1. Transcription in Prokaryotic (Bacteria)
The conversion of DNA into an RNA transcript requires an enzyme known as RNA polymerase
RNA polymerase
Catalyzes the formation of a phosphodiester bond between the 3’ end of the growing mRNA chain and the new ribonucleotides
Transcription must occur in the 5’ to 3’ direction
No primer is needed to begin transcription

2. Transcription in Bacteria
The template strand on DNA is what is “read” by RNA polymerase into a message.
The non-template strand is confusingly referred to also as the coding strand because
the DNA sequence in this strand is complementary to the message which is made into
Protein.

3. RNA Polymerase Structure
X-ray crystallography indicates that
Large globular enzyme
Several channels in the interior
Core enzyme
Just RNA polymerase
Active site for catalysis
Holoenzyme
Sigma plus RNA polymerase
Bacterial RNA Polymerase
Holoenzyme

4. Initiation of Transcription in Bacteria
Promoters
Sequences of DNA which “inform” RNA polymerase where to begin transcription
Located on non-template strand
40 to 50 base pairs in length
All have a TATAAT sequence
-10 box centered 10 bases away from the site where transcription begins (+1)
All have a TTGACA
-35 box located 35 bases away from the +1 site
Sequences inside promoter but outside -10 or -35 vary widely among genes

5. Terms Downstream Upstream
DNA located in the direction in which RNA polymerase will transcribe
Upstream
DNA located in the opposite direction in which RNA polymerase will transcribe
Upstream
Downstream
Promoter
TTGAC
TATAAT
Gene
-35
-10 +1

6. Initiation of Transcription in Bacteria
Sigma makes the initial contact with DNA that starts transcription in bacteria
Once sigma has bound to the DNA the helix opens and the template is threaded through a channel
NTPs enter a different channel and diffuse to the active site
Incoming NTPs base pair complementary with the template strand
A base pairs with U not T

9. Opens the helix
Steers the template and
non-template strands into
the correct channels

10. Termination of Transcription in Bacteria
DNA sequence functions as a transcription termination signal
Makes a stretch of RNA which as soon as it is synthesized can base pair with itself
Hairpin loop – RNA secondary structure
The formation of the hairpin disrupts the interaction between RNA polymerase and the RNA transcript resulting in the physical separation

11. Termination of Transcription in Bacteria

12. Transcription in Eukaryotic Cells
Transcription of eukaryotic cells is more complex and requires more proteins and regulators
Transcription factors
The initiation of transcription is accomplished by basal transcription factors
Function analogous to sigma
Can interact with DNA independently of RNA polymerase

14. Transcription Regulation
What we know from prokaryotes:
Several related genes can be transcribed together (ie. lac operon)
Need RNA Polymerase to recognize a promoter region
Why eukaryotes are different:
Genes are nearly always transcribed individually
3 RNA Polymerases occur, requiring multiple proteins to initiate transcription

15. Transcription Regulation
Typical prokaryotic promoter: recognition sequence + TATA box -> RNA Polymerase attachment -> transcription
Typical eukaryotic promoter: recognition sequence + TATA box + transcription factors -> RNA Polymerase II attachment -> transcription

16. Transcription Regulation
RNA polymerase interacts w/promoter, regulator sequences, & enhancer sequences to begin transcription
Regulator proteins bind to regulator sequences to activate transcription
Found prior to promoter
Enhancer sequences bind activator proteins
Typically far from the gene
Silencer sequences stop transcription if they bind with repressor proteins

18. Activator:
A transcriptional activator is a protein that increases gene transcription of a gene or set of genes.
Most activators are DNA-binding proteins.
Most activators function by binding sequence-specifically to a DNA site located in or near a promoter and making protein-protein interactions with the general transcription machinery (RNA polymerase and general transcription factors).
Transcription factor :
is a protein that binds to specific DNA sequences, thereby controlling the flow (or transcription) of genetic information from DNA to mRNA.
Transcription factors perform this function alone or with other proteins in a complex, by promoting (as an activator), or blocking (as a repressor) the recruitment of RNA polymerase (the enzyme that performs the transcription of genetic information from DNA to RNA) to specific genes

19. Repressor:
repressor is a DNA-binding protein that regulates the expression of one or more genes by binding to the operator and blocking the attachment of RNA polymerase to the promoter, thus preventing transcription of the genes. This blocking of expression is called repression.
Repressor proteins are coded for by regulator genes. Repressor proteins then attach to a DNA segment known as the operator. By binding to the operator, the repressor protein prevents the RNA polymerase from creating messenger RNA

20. Transcription in Eukaryotic Cells
Eukaryotic genes contain promoters
Promoters are much more diverse and complex
Many promoters recognized by RNA pol II include a TATA box at -30
Some promoters recognized by RNA pol II do not contain a TATA box
RNA pol I and RNA pol III interact with entirely different promoters

21. Transcription in Eukaryotic Cells
Unlike prokaryotic cells eukaryotic cells must process the mRNA in the nucleus before it can be made into a protein
Three major modifications are made
Intron splicing
Addition of poly A tail
Additon of 5’ cap

22. Exons & Introns Exons Introns Coding regions of DNA
Expressed as proteins
Introns
Non-coding regions of DNA
Intervening

23. Intron Removal
Introns are removed in a process known as intron splicing
Splicing occurs while transcription is still underway
Removed by ribozymes known as small nuclear ribonucleoproteins
snRNPs :
are RNA-protein complexes that combine with unmodified pre-mRNA and various other proteins to form a spliceosome, a large RNA-protein molecular complex upon which splicing of pre-mRNA occurs. The action of snRNPs is essential to the removal of introns from pre-mRNA, a critical aspect of post-transcriptional modification of RNA, occurring only in the nucleus of eukaryotic cells.

24. 1- Pre-mRNA splicing.
Small nuclear RNAs (snRNAs) form a complex called a spliceosome with small nuclear ribonucleoproteins (snRNPs) and other proteins.
2- The snRNAs bind to specific nucleotides in the introns of a pre-mRNA.
3- The RNA transcript is cut, releasing the introns and splicing the exons together, producing mature mRNA.

25. Adding a Cap and a Tail 5’ cap
The process of 5′ capping is vital to creating mature messenger RNA, which is then able to undergo translation. Capping ensures the messenger RNA's stability while it undergoes translation in the process of protein synthesis, and is a highly regulated process that occurs in the cell nucleus. Because this only occurs in the nucleus, mitochondrial and chloroplast mRNA are not capped.
Poly A tail
Polyadenylation is the addition of a poly(A) tail to an RNA molecule. The poly(A) tail consists of multiple adenine bases. In eukaryotes, polyadenylation is part of the process that produces mature messenger RNA (mRNA) for translation. It, therefore, forms part of the larger process of gene expression.

26. Three; each produces a different class of RNA
Comparing Transcription: Bacteria vs. Eukaryotes
Aspect
Bacteria
Eukaryotes
RNA polymerase
One
Three; each produces a different class of RNA
Promoter Structure
-35; -10
Complex and variable; often has -30
Proteins which contact promoter
Sigma; many types
Many basal transcription factors
RNA processing
None
Extensive
Intron splicing
Cap and poly A tail