Presented BY
HAMSA.D

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Central Dogma of Molecular Biology
• The flow of information in the cell starts at DNA, which replicates to form more DNA.
• Information is then ‘transcribed” into RNA, and then it is “translated” into protein.
• The proteins do most of the work in the cell.
RNA
• RNA plays a central role in the life of the cell.
• RNA can both store information (like DNA) and catalyze chemical reactions (like proteins).
• One theory for the origin of life has it starting out as RNA only, then adding DNA and proteins later. This theory is called the “RNA World”.
• RNA/protein hybrid structures are involved in protein synthesis (ribosome), splicing of messenger RNA, telomere maintenance, guiding ribosomes to the endoplasmic reticulum, and other tasks.
• Recently it has been found that very small RNA molecules are involves in gene regulation.
RNA Used in Protein Synthesis
• messenger RNA (mRNA). A copy of the gene that is being expressed. Groups of 3 bases in mRNA, called “codons” code for each individual amino acid in the protein made by that gene.
– in eukaryotes, the initial RNA copy of the gene is called the “primary transcript”, which is modified to form mRNA.
• ribosomal RNA (rRNA). Four different RNA molecules that make up part of the structure of the ribosome. They perform the actual catalysis of adding an amino acid to a growing peptide chain.
• transfer RNA (tRNA). Small RNA molecules that act as adapters between the codons of messenger RNA and the amino acids they code for.
RNA vs. DNA
• RNA contains the sugar ribose; DNA contains deoxyribose.
• RNA contains the base uracil; DNA contains thymine instead.
• RNA is usually single stranded; DNA is usually double stranded.
• RNA is short: one gene long at most; DNA is long, containing many genes.
Transcription
• Transcription is the process of making an RNA copy of a single gene. Genes are specific regions of the DNA of a chromosome.
• The enzyme used in transcription is “RNA polymerase”.
• The raw materials for the new RNA are the 4 ribonucleoside triphosphates: ATP, CTP, GTP, and UTP.
• As with DNA replication, transcription proceeds 5- to 3’: new bases are added to the free 3’ OH group.
• Unlike replication, transcription does not need to build on a primer. Instead, transcription starts at a region of DNA called a “promoter”.
OVERALL VEIW
Process of Transcription
• Transcription starts with RNA polymerase binding to the promoter.
• This binding only occurs under some conditions: when the gene is “on”. Various other proteins (transcription factors) help RNA polymerase bind to the promoter. Other DNA sequences further upstream from the promoter are also involved.
• Once it is bound to the promoter, RNA polymerase unwinds a small section of the DNA and uses it as a template to synthesize an exact RNA copy of the DNA strand.
• Cont…
• The DNA strand used as a template is the “coding strand”; the other strand is the “non-coding strand”. Notice that the RNA is made from 5’ end to 3’ end, so the coding strand is actually read from 3’ to 5’.
• RNA polymerase proceeds down the DNA, synthesizing the RNA copy.
• In prokaryotes, each RNA ends at a specific terminator sequence. In eukaryotes transcription doesn’t have a definite end point; the RNA is given a definitive termination point during RNA processing.
Reverse Transcription
• Most importantly, some RNA viruses, called “retroviruses” make a DNA copy of themselves using the enzyme reverse transcriptase.
• The DNA copy incorporates into one of the chromosomes and becomes a permanent feature of the genome. The DNA copy inserted into the genome is called a “provirus”. This represents a flow of information from RNA to DNA.
• Closely related to retroviruses are “retrotransposons”, sequences of DNA that make RNA copies of themselves, which then get reverse-transcribed into DNA that inserts into new locations in the genome.
• Unlike retroviruses, retrotransposons always remain within the cell. They lack genes to make the protein coat that surrounds viruses.
FOOT PRINTING
• Principles used in DNA sequencing, identifies the DNA sequence bound by a particular protein
PROKARYOTIC TRANSCRIPTION
PROKARYOTIC RNA POLYMERASE
a. E.coli has a single DNA directed RNA polymerase that synthesizes all types of RNA
b. It is a large and complex enzyme with molecular weight of about 4,50,000
c. Multienzyme has five core subunits
α – (two subunits ) – 36,500 Daltons
β - 1,51,000 Daltons
β’ – 1,55,000 Daltons
ω - 11,000 Daltons
d. RNA polymerase performs multifunctions
e. RNA polymerases, whether from E.coli or other organisms lack a proof reading 3’ 5’ exonuclease activity such as that found in many DNA polymerases
f. Enzyme does not require any primer
PROMOTORS OF PROKARYOTIC DNA
• RNA polymerase binds to specific sequences in the DNA called promoters
a. Consensus sequence of (5’)TATAAT(3’) at -10 region
b. Consensus sequence of (5’)TTGACA(3’) at -35 region
c. AT rich recognition element – up(upstream promotor) element occur between -10 and -60 in the promotors of certain highly expressed genes
STEPS IN PROKARYOTIC TRANSCRIPTION
• Initiation
• Elongation
• termination
Factors involved in initiation
1. Polymerase sits on non-specific DNA
2. Sigma subunit searches and finds promoter
3. Polymerase binds to -35 region
3. B and B’ bind tightly to DNA
4. DNA begins melting at -10 region
5. Complementary nt are added and bonded at +1
6. Sigma subunit falls off polymerase
Elongation
• Transcription occurs in 5’-3’ direction
• Asymmetrical
Termination
• Intrinsic Terminators
GC rich region in DNA
Allows hairpin in RNA
• Cause polymerase to stall
• All subunits of polymerase fall apart
• mRNA is free
• DNA recoils
• - rho dependent terminators (involving a protein factor)
- rho independent terminators (terminatiom occurs at hairpin structures
Rho-independent terminator
• Rho-independent terminator contains a short inverted repeat (~20 bp) and a stretch of ~8 A:T base pairs.When itis followed by a stretch of A:U base pairs ,they can form a stem-loop structure called "hairpin" by base-pairing itself.Hairpin is a factor to cause termination by disrupting the elongation complex.
• Rho -dependent terminators
• Have less well-characterized RNA elements, and requires Rho protein for termination
• Rho is a ring-shaped single-stranded RNA binding protein, like SSB
• Rho binding can wrest the RNA from the polymerase-template complex using the energy from ATP hydrolysis
• Rho binds to rut (r utilization) RNA sites
• Rho does not bind the translating RNA