Topic 7 and 8

Describe the different RNA’s and their function

Describe the role of promoters and terminators in transcription

• DNA sequences that signal the start of transcription are called Promoter

• DNA sequences signaling the end of transcription are called Terminator

• In bacteria, a subunit of the RNA polymerase, sigma factor, recognizes the promoter sequence • Once bacterial RNA polymerase starts synthesizing RNA, the sigma factor is released • Interaction of transcribed 3’ terminator sequence with RNA polymerase causes enzyme to let go of DNA template

• Promoter and Terminator regions have specific DNA sequences

• Promoter sequences have a distinct polarity (asymmetrical sequences) → determines which strand is used as template by RNA polymerase and which direction the strand is transcribed

Compare and contrast how RNA polymerase initiates transcription in eukaryotic and prokaryotic cells.

Bacteria use a single type of RNA polymerase for transcription

• Different gene architecture in bacteria (very short regions of non-coding DNA)

Eukaryotic cells have three RNA polymerases - I, II, and III

• eukaryotic RNA polymerases require support of accessory proteins = transcription factors

• plants, animals/humans (individual genes are spread out, long stretches between genes) → large variety of regulatory DNA sequences to allow for more complex regulation of transcription

• •Eukaryotic transcription factors assemble at the promoter region

• TFIID (with TBP (TATA-binding protein) subunit) binds to TATA box → causes local distortion of DNA double helix → helps to recruit/assemble other TFs and RNA polymerase II to form the transcription initiation complex

• Phosphorylation of the tail of RNA polymerase II by TFIIH enables release of polymerase from complex and starts RNA transcription

• Sequences in the promoter region promote binding of transcription factors and determine direction of transcription by RNA polymerase II

Describe the process of RNA processing

• RNA processing enzymes are recruited via the phosphorylated tail of RNA polymerase and occurs while RNA is being synthesized in the nucleus.

• RNA processing includes:

– capping (5’end)

• Includes addition of guanine nucleotide bearing a methyl-group via 5’-to-5’ triphosphate bridge

• Capping takes place before whole transcript is completed • In bacteria 5’ end is the first nucleotide of the transcript

– Polyadenylation (3’end)

• 3’ end of eukaryotic mRNA is trimmed (by cutting RNA chain) followed by addition of poly-A tail

– splicing

removes introns and ‘stitches’ exons together

• Special nucleotide sequences on either end of the introns (---AG---) with the pre-mRNA transcript serve as recognition element for splicing machinery

• Sequences are referred to as splice donor (5’ end) and splice acceptor (3’ end)

Describe the process of RNA splicing

Splicing is carried out by RNA molecules, small nuclear RNAs (snRNAs) that are packaged with protein to form small nuclear ribonucleoproteins (snRNPs)

• snRNPs recognize splice sites through complementary base pairing (between snRNAs and pre-mRNA)

• together with other proteins, for spliceosome

• Guided by splice donor and acceptor sequences, pre-mRNA introns are removed from DNA.

• A typical ‘lariat’ structure is formed by the reaction of the adenine nucleotide at the splice acceptor site

• Lariat release to join exons together.

• Alternative splicing increases coding potential to genes

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What enzyme couple tRNA to amino acid

Amino acyl- tRNA - Synthase - recognises anticodon of tRNA and attaches corresponding amino acid

Describe how mRNA message is decoded at the ribosome

Ribosome has small and large subunit

• Small is where tRNA is matched to mRNA codons

• Large is where amino acids are covalently linked into polypeptide chain

• Three binding sights in each ribosome, A site is where charged tRNA binds to mRNA, P site holds growing chain in place.

• Large subunit shifts to move spent ribosome into E site where it is ejected and process beings again.

• Ribosomes are made out o rRNAs

How is protein regulated in the cell

Proteases are specific enzymes that cut peptide bonds between amino acids

• Eukaryotic cells have protein-degrading machines called proteasomes

How do proteasomes know which protein to degrade? → Ubiquitin

• Proteins marked for destruction are covalently labelled with a ubiquitin-chain (a small protein)

How is transcription regulated?

• Many transcriptional regulators form dimers on DNA.

• specific protein-DNA interactions and specific protein-protein interactions are formed

Often a combination of activation and repression enables fine tuning of gene expression.

In Eukaryotes, Chromatin structure plays an important role and chromatin needs to be ‘opened up’ or remodelled

Transcription factors - repressors and activators that allow binding

Examples of generating specialized cell types

Single transcription regulators can trigger the formation of an organ. • The Ey gene

Fly develops eye on leg

Regulatory RNA’s

Micro RNA (Mi RNA) -regulate protein coding genes in many organisms including humans. One miRNA can regulate many genes

Small (or short) interfering RNA (siRNA) - RNA interference can be used for degradation of foreign RNAs

Long non-coding RNA (lncRNA) - used as a scaffold to attract proteins to specific sequences • LncRNA can also base pair with other RNAs