Bacterial genes can be divided into three parts: 1.

Promoter: a sequence upstream of the start of the gene which orients the RNA polymerase to start transcription at the start of the gene and in the right direction. 2. RNA-coding sequence: It is the DNA sequence transcribed by the RNA polymerase into the RNA transcript. 3. Terminator: specifies where transcription stops Initiation From comparisons of sequences upstream of coding sequences and from studies of the effects of specific base-pair mutations upstream of transcription initiation sites, two promoters in E. Coli have been shown: 1. - 35 box 2. - 10 box (Pribnow box) Complete RNA polymerase (holoenzyme) in bacteria is made of core enzyme and a sigma factor. The core enzyme is made of four subunits: 2 alpha, 1 beta, 1 beta prime. (There is only ONE RNA polymerase in E. Coli.) The sigma factor is only required for initiation, not elongation nor termination. The RNA polymerase, through the sigma factor, first binds to - 35 box and then fixes to all of promoter. The DNA is still annealed (closed promoter complex). The untwisted form of the promoter is called the (open promoter complex). Not all promoters have - 35 and - 10 recognition sequences. There are other sigma factors for other promoters that initiate transcription of genes under different conditions. (heat shock genes) Transcription of bacterial genes is regulated by the interaction of regulatory proteins with regulatory sequences upstream of RNA-coding sequence. There are two classes of regulatory proteins: activators and repressors. Elongation After 8 to 9 nucleotides have been polymerized, the sigma factor dissociates from the core enzyme. The RNA polymerase becomes more compact because of this. About 9 bases (9 hybrid base-pairs in untwisted region) of the new RNA strand remain bound to the enzyme as elongation proceeds with the remainder exiting in a single-strand form. RNA polymerase has two proofreading activities: 1. 3'5' exonuclease activity 2. Going backward one or more nucleotides and cleaving them.

Termination It is signaled by a terminator sequence. There are two types of terminators in E. coli: Rho - dependent terminator (type II) and Rho - independent terminator (type I). Type I: It consists of an inverted repeat sequence upstream of transcription termination point. This leads to the formation of a hairpin loop structure made of G-C neck region. This slows down the RNA polymerase and causes it to stop. A string of U nucleotides downstream destabilizes the base pairing between newly synthesized RNA and the DNA, and hence the polymerase dissociates. Type II: Rho protein (helicase) binds to the C rich terminator sequence upstream of the termination site and moves after the polymerase until it catches it and unwinds the hybrid base pairs causing the polymerase to dissociate. The helicase hydrolyses ATP for energy. (Rho also dissociates).

Transcription in eukaryotes:
Three RNA polymerases transcribe genes for four main RNAs. RNA polymerase I is in the nucleolus and is for rRNA (all except 5s). RNA polymerase II is in nucleoplasm and is for mRNA and most snRNA. RNA polymerase III is in nucleoplasm and is for tRNA, 5s rRNA and the rest of snRNA. Promoters Two regions of promoters: promoter proximal elements and core promoter. Core promoter: set of cis-acting sequence elements till 50 upstream needed for the transcription machinery to start RNA synthesis at the correct site. Best-characterized core promoter elements are the Inr (initiator) which is a short sequence that spans the transcription initiation site (+1) and the TATA box which is made of 7 nucleotides. Both of these specify where the transcription machinery assembles and where transcription begins. However other elements are needed to maximize transcription. Prompter-proximal elements: from - 50 till - 200. There are two well-defined ones: the CAAT "cat" box and the GC box. These play a role in determining the efficiency of the promoter. Various combinations of core elements and proximal elements determine how and when a gene is expressed. Enhancers Enhancers are also cis acting elements which can be downstream, upstream or in the gene. These maximize the level of transcription by getting close to the transcription machinery.

Transcription initiation RNA polymerases in eukaryotes cannot manage alone. A complex called PIC (pre initiation complex) or the complete transcription initiation complex should be assembled. PIC is made of GTFs (general transcription factors) and RNA polymerase II. First TFIID binds to the TATA box. This forms a binding site for other TFIIs. Next RNA polymerase II binds and finally TFIIH binds. TFIIH has helicase-like activity and hence unwinds the core promoter DNA initiating transcription. mRNA: Mature mRNA is made of 3 parts: - 5' UTR (leader sequence) - protein-coding sequence - 3' UTR (trailer sequence) In Prokaryotes, transcription and translation are coupled (no nuclear membrane). Also Prokaryotic mRNA is polycistronic whereas that of Eukaryotes is monocistronic. To process a precursor mRNA (pre mRNA) into a mature mRNA, there are three steps: 1. 5' capping (during transcription) 2. 3' poly A tail addition (at the end of transcription) 3. Intron splicing and exon ligation 5' capping: A 7-methyl Guanosine is added to the 5' side by a 5'-5' linkage by a capping enzyme. It's important to: 1. protect of 5' end mRNA from degradation by exonucleases 2. bind to the ribosome fro translation 3' poly A tail addition: Poly A tail = 50-250 adenines added from ATPs by PAP (poly(A) polymerase). The tail is not encoded by DNA, and it is not removed in the mature mRNA. mRNAs with the poly A tail are called poly(A) + mRNAs. It's important to: 1. protect 3' end of mRNA from degradation by exonucleases 2. transport the mRNA to the nucleus for translation It's signaled when the machinery passes the poly(A) site 20-30 nucleotides downstream of the poly(A) consensus sequence 5'-AAUAAA-3'. A number of proteins (CPSF, CstF, CFI and CFII) bind to and cleave the mRNA at the poly(A) site. Then, PAP does its job!

The post poly(A) site transcription terminate by the action of exonuclease which binds to it and degrades it until it catches up with the machinery destabilizing the enzyme-TF-DNA complex. Intron splicing The splicing takes place within the spliceosome. It's a complex of the pre-mRNA, snRNAs and proteins. The snRNAs with proteins form the snRNPs (snurps). There are five main snRNAs which are U1, U2, U4, U5 and U6. U4 and U6 form a snurp together with a protein whereas each of the rest form a snurp alone 1. U1 snurp binds to the 5' GU. 2. U2 snurp binds to the branch point sequence. 3. U4/U6 snurp interact with U5 snurp, and the combination binds to U1 and U2. This causes the intron to loop. 4. U4 leaves leading to active spliceosome. 5. The intron is excised at 5' and binds to the adenine of branch-point sequence by 5'-2' linkage giving lariat structure. 6. The spliceosome excises the intron at 3' end, and the snRNPs are released. In this event, there are RNA-RNA, RNA-protein and protein-protein interactions. There is Alternative Splicing, which is when different mRNAs are produced from the same gene.

Self-splicing protein-independent reaction for group I intron in Tetrahymena pre-rRNA The intron RNA sequence has enzymatic activity, but it is not retained at the end as protein enzymes. These RNA enzymes are called Ribozymes. Mechanism of group I self-splicing introns: 1. The pre-rRNA is cleaved at the 5' splice junction and Guanosine is added to the 5' end of the intron. 2. The intron is spliced at the 3' splice junction. 3. The two exons are spliced. 4. The excised intron circularizes into a lariat molecule, which is cleaved to produce a circular RNA and a short, linear piece of RNA. Group II introns also self-splice using a different mechanism.

RNA editing It is posttranscriptional insertion or deletion of nucleotides or conversion of one base to another. The functional RNA molecule has a base sequence that does not match the base-pair