to recognize different sets of promoters

Sigma Factors in E. coli σ70 (RpoD) - the "housekeeping" sigma factor, transcribes most genes in growing cells. σ54 (RpoN) - the nitrogen-limitation sigma factor σ38 (RpoS) - the starvation/stationary phase sigma factor σ32 (RpoH) - the heat shock sigma factor, it is turned on when exposed to heat σ28 (RpoF) - the flagellar sigma factor σ24 (RpoE) - the extracytoplasmic/extreme heat stress sigma factor σ19 (FecI) - the ferric citrate sigma factor, regulates the Fec gene for iron transport

Bacillus subtilis sigma factors
• More than ten alternative σ factors known: - Vegetative growth (B. subtilis σA and σF = E. coli σ70) - Heat shock (stress) - Sporulation - Phage infection • Sporulation cascade: - One early gene product is a σ factor (σF); expressed in forespore - σF specifies a new set of genes, including downstream σ factors in both the mother cell and the forespore. - The alternative σ factors turn on genes that lead to the development of a spore cell.